Intragonadal regulatory protein

A protein having a molecular weight of from about 10,000 to 18,000 daltons, isoelectric points of from about pH 4.0 to 6.5 and having the reversible biological effect of inhibiting aromatase activity in a biological system, and antibodies to the protein, modulate follicular development and spermatogenesis and provide for diagnostic tests of gonadal functions.

FIELD OF THE INVENTION 
This invention relates generally to the field of biochemistry, and more 
particularly to the chemistry and physiology of gonadal function. 
BACKGROUND AND SUMMARY OF THE INVENTION 
The term gonad refers to the mammalian reproduction system, and includes 
both the testis and the ovary. The function of these glands is dependent 
upon the interrelationship of a number of hormones and proteins, many of 
which are found, in varying amounts, in both sexes. 
The ovary is the essential female reproductive organ in which eggs are 
produced. In vertebrates there are commonly two ovaries, suspended from 
the dorsal surface of the broad ligaments, one on each side of the uterus. 
Adult human ovaries are composed of a fibrous vascular stroma in which are 
imbedded the Graafian follicles, which contain the eggs. The eggs are 
discharged by the bursting of the Graafian follicles on the surface of the 
ovary and are then immediately received into the mouth of the oviduct. 
Thereafter, the eggs flow through the fallopian tube to the uterus, 
covered by a mucous membrane known as the endometrium, where the 
fertilized ovum develops. 
The egg cells or ova are periodically matured in the ovaries at intervals 
of approximately four weeks. At the end of each four week period, one egg 
reaches maturity and passes into one of the fallopian tubes. The egg 
descends gradually and remains viable for a short while, and during this 
time fertilization may take place. 
Within the ovary, there is a layer of cells called the germinal epithelium. 
Here, the potential egg begins its existence and continues to develop 
until a primary follicle, i.e., a group of cells isolated from the main 
layer, is formed around the potential egg. During a lifetime, each human 
ovary forms between 200,000 and 400,000 follicles. Of all these potential 
eggs only a few develop into mature eggs, most of them degenerating. The 
primary follicle that does not degenerate increases in size, and the egg 
cell itself enlarges up to thirty times its original size. 
Other changes occur in the areas adjacent to the follicle. As the follicle 
matures, it moves toward the surface of the ovary and when the maturation 
process is complete, the follicle protrudes from the surface. At this 
time, ovulation occurs, i.e., the follicle bursts and the egg is expelled 
from the surface of the ovary. 
The deveIoping follicle produces sex hormones by metabolizing pre-hormones 
using a series of enzymes: 3.beta.-ol dehydrogenase, 
17.alpha.-hydroxylase, hydroxysteroid dehydrogenase, and aromatase. 
Aromatase is a central enzyme in the production of sex hormones referred 
to as estrogens (estradiol, estrone and estriol). 
Estrogenic hormones play a particularly important role in both the 
menstrual cycle and the reproductive cycle. Although 17.beta.-estradiol is 
the principal estrogenic hormone, a number of other estrogenic substances 
have been isolated, including estriol and estrone. These hormones induce 
the growth of the vaginal epithilium, secretion of mucous by the glands of 
the cervix and initiate the growth of the endometrium. 
The corpus luteum, which fills a ruptured Graafian follicle in the 
mammalian ovary, produces at least three hormones, progesterone, 
17.beta.-estradiol and relaxin. Progesterone acts to complete the 
proliferation of the endometrium, which was initiated by the estrogenic 
hormones, and to prepare it for the implantation of the ovum. 
This reproductive cycle is well regulated as long as the production and 
secretion of both the sex hormones from the ovaries and the gonadotropic 
hormones of the pituitary gland are within normal limits. The anterior 
lobe of the pituitary, by manufacturing and secreting the gonadotropic 
hormones, controls the production of the sexual hormones in the ovaries 
and stimulates the development of the reproductive organs and the 
maintenance of their structure. The ovaries, under control of the 
gonadotropic hormones, produce the female sexual hormones. In turn, the 
rate of production of gonadotropins by the pituitary is influenced by the 
production of sex hormones. The effects are mutual and the two glands 
maintain an exact balance in hormone production. 
More specifically, the follicle-stimulating hormone (FSH) from the 
pituitary stimulates the Graafian follicles, which thus produce estrogens. 
Estrogens not only inhibit FSH production through negative feedback on the 
pituitary, but also stimulate the pituitary to increase its production of 
luteinizing hormone (LH) through positive feedback. This hormone (LH) in 
turn brings about ovulation of the Graafian follicle. After the ova are 
discharged, the LH stimulates the empty follicle, now the corpus luteum, 
to produce progesterone. This hormone brings about the changes in the 
reproductive organs required for the development of the embryo. The 
progesterone then partly inhibits the pituitary from producing more LH. 
Thus, there is no further ovulation. The subsequent fall in progesterone 
then releases the pituitary inhibition allowing for the production of FSH 
to begin the process anew. 
When pregnancy occurs, the placenta of the embryo itself produces human 
chorionic gonadotropin (hCG), which stimulates the continuing production 
of progesterone from the corpus luteum, thus preventing menstruation and 
stimulating the continuing development of the uterus. This progesterone 
also inhibits further ovulation in spite of the presence of the hCG from 
the placenta. 
The gonadotropic hormones have been determined to be proteins, with 
variable amounts of carbohydrates, and their structures are known. The 
molecular weight of human LH is about 26,000, and that of human FSH is 
about 30,000. The cellular response to gonadotropic hormones is translated 
through cellular receptors. These receptors are located within the cell 
membrane and are specific for each gonadotropin, thus, LH only activates 
LH receptors and FSH only activates FSH receptors. 
Non-abortifacient means for the avoidance of pregnancy include oral 
contraceptive medications which contain estrogen and/or progesterone-like 
steroidal sex hormones. These medications, by raising the level of sex 
hormones in the blood stream, generate a cervical mucous which is hostile 
to spermatazoa. With increased levels of such hormones the endometrium 
tends to resist implantation of the fertilized ova. Further, the excess 
hormones provided by oral contraceptives directly inhibit the release of 
LH and FSH by the pituitary. As such, the ovarian cycle is disrupted and 
ovulation does not occur. The complications of such steroidal 
contraceptive medications, e.g., nausea, vomiting, weight gain, 
hypertension and tumor stimulation, adverse effects on calcium and 
phosphate metabolism, are well known and need not be discussed at length. 
However, these side effects result not only from the abnormally high 
levels of steroidal hormones in the blood stream, but from disruption of 
the hormonic homeostatis of the organism, i.e., the cycle of hormone 
adjustment between the ovary and the pituitary gland. 
Accordingly, it has been a desideratum to provide a contraceptive 
medication which permits the regulation of the ovarian process without an 
accompanying disruption of the hormonic homeostatis of the organism. 
While sex hormones are commonly referred to, as male (androgenic) and 
female (estrogenic) hormones, these substances, including the 
proteinaceous substances associated therewith, are each important in the 
regulation of both the male and the female reproductive systems. For 
example, the luteinizing hormone (LH) and follicle stimulating hormone 
(FSH) play important roles in the regulation of the testis. These 
gonadotropins are synthesized and released in the male pituitary under the 
regulation of a hypothalamic peptide (luteinizing hormone releasing 
hormone) which is synthesized in the hypothalamus. LH binds to the surface 
receptors on the Leydig cells and promotes increased testosterone 
synthesis. It should be noted that testosterone synthesis is also 
regulated by the prevailing estradiol concentration, with high estradiol 
levels decreasing testosterone synthesis. 
Spermatogenesis is a complex event where primitive germ cells (the 
spermatogonia) proceed through multiple cell divisions, in an orderly 
progression from spermatogonia to spermatocyte to spermatid, in order to 
form mature spermatozoa. In this progression, the intergenderal 
communality of the regulatihg hormones and proteins in the gonads is 
demonstrated by the fact that estrogen is a necessary component in the 
conversion of spermatogonia to spermatocytes. Further, the enzyme 
aromatase is a central enzyme in the conversion of androgens to estrogens. 
It should be noted that aromatase is a paracine (intragonadal) enzyme that 
has been shown to be critical to the regulation of fertility in both the 
male and female gender of mammalian species. 
It has also been a desideratum to provide a facile method for the 
regulation of fertility in both the male and female gender of mammalian 
species. It should be understood that the regulation of fertility, as 
employed herein, refers to a method and/or a composition of matter 
associated therewith, which enables either an increase or a decrease in 
mammalian fertility, that is, fertility control as opposed to mere 
contraception. 
The present invention accomplishes the foregoing objectives by providing a 
protein moiety which enables the intragonadal modulation of the level of 
aromatase in a mammalian host and thus can regulate the maturation of 
ovarian follicles and the production of viable ova and regulate 
spermatogenesis and the production of mature spermatozoa without 
disturbing the normal level of sex hormones in the body, and permit the 
evaluation and diagnosis of gonadal function and dysfunction. 
According to the invention a purified, non-steroidal proteinaceous material 
or moiety having a measurable molecular weight of up to about 20,000, an 
isoelectric point of from about pH 3.5 to about pH 7.0 is provided, and is 
characterized by having the biological effect of inhibiting aromatase 
activity as defined by the extent of the conversion of androgens to 
estrogens. The protein inhibits intragonadal aromatase activity, modulates 
the intragonadal activity of 3.beta.-ol dehydrogenase, inhibits the 
development of granulosa cell LH receptors and prevents the maturation of 
mature ova and the production of mature spermatozoa. The term protein 
moiety, as used herein, refers to a protein, proteins or functional 
operative groups thereon which produce the described results. The 
production and activity of the protein moiety is interspecies, and is 
effective in both monotocous and polytocous mammals. Further, methods are 
provided for the isolation, purification and production of the protein, 
and for the use thereof in the regulation of fertility control. 
In another aspect of the invention, antibodies are provided which inhibit 
the natural production of the protein with a corresponding increase in 
aromatase activity resulting in the promotion of follicular development, 
ovum maturation and spermatogenesis. Thus, mammalian fertility may be 
controlled substantially independently of exogenous sex hormones and 
without modulating extragonadal hormone levels. In yet another aspect of 
the invention, methods are provided for the use of antibodies to the 
protein for the quantification of the level of the protein in body fluids, 
thus permitting the evaluation and diagnosis of gonadal function and 
dysfunction. 
For convenience, the intragonadal and follicular regulating protein is 
referred to herein as FRP.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
The embodiment hereinafter set forth relates to the isolation and 
purification of the intragonadal protein moiety and its use in the 
inhibition of the activity of the aromatase enzyme, modulation of 
3.beta.ol dehyrogenase activity, regulation of mature ova and sperm 
formation and the production of the protein moiety by cell cultures. 
The protein moiety is isolated from the ovary or the testis by methods such 
as salt fractionation and dialysis, and chromatography. Granulosa or 
Sertoli cells may be cultured to produce substantial amounts of the 
protein moiety, which is then similarly purified. 
Generally, gonadal fluid may be obtained by compression of appropriate 
mammalian gonads, and purified by chromatographic separation to yield FRP, 
although initial salt fractionation and subsequent electrophoretic 
separation vastly improve the separation procedure. 
More specifically, FRP may be extracted from biological fluids including 
ovaries, testis, rete testis fluid, lymph, blood, and ovarian follicular 
fluid from mammalian species. To illustrate, a preferred process for the 
separation and purification of FRP as set forth in the Examples is 
summarized as follows: 
1. Biological fluid is mixed with saturated 
ammonium sulfate to achieve a 30-55% concentration. 
2. The precipitate from step 1 is re 
suspended in TRIS buffered saline (20 mM, pH 7.5) 
and dialyzed against 3 changes of distilled water (36 
hours, 4.degree. C.) to remove the remaining ammonium sulfate. 
3. The retentate from step 2 is passed through a hydroxylapatite column 
which is subsequently washed with 2 column volumes of TRIS buffered saline 
(20 mM, pH 7.5). The column is then eluted in a stepwise fashion with 0.1 
mM phosphate buffer followed by 0.5M phosphate buffer . 
4. The eluent from the 0.5M phosphate buffered saline is passed through a 
Sephacryl G-200 column and eluted with TRIS buffer (20 mM, pH 7.5). 
5. The eluent from step 4 which elutes in the &lt;40,000 molecular weight 
range is charged onto a Matrix gel Orange A column which was previously 
equilibrated with TRIS buffer (20 mM, pH 7.5). The Matrix gel Orange A 
column is washed with 3 column volumes of TRIS buffer and then eluted with 
TRIS buffer (20 mM, pH 7.5) to which has been added 0.5M KCl. 
6. The TRIS +0.5M KCl eluant from step 5 after dialysis is charged onto a 
hydrogen ion exchange column previously equilibrated with imidazole buffer 
(0.25 M, pH 7.4). The column is eluted with buffer over the pH range 7.4 
to 3.6. 
7. The eluent fractions corresponding to about pH 7.4 to pH 3.6 from step 6 
are eluted through a TSK 3000 column with TRIS buffer (pH 7.5, 20 mM) by 
high performance liquid chromatography. The fraction corresponding to 35 
to 37 minutes in FIG. 8 (analytical column) and 77.8 to 78.0 minutes in 
FIG. 9 (preparative column) contain FRP. 
8. The TRIS +0.5M KCl eluent from step 5 is layered on an agarose gel or 
Sephadex G-75 column which have been previously charged with ampholines 
(pH 3-8). A current is passed through the support matrix of sufficient 
length and wattage to allow equilibration of proteins at their isoelectric 
points. The fractions recovered from these electrophoretic separations 
corresponding to isoelectric points of from pH 3.5 to ph 7.0 contain FRP. 
9. The TRIS +0.5M KCl eluent from step 5 can be charged onto a hydrophobic 
reverse phase column. The column can be eluted with increasing 
concentrations of organic reagents (acetonitrile, trifluroacetic acid). 
FRP can be separated from the other components of step 5 eluent in this 
fashion using differential solubility to organic solvents. 
The biological effect of the moiety was assessed in laboratory animals by 
determining variations in ovarian weight and analyzing body fluids for 
steriod hormones and enzyme activities. Aromatase inhibition was 
determined by incubating granulosa cells in the presence of a substrate 
and FRP and measuring the amount of estrogen formed. The 3.beta.ol 
dehydrogenase was found to be modulated by incubating granulosa cells in 
the presence of a substrate and GRP and measuring the amount of 
progesterone formed. The addition of labelled LH and FSH to in vitro 
granulosa cell cultures showed a decrease in LH receptors in the cells, 
and no change in FSH receptors grown in the presence of FRP. 
Regularly ovulatory primates were exposed to FRP and in all instances the 
ovarian cycle was modified, along with follicular growth, as evidenced by 
estrogen and progesterone levels, without the concomitant alteration of LH 
and FSH levels. In subsequent cycles, the primates had timely menstrual 
onset and showed no toxic treatment effects. Male animals were injected 
with FRP and exhibited reduction of spermatogenesis. genesis. 
Pharmaceutically effective amounts may be administered to individuals 
orally, by injection, membrane absorption or other means which will be 
apparent to those skilled in the art. A particularly advantageous method 
of administration comprises the introduction of an effective amount of the 
moiety to the absorbent mucous membranes. 
Since FRP inhibits aromatase and normal follicle maturation, it plays a 
central role in the ovulatory process. Accordingly blockage of the GRP 
activity allows for the maturation of multiple follicles to ovulatory 
status. The protein may be coincubated with hybrid cells in vitro to 
produce antibodies to proteins from different species. The antibody 
produced by the hybrid cultures and collected by chromatography may be 
injected into females thus blocking the action of the follicular protein 
and allowing for development and eventual ovulation of one or more 
follicles. 
Agricultural uses of a similarly prepared antibody allow for the maturation 
of multiple follicles in livestock which could be collected, fertilized in 
vitro and reinserted into a properly prepared surrogate. FRP antibodies 
may also be employed to quantify the level of existing FRP in body fluids 
for a wide variety of diagnoses. 
EXAMPLES 
The following examples will describe in detail the identification, 
separation, activity and effects of the purified intragonadal regulatory 
protein, according to the following outline: 
I. Identification of the Sources and Physical Characteristics of FRP. 
A. Ovarian Venous Blood (Example One). 
B. Human Follicular Fluid (Example Two). 
C. Porcine and Bovine Follicular Fluid (Example Three). 
D. Granulosa Cells and Cell Culture (Example Four). 
E. Testis Fluid (Example Five). 
F. Sertoli Cells and Cell Culture (Example Six). 
II. Biological Activity 
A. Aromatase Inhibition (Example Seven). 
1. Non-Competitive Inhibition (Example Eight). 
B. Modulation of 3.beta.-ol-dehydrogenase Activity (Example Nine). 
C. Inhibition of LH-hCG Receptor Formation (Example Ten). 
D. Inhibition of FSH Augmented Adenylate Cyclase Activity (Example Eleven). 
E. Reduction of Follicular Atresia (Example Twelve). 
III. Whole Animal Studies 
A. Inhibition of Primate Ovarian Cycles (Example Thirteen). 
B. Inhibition of Spermatogenesis (Example Fourteen). 
IV. Preparation of FRP Antibodies (Example Fifteen) and use in diagnostic 
procedures 
I. Identification of the Sources and Physical Characteristics of FRP 
EXAMPLE ONE 
Identification of Follicular Inhibitory Protein(s) in Ovarian Venous Blood 
Ovarian venous blood (5 ml) was collected from six women (aged 26, 28, 31, 
36, 37 and 41 years) undergoing laparotomy for indications not related to 
ovarian dysfunction on days 12-14 after the onset of the last menstrual 
period. Patients 1-3 maintained regular menstrual cycles, while patients 
4-6 were anovulatory, as evidenced by oligomenorrhea and a lack of large 
antral follicles in the ovarian cortex. A 25-gauge needle was inserted 
into the venous drainage within the infundibulo-pelvic ligament, and 
free-flowing blood was aspirated. In addition, the locus of the 
preovulatory follicle was determined by direct visual inspection. 
Peripheral serum (10 ml) was collected concurrently from an antecubital 
vein. Serum was separated by centrifugation (800 x g for fifteen minutes) 
of the clotted specimen and stored frozen (-35.degree. C.) until 
fractionation. Concentrations of 17.beta.- estradiol in ovarian (1340, 
886, and 470 pg/ml) and peripheral (248, 261, and 201 pg/ml) venous 
samples collected from patients 1, 2 and 3, respectively, were consistent 
with the preovulatory 17.beta.-estradiol levels reported for normal women. 
In addition, comparable samples from anovulatory patients 4-6 contained 
low levels of 17.beta.-estradiol in both ovarian (200 pg/ml) and 
peripheral (50 pg/ml) sera. Slowly thawed serum was fractionated by the 
dropwise addition of an equal volume of saturated ammonium sulfate under 
persistent agitation at 4.degree. C. After twelve hours, the precipitate 
was resuspended (2:1, vol/vol) with 10% ammonium sulfate. After twelve 
hours of additional agitation and centriguation (3000.times.g for 30 
minutes), the supernatant was dialyzed with 10,000 molecular weight 
exclusion membranes against Dulbecco's phosphate-buffered saline (PBS; 
0.025 M; pH 6.8) for 16 hours. The retentate was passed through a 
Concanavalin A linked Sepharose 4B (Con A ) column (5 ml; Pharmacia, 
Piscataway, N.J.) which was washed with 5 vol 0.5 M NaCl, pH 7.4, then 
further eluted with 0.5 M .alpha.-methyl-D-mannoside in PBS at a flow rate 
of 20 ml/h at 4.degree. C. 
Additional fractionations were performed where indicated on a Sephadex G-25 
(superfine) column (1.6.times.50 cm; Vo =60 ml; 5 ml/h; 4.degree. C.) with 
PBS. All Sephadex molecular weight seiving was performed using a reverse 
flow technique. Both Sephadex G-50 and G-25 were prepared according to the 
instructions of the manufacturer and equilibrated in the elution buffer. 
To increase resolution, the smallest of the Sephadex beads were removed by 
direct pipetting of the surface before degassing and column packing (10 mm 
H.sub.2 O). Elution profiles were determined using an ISCO absorbance 
meter at 254 nm. 
The activity was assessed in twenty-three-day-old Sprague-Dawley rats 
(45-55 g) 2 days after hypophysectomy which were kept at 25.degree. C. 
with intervals of fourteen hours of light and ten hours of darkness. 
Animals were caged in groups of three and given rat chow and water ad 
libitum. Silastic implants containing DES (diethylstilbestrol) were 
prepared as follows. Ten grams of Silastic (TM) polymer were mixed with 3 
g DES for thirty minutes at 16.degree. C.; thereafter, four drops of 
stannous octoate catalyst were added, with an additional ten minutes of 
mixing. The material was passed through a Luer-lock syringe (id, 1 mm) 
into a steaming (95 C) 0.9% NaCl water bath and annealed for two hours. 
DES-containing Silastic implants (1.times.5 mm) were inserted sc in the 
hypophysectomy incision forty-eight hours before assay. The assay design 
consisted of three rats at each dose of reference preparation and unknown. 
Forty-eight hours after hypophysectomy, animals were given either varying 
concentrations of gonadotropins (LH:FSH, 1:1) dissolved in 0.15 M NaCl 
with 1% bovine serum albumin and/or equal volumes of test fractions in two 
divided daily doses. Twenty-four hours after the initial injection, 
animals were sacrificed by decapitation, and ovaries were removed, trimmed 
and weighed on a Roller-Smith balance. Rat trunk serum 17.beta.-estradiol 
determinations were performed by methods described in Goeblesmann et al., 
in Leprow et al (Eds.) Vasectomy: Immunological and Pathologic Effects in 
Animals and Man, Academic Press, New York, p. 165. Control determination 
of chromatography fractions containing inhibitory activity was performed 
by heating (56.degree. C.; for thirty minutes) or trypsin digestion (20 
mg/100 ml) of representative samples for 4 hours. 
FIG. 1 compares absorbency, at 254 nm, of the Sephadex G-50 fractions from 
a preovulatory ovarian venous sample of patient 1 to the bioassay results 
of the sample, as determined by rat ovarian weight and serum 
17.beta.-estradiol concentrations. An initial peak rose to 28 absorbency 
units, followed by a gradual downward slope, with the emergence of a 
smaller second peak (V.sub.e /V.sub.o =1.42-1.55). When these eluents were 
tested in the bioassays, the combined rat ovarian weights ranged from 
57-100 mg, and rat serum 17.beta.-estradiol levels ranged from 70-230 
pg/ml throughout the initial fractions. Thereafter, fractions with a 
V.sub.e V.sub.o of 1.42-1.55 corresponded to an inhibition of hMG-induced 
ovarian stimulation in the bioassay, as evidenced by a decrease in ovarian 
weight (59.+-.0.5 mg) and a significant (P&lt;0.01, by paired t test) 
decrease in serum 17.beta.-estradiol to levels less than 25 pg/ml. As a 
consequence, these fractions were pooled and processed for dose response 
activity. Peripheral and ovarian venous blood collected from the ovary 
contralateral to the site of ovulation in patient 1 demonstrated similar 
G-50 elution profiles (data not shown). However, when representative 
fractions were tested by bioassay, no reduction in ovarian weight or serum 
17.beta.-estradiol was found. Further, ovarian venous blood preparations 
from the anovulatory patients also failed to suppress the response of the 
ovaries to hMG stimulation. However, ovarian venous sera from the 
ovulatory ovary of patients 2 and 3 had a similar Sephadex G-50 elution 
profile. Fractions with a V.sub.e V.sub.o of 1.48-1.60 suppressed the 
response of rat ovarian weight (57.4.+-.2.1 vs. 81.2.+-.4.5 mg; P&lt;0.05) 
and serum 17.beta.-estradiol concentrations (25 vs. 68-120 pg/ml; P&lt;0.01) 
to hMG stimulation. When active fractions from the G-50 eluents of 
patients 1-3 were heated or trypsin digested, they lost their ability to 
suppress ovarian weight or 17.beta.-estradiol secretion in response to hMG 
stimulation. 
FIG. 2 depicts the dose-response curve of ovarian weight suppression in the 
DES-treated rat ovaries by active Sephadex G-50 fractions (V.sub.e 
/V.sub.o =1.42-1.55) derived from patient 1. Analysis of the rat ovarian 
weight and serum 17.beta.-estradiol concentrations revealed (insert: 
central tendency ovarian weights), a linear dose-response pattern. When 
these same fractions were treated with heat or trypsin, no suppression of 
ovarian weight was present. 
The isoelectric point of active fractions eluted from the Sephadex G-50 
column was estimated by ampholyte displacement chromatography. Pooled 
aliquots (1 ml) of active fractions were layered on a Polybuffer Exchanger 
94 (25 ml; equilibrated to pH 7.4 with 0.025 M Imidazole HCl) column 
(0.6.times.30 cm). Fractions were eluted with Pharmalyte(.TM.) 
Polybuffer(.TM.) 74-HCl adjusted to pH 4.0 with HCl 1 N at 10 ml/h at 4.C 
Fractions that eluted at a pH greater than 7.4 were collected and 
rechromatographed in the same Pharmalyte column reequilibrated to pH 9.4 
with ethanolamine 0.025 M and eluted with Polybuffer 96 adjusted to pH 6.0 
with 1 N KOH. 
FIG. 3 compiles the active Sephadex G-25 fractions (V.sub.e /V.sub.o 
=1.20-1.25) pooled and developed by ampholyte displacement chromatograms 
from patient 1 for elution ranges pH 9-4. The bioassay results from 
ovarian weight and serum 17.beta.-estradiol concentrations from 
representative fractions suggested that the isoelectric point of the 
active Sephadex G-50 eluents from patient 1 were between pH 6.2-6.5. 
Fractions corresponding to a pH of 6.1-6.5 manifest inhibitory activity in 
excess of 50% tested in the bioassay with hypophysectomized, 
twenty-three-day-old, DES-treated rat ovaries receiving hMG therapy 
(ovarian weight). Ampholyte displacement chromatography of the fractions 
with follicle-inhibiting activity was obtained on Sephadex G-25 gel 
filtration fractions with V/V.sub.o =1.20-1.25. 
FIG. 4 depicts the K.sub.av values for the molecular weight standards and 
active Sephadex G-50 fractions from patients 1-3. Estimations of molecular 
weight ranged from 14,000-16,500 for patient 1, from 14,000-17,000 for 
patient 2, and from 16,000-18,000 for patient 3. 
Thus, it is seen that at least one protein which suppresses the follicle 
response to gonadotropins is secreted by the preovulatory ovary. 
Specifically, a heat- and trypsin-labile substance is secreted directly 
into the venous drainage from the ovary containing the dominant follicle 
which suppresses the follicular response to gonadotropins. That this 
protein is not secreted in large amounts by anovulatory ovaries was 
evidenced by the failure of the bioassay to detect inhibitory activity in 
the venous drainage of the contralateral ovary of patients 1-3 as well as 
the ovarian venous effluents from three anovulatory women. This potential 
intra- and/or interovarian regulator of folliculogenesis mediates 
dominance of the preovulatory follicle by an active process, such that 
after the selection of the dominant follicle, the gonadotropin 
responsivity of other follicles on the same and contralateral ovaries is 
suppressed. 
EXAMPLE TWO 
Identification of Follicular Regulatory Protein(s) in Pooled Human 
Follicular Fluid 
To evaluate the role of nonsteroidal, follicular fluid proteins in 
folliculogenesis, the 10-55% saturated ammonium sulfate fraction of pooled 
human follicular fluid was dialyzed against 0.025 M Tris/HCl(pH 7.5) using 
10,000 molecular weight exclusion membranes, then passed through agarose 
immobilized textile dye. Activity was determined by test fraction 
inhibition of human menopausal gonadotropin (2 U human LH/FSH day) induced 
ovarian weight gain, and serum estradiol increase in hypophysectomized, 
diethylstilbesterol-treated, twenty-five-day-old female rats. 
Specifically, human follicular fluid was aspirated from regularly 
menstruating women (aged twenty-five to thirty-five years) undergoing 
ovarian hyperstimulation during participation in an in vitro fertilization 
protocol by treatment with clomiphene citrate (150 mg/day for five days, 
beginning three-eight days after onset of spontaneously occurring menses) 
and hCG (5000 IU forty-eight hours before aspiration). Sera were collected 
daily and estrogen concentrations were determined. When serum estrogen 
concentrations exceeded 800 pg/ml, patients underwent laparoscopy for 
aspiration of follicles in excess of 20 mm in diameter. The follicular 
aspirate was immediately centrifuged (800 x , the granulosa cells were 
removed and the aspirate was then frozen (-57.degree. C.). Follicular 
aspirates from twenty such patients were pooled and provided the hFF used 
throughout this study. 
Pooled hFF was slowly thawed and fractionated by dropwise addition of an 
equal volume of saturated ammonium sulfate (SAS) during persistent 
agitation at 4.degree. C. After a twelve hour incubation at 4.degree. C., 
the precipitate was pelleted, the supernatant was discarded, and the 
pellet was resuspended (2:1, vol/vol) with 10% SAS. An additional twelve 
hours of agitation was followed by centrifugation at 3,000 x for thirty 
minutes. The resulting supernatant was dialized using 10,000 molecular 
weight exclusion dialysis membranes against three changes of 0.025 M 
Tris/HCl, pH 7.5, (buffer A) for sixteen hours. Insoluble material was 
removed from the retentate by centrifugation (3,000 x for 30 minutes). 
A series of agarose (triazine ring) immobilized textile dyes (Dye-Matrix 
Screening Kit, Amicon, Lexicton, Mass.) were prepared according to the 
manufacturer's instructions. Columns (9.times.32 mm, 2 ml bed volume) 
containing Matrix Gel Blue A (triozinyl dye Cibacron Blue 3GA), Red A 
(Procion Red HE3B), Blue B (Cibacron Brilliant Blue FBR-P), Orange A, or 
Green A were equilibrated with 20 mM Tris/HCl (pH 7.5), then charged with 
0.5 ml aliquots of the dialyzed retentate. Unbound material was eluted 
with 10 ml buffer A. The bound protein was eluted with 1.5 M KCl in buffer 
A. Eluent fractions were dialyzed overnight against buffer A, and protein 
concentration determined. 
Eluents containing active material (Orange A-bound fractions) were further 
separated (10 ml/h, 4.degree. C.) on a Sephadex G-50 (superfine) column 
(1.6.times.50 cm, V.sub.O =60 ml, 5 ml/h, 4.degree. C.) with buffer A. 
Elution profiles were determined using an ISCO absorbance meter at 254 nm. 
For estimation of molecular weight by gel filtration, the same Sephadex 
G-50 column used for purification (2.6.times.70 cm, V.sub.t =280, V.sub.O 
=90 ml) was equilibrated and developed with molecular weight standards 
ribonuclease-A, chymotrypsin, and ovalbumin in buffer A at 10 ml/h, 
4.degree. C. Fractions were then assessed for activity in the bioassay. 
K.sub.av for each standard and active fraction was calculated using 
V.sub.t =280, V.sub.O =90 (K.sub.av =V.sub.e -V.sub.O /V.sub.t -V.sub.O). 
Additional aliquots from the 10-55% SAS fraction of the hFF (10 ml) were 
passed through a Concanavalin A-linked Sepharose 4B (Con A) column which 
was washed with 5 vol of 0.2 M NaCl, 0.05 M Tris/HCl (pH 7.4), then 
further eluted with 0.2 M .alpha.-methyl-D-mannoside in buffer A at a flow 
rate of 20 ml/h. Both Con A-bound and -unbound fractions were assessed for 
activity in the bioassay. Chromatography fractions containing inhibitory 
activity were heated (56.degree. C., 1 h) or trypsin digested (10 mg/100 
ml) for three hours, and then retested for bioactivity. 
Both Orange A-bound and 10-55% SAS hFF were further purified by isoelectric 
focusing using a Sephadex G-15 support matrix. The apparatus consisted of 
a 4.times.30 cm water-jacketed glass column containing a 2.5.times.20 cm 
G-15 Sephadex bed supported by a 2.5.times.8 cm Teflon elution plug under 
a 25-m Millipore filter. The column was previously equilibrated with 
two-bed volumes of a solution containing carrier ampholytes (4% of pH 3-10 
and 4% of pH 2-4) in 12.5% glycerol. Cytochrome C was used as an internal 
marker protein (pI=10.5). The fractions were then washed into the column 
with 20 ml ampholyte-glycerol solution. A second Millipore filter was 
placed on the top of the Sephadex bed. A 10-ml polyacrylamide solution of 
14% acrylamide, 0.3% Bis, and 50 .mu.l 
N,N,N',n'-tetramethylethylenediamine (polymerized by the addition of 0.1 
ml of 10% ammonium persulfate) was then poured over the filter. Upon 
completion of polymerization (twenty minutes), the column was inverted, 
the Teflon plug was removed, and a second acrylamide plug was layered over 
the bottom filter. After polymerization of the bottom plug, the column was 
returned to its upright position and lowered into anode buffer containing 
1% sulfuric acid. The remaining upper portion of the column was filled 
with 1% ethanolamine. The column was cooled by recirculating water at 
1.degree.-4.degree. C. throughout the procedure. Isoelectric focusing was 
initiated at 800 constant V (16 mA) and allowed to proceed to equilibrium 
as monitored by an eventual decline in the milliamperage to 2.5 mA (8-12 
h). Thereafter, pooled column fractions (3 ml) were dialyzed against 
buffer A to remove ampholytes before bioassay. 
Since an apparent isoelectric point was reproducibly determined in material 
isolated from the human ovarian vein blood by ampholyte displacement 
chromatograph in Example One, this procedure was employed with the 10-55% 
SAS-dialyzed hFF. Pooled aliquots (10 ml) were layered one a Polybuffer 
Exchanger 94 adjusted to pH 7.4 with 0.025 M Imidazole buffer, 10 ml/h, 
4.degree. C. The column was eluted with Polybuffer 74 adusted to pH 4.0 
with lN HCl. Fractions that eluted at a pH greater than 7.4 were collected 
and rechromatographed in the same Pharmalyte column reequilibrated to pH 
9.4 with ethanolamine 0.025 M, and eluted with Polybuffer 96(.TM.) 
adjusted to pH 6.0 with 1 N KOH. 
The rats used in the bioassay were hypophysectomized and implanted as 
described in Example One, and serum estradiol-17.beta. concentration was 
determined as described previously. 
Control determinations (no injected test fractions) for unstimulated 
ovarian weight were 34.7.+-.3.2 (SEM) mg/rat and for LH-FSH-stimulated 
were 192.0.+-.30.5 mg/rat. Control levels of trunk serum estradiol were 
12.5 .+-.0.7 pg/ml for unstimulated rats, and 118.5 .+-.21 pg/ml for 
LH-FSH-stimulated. Where indicated, 100% inhibition equals ovarian weight 
and/or serum estradiol concentration of mean unstimulated control values. 
Zero percent inhibition equals ovarian weight or serum estradiol 
concentration of LH-FSH-stimulated control rats. These results are similar 
to those which have been obtained with the previous application of this 
bioassay procedure in Example One. Tests of statistical significance wer 
performed by Student's t test and Duncan's multiple range analysis. 
Protein separation was performed using a Waters HPLC/ GPC Model 244 Liquid 
Chromatograph equipped with a 0.75.times.50 cm TSK 3000 SW gel exclusion 
column. A 100 -ul aliquot of the dialyzed, Orange A-dye-matrix eluent was 
separated on each high performance liquid chromatographic (HPLC) run. The 
proteins were eluted from the TSK column using an isocratic gradient of 
0.02 M PBS (pH 7.0) at a flow of 0.5 ml/min. The protein peaks were 
detected by absorbance at 280 nm with a Waters variable wavelength 
detector (Model M-450) and molecular weight estimates of the specific 
follicular fluid proteins were performed using highly purified molecular 
weight chromatography standards of ribonuclease-A, chymotrypsin, 
ovalbumin, and bovine serum albumin (BSA). 
Table 1 summarizes the results of Dye-Ligand matrix chromatography of the 
10-55% SAS-dialyzed hFF fraction. Although Orange A bound only 17% of the 
charged protein, 1.5 M KCl eluted bioactive material that contained the 
greatest inhibition of the hMG-induced rat ovarian weight gain (89.+-.6.8% 
SEM; P&lt;0.05) when compared to the other bound fractions. 
TABLE 1 
______________________________________ 
FF protein recovery and inhibition of rat ovarian 
weight in response to exogenous gonadotropin 
timulation by the 10-55% SAS pooled hFF fraction 
developed through Dye-Matrix chromatography 
Unbound Bound 
% Protein % Protein 
% 
Dye-Matrix 
Recovered % Inhibition.sup.a 
Recovered 
Inhibition.sup.a 
______________________________________ 
Control 96 68 .+-. 3.2 
4.6 0 
Blue A 35 10 .+-. 4.7 
63 0 
Blue B 87 11 .+-. 8.1 
21 14 .+-. 3.2.sup. 
Red A 38 0 63 0 
Orange A 
86 0 17 89 .+-. 6.8.sup.b 
Green A 31 0 68 0 
______________________________________ 
Bound material was eluted with 1.5 M KCl in TrisHCl (0.025 M, pH 7.5). 
.sup.a Values are the means .+-. SEM. 
.sup.b Significantly greater inhibition of rat ovarian weight response to 
gonadotropin stimulation compared to other Dyematrix eluents (P &gt; 0.05, 
Student's t test). 
FIG. 5 depicts the chromatographic elution profile of hFF developed through 
Sephadex G-50 at 254 nm after SAS 10-55% cut, dialysis (10,000 molecular 
weight), and elution of Orange A-bound material with 1.5 M KCl. Fractions 
(2 ml) were tested in the LH-FSH-stimulated hypophysectomized, immature, 
DES-treated rat for inhibition of ovarian weight, and serum estradiol 
concentration (mean.+-.SEM). An initial peak in absorbance can be seen at 
V.sub.e -V.sub.O ratio of 1.0-1.1, which after descending, reaches a 
gradual ascending second plateau (V.sub.e -V.sub.O of 1.58-1.68). 
Biological activity was determined as inhibition of ovarian weight and 
trunk serum estradiol levels. The same column was then equilibrated with 
molecular weight standards and developed with buffer A, allowing for 
molecular weight estimation (K.sub.av =log V.sub.e -V.sub.O /V.sub.t -VO) 
for fractions containing inhibitory activity. Molecular weight of eluents 
containing inhibitory activity was estimated to be 13,000-18,000. 
Eluents from the isoelectric focusing of hFF after SAS (10-55%), dialysis 
and Orange A dye matrix chromatography (1.5 M KCl eluate) (FIG. 6) were 
evaluated for activity in the bioassay. Only fractions in the pH range of 
3.5-4.5 contained clear inhibition of ovarian weight and trunk 
17.beta.-estradiol levels (not shown). When isoelectric point 
determination was performed using ampholyte displacement chromatography 
(FIG. 7), inhibition of rat ovarian weight was found in the pH range 
3.5-4.5. In addition, a second area of inhibition in the bioassay was 
noted at (pH 6.0-6.5.). 
FIG. 8 depicts the HPCL elution profile of the Orange A-bound extracted hFF 
material eluted through a TSK 3000 analytical molecular weight exclusion 
column. FIG. 9 indicates the elution profile of the same material eluted 
through a TSK 3000 preparative molecular weight exclusion column. Each of 
these elution profiles displays unique chromatographic patterns of FRP. 
In FIG. 8, the HPLC eluent was divided into ten fractions based on peak 
absorbances [0: 18.5-21.0 minutes (void volume); 1: 21.0-23.0 minutes; 2: 
23.0-25.0 minutes; 3: 25.0-28.5 minutes; 4: 28.5-32.2 minutes; 5: 
32.2-34.0 minutes; 34.0-40.0 minutes; 7: 40.0-45.0 minutes; 8: 45.0-49.0 
minutes; 9: 49.0-53.0 minutes]. Aromatase inhibiting activity elutes at 
40-45 minutes. These fractions corresponded to the following molecular 
weight ranges: 1, 100,000; 2, 100,000; 3, 70,000-100,000; 7: 5,500-18,000; 
8: 2,500-5,500; 9, 2,500. The retention times of peak absorbances after 
HPLC elution, when correlated to molecular weight standards, were highly 
reproducible (13 runs). 
When HPLC fractions were tested in the bioassay (see FIG. 10), inhibition 
of ovarian weight gain and serum estradiol elevation were evident in rats 
injected with the 5,500-18,000 molecular weight fraction [78+8% (SEM) and 
89+10%, respectively (P&lt;0.01)]as compared to the other fractions. FIG. 11 
shows a polyacrylamide gel, with sodium dodecyl sulfate, of the fractions 
shown in FIG. 8 after HPLC. Columns 1-5 in FIG. 11 correspond, 
respectively, to the columns 3-7 in FIG. 8. The FRP is shown at the bottom 
of column 5, with a molecular weight of 15,000 to 16,000 when compared to 
the molecular weight standards to the right of column 5. The standards 
include ribonuclease A at 14,000, and chymotripsinogen A at 22,000. 
In Example One, there is described a protein, in the venous drainage of the 
human ovary which contains the preovulatory follicle, which suppresses the 
follicular response to gonadotropins. That this protein was not secretedin 
large amounts by anovulatory ovaries was evidenced by failure of the 
bioassay to detect inhibitory activity in the venous drainage of the 
contralateral ovary in ovulatory patients as well as both ovarian vein 
effluents from anovulatory women. In the present example, a comparable 
isolation procedure has shown a similar biological activity in human 
follicular fluid (hFF). The rat bioassay employed to identify material in 
hFF which inhibits LH-FSH-mediated follicular stimulation was the same as 
that reported for human ovarian vein blood extracts. Follicular inhibitory 
activity in hFF had a molecular weight, determined by HPLC size exclusion 
chromatography (10,000-18,000) that is similar to that of follicular 
inhibitory activity recovered from human ovarian vein serum 
(14,000-17,000). The indicated isoelectric point for inhibitory activity 
in human ovarian vein extract, as determined by chromatofocusing (pH 
5.8-6.4), is similar to the hFF extract reported here. 
The follicular inhibitory substance reported here was derived from 
follicular aspirates of women during spontaneous ovarian cycles whose 
follicles were hyperstimulated by clomiphene and hCG therapy. 
Consequently, no conclusion can be drawn regarding this material in 
normally developing follicles. However, since a similar activity has been 
identified in the ovarian vein serum draining the spontaneous preovulatory 
ovary (Example One) these data indicate this inhibitory protein to be a 
product of the dominant follicle itself. 
EXAMPLE THREE 
Gonadal Regulatory Protein Fractions in Porcine Follicular Fluid 
Example Two details the isolation of a protein fraction (FRP) from human 
follicular fluid, which suppresses follicular response to gonadotropins. 
The present example employs an identical isolation, bioassay and HPLC 
procedures with respect to porcine follicular fluid (pFF). Isoelectric 
focusing demonstrated inhibitory activity at about pH 4.2-4.5 and pH 6.27 
and peak activity was found in the molecular weight range 12,000-18,000 
daltons. 
Granulosa cells were isolated from rat ovaries and FSH binding was 
determined by the procedure of Erickson, as described in Example Four. 
Aromatase activity of the rat granulosa cells was determined in a 
procedure similar to that described therein. 
Table 2 sumarizes the results of dye-ligand matrix chromatography of the 
10-55% saturated ammonium sulfate porcine follicular fluid fraction. 
Although Orange A bound only 13% of the charged protein, 1.5 M KCl elution 
recovered bioactive material which contained the greatest inhibition of 
hMG-induced rat ovarian weight response p&lt;0.05) when compared to the other 
dye-ligand eluents. When these same fractions were treated with heat 
(56.degree. .degree. C..) or trypsin (10 mg%), no suppression in ovarian 
weight was found. 
TABLE 3 
______________________________________ 
Protein Recovery and Inhibition of Ovarian Weight 
Response From Dyematrix Chromatography of the 10-55% 
Saturated Ammonium Sulfate Dialyzed Porcine Follicular 
Fluid Fraction 
Unbound 
% Bound 
Dyematrix 
Protein % Inhibition 
% Protein 
% Inhibition 
______________________________________ 
CONTROL 94 78 .+-. 6.1 
8.8 0 
BLUE A 34 22 .+-. 8.3 
65 0 
BLUE B 81 0 20 39 .+-. 7.4 
RED A 33 12 .+-. 4.2 
65 0 
ORANGE A 88 0 13 84 .+-. 7.4 
GREEN A 37 0 63 22 .+-. 3.8 
______________________________________ 
*Significantly greater inhibition (p .05 Student's T) of hMGinduced rat 
ovarian weight response compared to other dyeligand eluents. 
FIG. 14 depicts the chromatographic elution profile of pFF at 280 nm 
developed through Sephadex G-100 following the 10-55% SAS cut, dialysis, 
and elution of Orange A-bound material with 1.5 M KCl. An initial peak can 
be seen at a V.sub.e V.sub.o of 1.1 to 1.17, thereafter trailing, followed 
by a gradual ascending second plateau at V.sub.e /V.sub.o 1.7 to 1.9. When 
biological activity was assessed in the HIFR-hMG bioassay, 95% inhibition 
of both ovarian weight and trunk serum estradiol-17-B levels were found at 
V.sub.e V.sub.o of 1.5. The same column was then equilibrated with 
molecular weight standards and developed with, 0.025 M PBS, pH 7.4, 
allowing for estimation of the K.sub.av (V.sub.o -V.sub.e /V.sub.e 
-V.sub.o) of fractions containing inhibitory activity. 
When eluents from the isoelectric focusing (IEF) of the Orange A bound pFF 
extraction (FIG. 12) were evaluated for bioactivity by rat bioassay, only 
fractions within the range of pH 3.7 to 4.0 contained obvious inhibition 
of both rat ovarian weight response and trunk serum estradiol-17B levels 
(83.6+9.4% and 47.2% respectively). Serial dilutions of the extracted PFF 
fractions eluted from the Orange A dye-matrix with 1.5 M KCl recovered 
from the isoelectric focusing column were tested for activity in the 
hypophysectomized immature DES-treated female (HIFR-rat) bioassay. A dose 
response relationship was apparent (FIG. 13) with fractions in the pH 
3.5-4.0 range, while other pH ranges from the IEF column were without 
inhibitory activity. 
When aliquots of this Orange A matrix column were separated by hydrogen ion 
chromatography (chromatofocusing), fractions corresponding to pH 4.0-4.5 
(FIG. 15, fraction 7) and pH 6.0-6.5 (FIG. 15, fraction 4)inhibited 
microsomal aromatase activity. When fractions 4 and 7 are further purified 
by HPLC molecular weight separation, aromatase inhibitory activity (FRP) 
is present in the fractions corresponding to 15,000-16,000 daltons. 
When aliquots from the ammonium sulfate 10-55% cut eluted from the Orange A 
dye matrix were passed through Concanavalin-linked Sepharose 4B (eluted 
3.times.Vo with PBS followed by 0.2 M alpha-methyl mannoside), no 
inhibitory activity in the rat bioassay was noted in the Con A bound 
fraction (i.e. eluted with mannoside) with only marginal recovery of 
activity (20-30% inhibition of ovarian weight response) in the material 
eluted in the unbound PBS fraction (data not shown). 
FIG. 16 depicts the HPLC elution profile of the Orange A-bound extracted 
PFF material after separation through the Sephadex G-100 column (V.sub.e 
V.sub.o 1.3-1.7). The HPLC eluent was divided into five fractions based on 
peak absorbances (1: retention time --25.0-27.8 minutes; 2: 27.8-31.0 
minutes; 3: 31.0-36.0 minutes; 4: 36.0-39.0 minutes; 5: 39.0-43.0 
minutes). These fractions corresponded to the followino molecular weight 
ranges: 1: 100,000-74,000; 2: 74,000-36,000; 3: 36,000-18,000; 4: 
18,000-12,000; 5: 12,000-5,800. The retention times of peak absorbances 
after HPLC elution, when correlated to molecular weight standards, were 
highly reproducible (five runs). 
When HPLC fractions were tested in the bioassay (see FIG. 17), inhibition 
of ovarian weight gain was evident in rats injected with fraction 3 
(74.+-.8%; p&lt;0.01) and fraction 4 (51.+-.9%, p&lt;0.05) as compared to the 
other three fractions. Inhibition of androstenedione conversion to total 
immunoreactive estrogen by granulosa cells harvested from the 
HPLC-fraction treated rats ovaries (n=6 ovaries/HPLC fraction) was evident 
in HPLC fraction 3 (11.1.+-.3 pg/10,000 cells/ml). When no test fractions 
were injected into the HIFR-hMG bioassay rat prior to the aromatase assay, 
a range of 30-60 pg of total immunoreactive estrogen/ 10,000 cells/ml was 
present in the control incubates. 
FIG. 18 shows the results of FSH binding studies performed on the granulosa 
cells removed from the HIFR-hMG rats used in the bioassay of the 
Sephadex-G100 fraction which contained inhibitory activity. No difference 
in FSH binding to the rat granulosa cells was evident between the control 
and inhibitor treated HIFR-hMG rats. 
Subsequent procedures, identical to those described in this exmple 
regarding porcine follicular fluid, have isolated FRP having a molecular 
weight range of about 12,000 to 18,000 daltons and an isoelectric point of 
from pH 4.0 to 6.5, and inhibiting aromatase activity as described above, 
from bovine follicular fluid. 
EXAMPLE FOUR 
Identification of Regulatory Protein(s) in Human Granulosa Cell Secretions 
Follicular fluid was aspirated from regularly menstruating women 
(twenty-five to thirty-five years old), undergoing clomiphene citrate (150 
mg/day for five days, beginning three-eight days after the onset of 
spontaneously occurring menses) and hCG (4000 IU, 36 hours before 
aspiration) therapy during participation in an in vitro fertilization 
protocol. Serum was collected daily, and estrogen concentrations were 
determined. When serum estrogen concentrations exceeded 800-1000 pg/ml, 
patients underwent laparoscopy for aspiration of follicles in excess of 20 
mm in diameter. Follicular aspirates were immediately centrifuged (800 
.times.g), granulosa cells were removed for culture, and the supernatant 
was frozen . Follicular aspirates from seven patients were evaluated. 
The aspirated follicular fluid volume, number of viable granulosa cells, 
and follicular fluid steriod concentrations from the largest follicle 
recovered from the seven patients are depicted in Table 2. All of the 
antral fluids contained high concentrations of progesterone (7-12 ug/ml), 
indicating premature luteinizations of the follicles (30-32) as a result 
of the clomiphene/hCG therapy. Approximately 100,000 viable granulosa 
cells were obtained from each follicle. 
TABLE 2 
______________________________________ 
Follicular fluid aspirate 
Pat- Vol- Viable Steroid conc. (ng/ml) 
ient ume granulosa Estra- 
Es- Proges- 
17-Hydroxy- 
No. (ml) cells diol trone 
terone progesterone 
______________________________________ 
1 12.0 1.0 .times. 10.sup.5 
396 37 12,377 212 
2 7.1 204 3 11,761 889 
3 12.2 2.6 .times. 10.sup.5 
440 220 12,746 82 
4 12.0 1.7 .times. 10.sup.5 
296 10,132 176 
5 5.3 0.75 .times. 10.sup.5 
327 7,500 667 
6 5.3 0.7 .times. 10.sup.5 
1,740 411 9,197 1,091 
7 8.8 10.5 .times. 10.sup.5 
708 492 11,371 1,200 
______________________________________ 
Individual hFF samples were slowly thawed and fractionated by dropwise 
addition of an equal volume of saturated ammonium sulfate during 
persistent agitation at 4.degree. C.. After a twelve hour incubation at 
4.degree. .C, the precipitate was recovered by centrifugation and 
resuspended (2:1, vol/vol) in 10% ammonium sulfate. An additional twelve 
hours of mixing was followed by centrifugation at 3,000 x g for thirty 
minutes. The resulting supernatant was dialyzed (10,000 molecular weight 
exclusion membranes) against phosphate-buffered saline (PBS) (0,025 M; H 
6.8) for sixteen hours at 4.degree. C. and then lyophilized. The retentate 
(500 mg in 0.5 ml aliquots) was passed through a column (9.times.32 mm; 
bed volume, 2 ml) containing agarose-immobilized Orange A (Dye matrix, 
Amicon), which had been equilibrated with 20 mM Tris-HCl, pH 7.5. Unbound 
material was eluted with 10 ml 20 mM Tris-HCl, pH 7.5 and bound material 
was eluted with 10 ml 1.5 M KCl in 20 mM Tris-HCl pH 7.5. Bound eluent 
fractions were dialyzed overnight against PBS or distilled water. Protein 
concentrations were determined by the method of Lowry et al., J. Biol. 
Chem. 143:265. 
Rats were prepared for bioassay as described in the previous examples. 
Results of control determinations (no injected test fractions) were 34.8 
+3.2 mg/rat for unstimulated ovarian weight and 122.0+13.5 mg/rat for 
FSH-stimulated ovarian weight. Control levels of trunk serum estradiol 
were 12.5+0.7 pg/ml for unstimulated and 118.5+21 pg/ml for LH/FSH 
stimulated. Where indicated, 100% inhibition equals the ovarian weight 
and/or serum estradiol concentration of mean unstimulated control values. 
Zero percent inhibition equals the ovarian weight or serum estradiol 
concentration of LH/FSH-stimulated control rats. These results are similar 
to those obtained during the previous examples. Tests of statistcal 
significance were performed by Student's t test and Duncan's multiple 
range analysis. 
FIG. 19 depicts the effect of extracted human follicular fluid (ehFF and 
respective granulosa cell culture media (24, 48, and 72 hours) on the 
inhibition of LH/FSH (2 IU)-stimulated rat ovarian weight augmentation and 
serum estradiol secretion (2 ml/rat). Each value represents the 
mean.+-.SEM of three rats. 
All four follicular fluid extracts (ehFF) contained inhibitory activity, as 
evidenced by inhibition of both rat ovarian weight (40-65%) and trunk 
serum estradiol concentrations (85-100%). Bioassay of culture media from 
the respective granulosa cell cultures collected twenty-four, forty-eight 
and seventy-two hours after plating indicated that inhibitory activity was 
present during the first forty-eight hours of incubation. Variability in 
the initial twenty-four hour determination of patients 2 and 3 may relate 
to initial plating efficiency. Importantly, all bioassay determinations 
were made after complete medium changes each twenty-four hours. No 
inhibitory activity was noted after seventy-two hours of culture. 
FIG. 20 depicts the compiled (mean .+-.SE) bioassay results of culture 
media (containing FRP) derived from granulosa cell cultures of patients 5, 
6 and 7. All media tested without gonadotropins added to the culture 
contained inhibitory activity of both ovarian weight and trunk serum 
estradiol concentrations exceeding 75% throughout the first two days of 
culture. Thereafter (days 3 and 4), the inhibitory activity of media from 
unstimulated cultures declined (43% and 37%, and 18% and 12% for ovarian 
weight and serum estradiol levels, respectively). After coincubation of 
the granulosa cells with varying doses of LH/FSH (0.3, 1.0 and 3.0 U), 
inhibitory activity, as determined in the HIFR-hMG bioassay, was markedly 
suppressed in all cultures after the first twenty-four hours (&gt;20%). 
When progesterone concentrations were determined for these culture media , 
an inverse correlation between inhibitory activity in the bioassay and 
culture medium progesterone concentrations was apparent. The unstimulated 
cultures (no added LH/FSH) had the least progesterone throughout the four 
days of culture (&lt;10 ng/ml), but contained inhibitory activity (FIG. 20), 
albeit in decreasing amounts as culture duration continued. However, after 
LH/FSH was added to the granulosa cell cultures in increasing amounts, the 
progesterone concentrations rose in a dose-and time-dependent pattern, 
while bioassay inhibitory activity declined to essentially the limits of 
bioassay detectability (FIG. 20). 
High performance liquid chromatography 
The high performance liquid chromatographic (HPLC) separation of follicular 
fluid steroids was performed. 
Protein separation was performed using the same Waters HPLC/GPC Model 244 
liquid chromatograph equipped with two Waters I-125 gel exclusion columns 
connected in series. A 10-.mu.l aliquot of the dialyzed, Orange A dye 
matrix-bound eluent was separated on each HPLC run. The proteins were 
eluted from the I-125 columns using an isocratic gradient of 0.02 M PBS, 
pH 7.0, at a flow of 0.5 ml/minute (800 psi). The protein peaks were 
detected at 280 nm, and molecular weight estimates of the specific 
follicular fluid proteins were performed using highly purified molecular 
weight chromatography standards of ribonuclease-A, chymotrypsin, 
ovalbumin, and BSA. 
Granulosa cells were cultured for up to four days in twenty-four hour 
intervals using 35.times.10-mm tissue culture dishes and 2 ml medium 199 
containing 25 mM Hepes supplemented with 100 U/ml penicillin, 100 .mu.g 
streptomycin sulfate, and 15% fetal calf serum (medium A). Cultures were 
maintained in a humidified, 95% air-5% CO.sub.2 incubator at 37.degree. C. 
After each twenty-four hour incubation period, spent medium was collected 
and stored frozen at -20.degree. C. until bioassay was performed. Where 
indicated, human menopausal gonadotropin FSH-LH,1:1) was added to specific 
cultures at the time of complete medium change. At the end of each 
twenty-four hour incubation period, spent medium was collected for 
bioassay, and 2 ml fresh medium were added. The final cell pellets were 
dispersed in medium A, and aliquots (0.5 ml) of the cell suspension were 
diluted with 0.05 ml trypan blue for quantitation of viable cells in a 
hemocytometer. Initial plating density was 0.5.times.10.sup.5 granulosa 
cells/plate. 
Granulosa cells were isolated from rat ovaries and were collected by 
centrifugation at 800 x g at 4.degree. C. for ten minutes. FSH binding was 
determined using a modification of known techniques. Rat FSH, provided by 
the National Pituitary Agency, was labeled by the chloramine-T procedure. 
Cells were resuspended in appropriate volumes of PBS-01% gelatin 
(PBS-gel), pH 7.0. All assays were run with three concentration of labeled 
hormone (100 .mu.l), buffer (PBS-0.1% gel; 100 .mu.l) and 100.mu.l cells. 
Reactions were initiated by the addition of granulosa cells and were 
carried out for four hours at 25.degree. C. Reactions were terminated by 
adding 1 ml cold PBS, followed by centrifugation at 30,000 x g for ten 
minutes. The supernatant was carefully aspirated, and the pellet was 
rewashed with 1 ml PBS. The final pellet was counted in a .gamma.-counter. 
Specific binding was calculated as the difference between binding in the 
presence (nonspecific) and absence (total) of an excess of unlabeled 
hormone. 
FIG. 21 depicts the FSH binding studies performed o the granulosa cells 
removed from the HIFR-hMG-treated rat ovaries used in the bioassay 
experiments shown in FIG. 19. Specific binding of rat FSH (rFSH) to 
granulosa cells was determined by incubating three concentrations of 
labeled rFSH in the presence and absence of excess unlabeled rFSH. Rat 
granulosa cell specific FSH binding was similar whether the rat received 
injections of spent medium from human granulosa cell cultures or saline. 
However, a marked difference in the ovarian weight and trunk serum 
estradiol concentrations of these rats was present. 
Replicate 0.1-ml portions of each rat granulosa cell suspension were 
pipetted into 12.times.75-mm polystyrene tubes. Androstenedione, the 
referent aromatase substrate, was added in 0.1 ml medium A (final 
concentration, 1.0.times.10.sup.-7 M). All incubations were performed in 
triplicate for three hours at 37.degree. C. in a shaking water bath (120 
cycles/minute). The reaction was stopped by transferring the tubes to an 
iced water bath before centrifugation for five minutes at 1000 x g. The 
supernatants were decanted and stored at -20.degree. C. until measurements 
of estradiol and estrone were performed. Control incubations (no 
androstenedione added) were processed in the same way. Blank estrogen 
values obtained for the controls were subtracted from the corresponding 
values for incubations in the presence of androstenedione. Aromatase 
activity was expressed as estrogen production (nanograms per viable 
granulosa cell). 
Rat granulosa cell aromatase activity is shown in FIG. 22 and was markedly 
inhibited (P&lt;0.01) by treatment with spent media from all four days of 
culture (2.4.+-.0.4, 2.9.+-.0.7, 2.4.+-.0.4, and 2.1.+-.0.2 pg 
estrogen/10,000 viable cells/ml, respectively, compared to saline control 
4.6.+-.0.2 pg estrogen/10,000 viable cells/ml. Control determinations were 
performed on rat granulosa cells collected from HIFR-hMG-treated rats 
which did not receive culture medium injections. Inhibition of relative 
estrogen production by rat granulosa cells in the presence of 10.sup.-7 M 
androstenedione was seen throughout all four days of human granulosa cell 
culture medium treatment. Taken together, these data indicate that 
although no marked inhibition of rat granulosa cell FSH binding was 
induced by the human grandulosa cell culture medium, a clear disruption of 
aromatase activity occurred, which may account for the decreased rat 
ovarian weight and serum estradiol concentrations. 
Using the same purification techniques and bioassays described above, a 
similar protein or proteins have been identified in the culture media 
derived from the granulosa cells of human follicles. This data indicates 
that human granulosa cells secrete a protein that inhibits follicular 
response to gonadotropins. 
All of the antral fluid's steroid concentrations suggested premature 
luteinization. After extraction, all seven follicular fluids contained 
inhibitory activity, as evidenced by reduction of both rat ovarian weight 
(45-85%) and trunk serum estradiol concentrations (85-100%) in the 
HIFR-hMG bioassay. Bioassay of these follicles' granulosa cell culture 
media indicated inhibitory activity present during the first forty-eight 
hours, while no inhibitory activity was noted after seventy-two hours of 
culture. Spent culture media derived from 9ranulosa cells cultured without 
additional 9gonadotropins contained inhibitory activity in the HIFR-hMG 
bioassay throughout the first two days in vitro. Thereafter (days 3 and 
4), inhibitory activity of media from unstimulated cultures declined. 
After coincubation of the granulosa cells with varying doses of LH/FSH, 
inhibitory activity was markedly suppressed even after the first 
twenty-four hours. An inverse correlation was apparent between inhibitory 
activity in the bioassay and the culture medium progesterone level. 
Although FSH binding of granulosa cells derived from rats used in the 
HIFR-hMG bioassay was similar with or without injection of test fractions, 
their aromatase activity was markedly inhibited by treatment with human 
granulosa cell culture medium. These data indicate that although no marked 
inhibition in rat granulosa cell FSH binding was induced by the human 
granulosa cell culture medium, a clear disruption of aromatase activity 
occurred, which accounts for the decreased rat ovarian weight and serum 
estradiol concentrations found in the bioassay. Disruption of 
gonadotropin-mediated aromatase induction by an intrafollicular protein 
may, in part, modulate the local balance between C-19 steroid aromatase 
and 5-.alpha.-reductase enzymic activities in individual follicles. 
A variety of nonsteroidal regulators of ovarian function have been 
identified in a variety of species, including oocyte maturation inhibitor, 
luteinizing inhibitor, folliculostatin or inhibin, and FSH binding 
inhibitor. The biophysical characteristics of the intraqonadal protein of 
this invention are different from such substances. This material elutes 
through gel exclusion chromatography with a molecular weight of 
12,000-18,000, binds to Orange A dye matrix, and has an apparent 
isoelectric point of from pH 4.0-4.5 to 6.0-6.5. These observations 
indicate that in addition to the intrafollicular steroidal mileau a 
variety of nonsteroidal compounds, secretory products of the granulosa 
cells or other ovarian compartments, contribute to the regulation of 
folliculogenesis. 
EXAMPLE FIVE 
The presence of the gonadal regulating protein in Testis 
Bovine testis were homogenized and the homogenate precipitated with 55% 
saturated ammonium sulfate. The precipitate was resuspended and dialyzed 
against distilled water. The retentate was eluted though Sephadex G-100 
with TRIS buffer (20 mM, pH 7.5). The eluant corresonding to a molecular 
weight range of 12,000-18,000 was collected and charged into an Orange A 
dye matrix column. The column was eluted with TRIS buffer containing 0.5 M 
KCl (20 mM, pH 7.5). This eluant contained FRP as evidenced by the 
inhibition of porcine qranulosa cell aromatase activity (FIG. 23; bTEc and 
bTE-OAB columns) and rat serum estradiol levels (FIG. 24; BTE +FSH and 
BTE-OAB +FSH columns). Further, this activity was contained in testicular 
extract fractions with isoelectric points of from pH 4.1-4.5 and 6.0-6.5. 
This demonstrates that the testis and its secretory product, rete testis 
fluid, also contains FRP. 
EXAMPLE SIX 
Extraction of FRP from Sertoli Cell Cultures. 
Granulosa cells secrete FRP, and Sertoli cells are the embryonic homologue 
in testis of the granulosa cells in the ovary. Further, the biological 
activities of granulosa cells are present in the Sertoli cells insofar as 
they have been identified and are relevant to gonadal function Thus, the 
Sertoli cell provides an additional, readily available source of FRP. 
Accordingly, Sertoli cells may be grown in cell culture and the recovered 
culture media is extracted by the procedures described in the foregoing 
examples, principally salt participation, chromatographic procedures and 
electrophoresis. 
II. Biological Activity 
EXAMPLE SEVEN 
The Effect of the Gonadal Regulatory Protein as an Aromatase Inhibitor 
In Examples One through Six, protein(s) in human ovarian venous effluent, 
human and porcine follicular fluid, bovine testis fluid and spent media 
from human granulosa and rat Sertoli cell cultures inhibited rat ovarian 
weight gain in response to gonadotropin stimulation. Gonadal fluid 
extracts containing this protein were found to have a molecular weight of 
12,000-18,000 and an isoelectric point of from about pH 4.0-4.5 to pH 
6.0-6.5. As inhibin, another protein secreted by human granulosa cells, 
increases with follicular maturation and decreases with luteinization, 
individual human follicles from untreated as well as hMG and clomiphene 
treated women were assessed for FRP activity, and that activity was 
correlated with the follicles' follicular fluid steroid and inhibin 
concentrations. 
Follicular aspirates were obtained from women (aged twenty-four-thirty-two 
years) who were participating in an in vitro fertilization protocol. All 
patients had regular ovulatory menstrual cycles based on monthly vaginal 
bleeding and at least a single luteal phase serum progesterone in excess 
of 3 ng/ml. When serial ultrasonographic examinations (ADR Model 2140 
real-time sector scanner with a 3.5 mHz rotating head transducer) 
demonstrated a follicular diameter in excess of 18 mm laparoscopy was 
performed for aspiration of all follicles greater than 16 mm in diameter. 
Follicular aspirates were immediately transferred to an adjacent 
laboratory for removal of granulosa cells by centrifugation (600 x G, 15 
minutes) and storage of follicular fluid (-37.degree. C.) until assay. 
Follicular fluid concentrations of estradiol, progesterone, 17-hydroxy 
progesterone, androstenedione and testosterone were determined by 
established radioimmunoassay techniques. 
FRP was isolated from individual follicular fluid samples substantially as 
described in Example Two. 
Aromatase Activity 
Porcine granulosa cells were collected from fresh ovaries obtained at the 
local slaughterhouse. After washing in serum free HAMS-HEPES tissue 
culture media, 5.times.10.sup.5 cells in 0.2 ml of medium were pipetted 
into 12.times.75 mm polystyrene tubes. Triplicate 200 ul portions of each 
follicular fluid preparation at three different protein concentrations 
(700-10 ug/ml) were tested. Each tube then received 100 .mu.l of FSH(10n 
g) in culture medium and was incubated at 37.degree. C in a shaking water 
bath for three hours. An atmosphere of 90% N.sub.2, 5% O.sub.2 and 5% 
CO.sub.2 was maintained throughout the incubation. The incubation was 
stopped by the addition of 0.5 ml Hams-Hepes media and centrifugation at 
1000 x g for five minutes. The granulosa cells were then resuspended in 
0.5 ml Hams-Hepes media, whereupon 100 .mu.l of cells were assayed for 
armoatase activity. Androstenedione, the referent aromatase substrate, was 
added in 0.1 ml medium (final concentration, 1.0.times.10.sup.-7 M). All 
incubations were performed in duplicate for three hours at 37.degree. C. 
in a shaking water bath (120 cycles/ minute). The reaction was stopped by 
transferring the tubes to an iced water bath before centrifugation (five 
minutes at 1000 x g). The supernatants were decanted and stored at 
-20.degree. C. until measurements of estrogen were performed. Control 
incubations (no androstenedione added) were processed in the same way. 
Blank estrogen values obtained for the controls were subtracted from the 
corresponding values for incubations in the presence of androstenedione. 
Aromatase activity was expressed as estrogen production (nanograms per 
viable granulosa cell). 
Inhibin Activity 
Ten percent (weight/volume) activated charcoal (Norite A) was added to the 
individual follicular fluids, and stirred continuously overnight at 
4.degree. C., followed by centrifugation (1000 x G, twenty minutes) and 
sterile filtered to remove the charcoal. The charcoal-stripped follicular 
fluid contained 10 pg/ml of androstenedione, progesterone, and estradiol 
as determined by radioimmunoassay. Inhibin activity was determined using 
the degree of inhibition of basal (i.e. non-LHRH stimulated) twenty-four 
hour FSH secretion by dispersed rat anterior pituitary cells in primary 
monolayer. For each cell culture, anterior pituitary glands were obtained 
from 20 cycling female Sprague-Dawly rats (250-300 gm body weight). During 
the thirty minute interval required for removal of all the pituitary 
glands, each gland was placed in medium (pH 7.4, 20.degree. C.). Pituitary 
glands were finely minced with scissors and incubated in a mixture 
containing 1% viokase, 3.5% collagenase, and 3% bovine serum albumin in 
medium buffer at 37.degree. C. for thirty to forty-five minutes. The 
dispersed cells were counted using a hemocytometer and pituicyte viability 
was determined by 1% trypan blue exclusion. Typically, more than 90% of 
the dispersed cells were viable. The cells were diluted to a concentration 
of 2.5.times.10.sup.5 viable cells per ml growth medium. Growth medium 
consisted of HAMS F10 containing 10% fetal ca1f serum with penicillin, 
fungiezone and streptomycin (50 u/ml and 50 mg/ml and 50 mg/ml 
respectively). Cells were added to tissue culture dishes in a volume of 
2.0 ml growth media, and attachment of the cells to the well surface was 
completed by two days. After cell attachment, the original growth media 
was discarded and the cells were washed twice with additional HAMS 
balanced salt solution. Thereafter, three concentrations of 
charcoaltreated follicular fluid (.02%, 0.2%, 2%) were added to the plate. 
A lyophillyzed porcine follicular fluid standard (PFF1 KT-1, provided by 
CP Channing) was resuspended in water and tested in each assay at 0.003%, 
0.016% 0.08% 0.04%, and 2% concentrations. Twenty-four hours later, the 
spent culture media was assayed in duplicate using the NIH-RIA kit for 
rFSH. 
Data Analysis 
The mean estrogen concentration in the serum control tube for each FRPassay 
was set at 100%, and the response in each test was expressed as a 
percentage of the control estrogen concentration. The coefficient of 
variation for each group of three replicate tubes was calculated. If the 
coefficient was 15%, the estrogen assay was repeated and/or one value of 
the three was discarded. A curve was constructed in which the percent 
inhibition of estrogen in each well was plotted vs the protein 
concentration of follicular fluid added. Unknown values were determined by 
plotting the experimentally determined values at three different protein 
concentrations (700-10 ug/ml) on a log-linear graph and extrapolating the 
value at 50 ug/ml. The percent inhibition of estradiol at 50 ug of unknown 
follicular fluid was read off the standard curve and expressed as percent 
aromatase inhibition for that follicle. 
For the inhibin assay, the response in each test plate was expressed as a 
percentage of the control FSH concentration, which was set at 100%. The 
coefficient of variation of each group of three replicate plates was 
calculated. If the coeffient was 15% the assay was repeated. A standard 
curve was constructed in which the percent inhibition of FSH in standard 
wells was plotted vs the log of the standard added. Least squares linear 
regression was used to construct a standard curve in the linear portion of 
the dose response curve. Unknown follicular fluid inhibin values were 
determined by plotting the experimentally determined values (0.02%, 0.2%, 
2%) on a log-linear graph and extrapolating the value at 1%. The percent 
inhibition of rFSH at 1% of unknown follicular fluid was read off the 
standard curve and expressed as Channing units (1 CU=1 unit of inhibin 
standard =the inhibition of rFSH in rat pituicyte culture by 1 nl of 
charcoal treated, ethanol extracted porcine follicular fluid). 
Tests for statistical significance were performed by one-way analysis of 
variance and Duncan's new multiple range test. Correlation between 
follicular fluid steroid concentration and FRP activity was performed 
using regression analysis with tests of statistical significance performed 
by t test corrected for N. 
Patient Outcome 
Seven patients underwent follicular aspiration during an untreated 
spontaneously occurring ovarian cycle. At the time of laparscopy, only one 
antral follicle was seen in each patient which was aspirated. Nine 
patients received clomiphene citrate therapy (150 mg/day, cycle days 5-9), 
providing a total of twenty-four follicles with diameters &gt;16 mm. All 
except one patient had multiple follicles aspirated. Six patients who 
underwent hMG therapy (150 IU LH/150 IU FSH administered daily beginning 
on cycle day 3 until follicle aspiration), provided twenty-three 
follicles. Care was taken to aspirate all follicles with diameters in 
excess of 16 mm. There was no difference between treatment groups in 
follicle size which averaged 18.7.+-.0.9 mm (x.+-.SEM, range 16-24 mm). At 
the time of aspiration, serum estradiol levels averaged 1456 pg/ml .+-.285 
pg/ml for all patients studied (range of 310-3200 pg/ml) with no 
significant difference between treatment groups. 
Validation 
To establish the number of porcine granulosa cells for the FRP assay, the 
following porcine granulosa cell concentrations were used 
0.5.times.10.sup.6, 1.times.10.sup.6, 2.times.10.sup.6 cells/ ml. The 
total amount of estrogen produced following a three-hour incubation was 
55.+-.9, 140.+-.27, 375.+-.48 pg/culture dish, respectively. Consequently, 
2.times.10.sup.6 porcine granulosa cells were used in each subsequent 
assay. To evaluate the effects of porcine FSH (NIH P-2 reagent) and 
follicular protein (100 ug), porcine granulosa cell cultures were prepared 
with or without porcine FSH added to the media (0.5 ml final volume), 
incubated at 37.degree. C. for three hours in a shaker bath with 95% 
O.sub.2 and 5% CO.sub.2, after which androstenedione (10.sup.-6 M) was 
added in 0.5 ml of growth media. Cultures were incubated for three hours 
at 37.degree. C. in a shaker bath then centrifuged (1000 x G fcr fifteen 
minutes) and media collected for estrogen determination. Without FSH, the 
estrogen production was 439.+-.41 pg/ml; with FRP added, the estrogen 
production was 22.4.+-.41.7 pg/ml. When 2 units/ml of porcine FSH were 
added without FRP 728 pg/ml estrogen were produced. Addition of FRP 
produced a dose-response relationship 1000 ug FRP: 200.26 pg/ml; 200 ug 
FRP: 306.37 pg/ml; 50 ng FRP: 345.41 pg/ml; 10 ug FRP: 334.+-.18 pg/ml of 
estrogen. Accordingly, individual patient values were extrapolated to 50 
ng/ml of FRP for comparisons of activity. 
The FRP in elution profiles from Orange A were analyzed using three 
different concentrations of KCl: 0.17 M KCl yielded 3.1 mg protein/ml 
which had a 37% inhibition of aromatase, 0.5 M KCl eluted 6 mg protein/ml 
which had an 84% inhibition of aromatase and 1.5M KCl eluted 0.05 mg 
protein/ml which had a 6.7% inhibition of aromatase. Accordingly, the 0.5 
M KCl fraction was used to elute the active material from the Orange A 
bound column. KCl (0.5M) was found to have no effect in the granulosa cell 
aromatase assay: control (without KCl, 3 determinations, 710.+-.41 ng/ml. 
with KCl, 712.+-.38 ng/ml). Duration of incubation time was assessed with 
and without FSH. Two hour assay incubations yielded 2 ng estrogen/ ml; 
twenty-four hours: 5.3 ng estrogen/ml. With 2 ng FSH added, 39 ng 
estrogen/ml at two hours and 6 ng FSH/ml produced 8 ng estrogen/ml at 
twenty-four hours. With 1 ng FSH/ml, 35.+-.1.8 ng estrogen/ml were 
produced at two hours and 4.7.+-.0.9 ng estrogen/ml at twenty-four hours. 
Accordingly, a three-hour incubation was used to determine specific FRP 
activity. 
When FRP preparation was heated to 56.degree. C. .times.1 hours, the 
inhibition of granulosa cell aromatase was lost. The FRP activity levels 
(% inhibition of porcine granulosa cell aromatase by 50 ug of follicular 
fluid) for untreated patients was 14.16.+-.5.32% (X.+-.SEM). For patients 
receiving hMG, FRP levels were 18.09.+-.3.46%, and for patients receiving 
clomiphene, 13.7.+-.5.36%. There was no statistically significant 
difference in the values between the three treatment groups. The amount of 
estradiol in the follicular fluid of untreated patients was 2.59.+-.1.2 
ug/ml, for patients receiving hMG therapy, 0.34.+-.0.5 ug/ml, for patients 
receiving clomiphene, 1.31.+-.0.34 ug/ml. These values were all 
significantly different (p &lt;0.05, untreated vs clomiphene, clomiphene vs 
hMG; p&lt;0.01 untreated vs hMG). Progesterone values for untreated patients 
were 9.84.+-.3.35 ug/ml, for hMG-treated patients, 5.18.+-.1.1 ug/ml, and 
for clomiphene-treated patients, 11.3.+-.2.3 ug/ml These differences were 
siqnificant for the unstimulated and hMG-treated patients (p &lt;0.05) and 
hMG vs clomiphene treated patients (p &lt;0.01). The 17-hydroxyprogesterone 
concentrations for patients receiving no additional therapy were 
1.66.+-.0.25 ug/ml, for those receiving clomiphene therapy 2.6.+-.0.3 
ug/ml, and for those receiving hMG therapy: 0.76.+-.0.11 ug/ml. All these 
values were significantly different (hMG vs clomiphene p&lt;0.01; hMG vs 
unstimulated p&lt;0.01; unstimulated vs clomiphene p&lt;0.025). Follicular fluid 
androstenedione concentrations in untreated patients were 61.9.+-.43 
ng/ml. For hMG and clomiphene treated patients they were 85.5.+-.37, and 
84.8.+-.43 ng/ml, respectively. Follicular fluid testosterone levels from 
untreated patients were 7.34 ng/ml.+-.3.7. For the treated patients, there 
was no difference in the follicular fluid testosterone concentrations in 
patients receiving either hMG (7.09.+-.2.14 ng/ml) or clomiphene 
(6.14.+-.1.8 ng/ml). 
Correlation of FRP vs Follicular Fluid Steroids 
There was a positive correlation between follicular fluid estradiol 
concentrations and FRP protein activity in untreated patients (r=0.689, 
p.&lt;0.01). For patients receiving hMG therapy, there was no significant 
correlation between FRP activity and follicular level estradiol 
concentrations (r=0.490, p&lt;0.1). For patients receiving clomiphene 
therapy, the correlation between FRP activity and follicular fluid 
estradiol was described by two populations using a second degree 
regression analysis (r.sub.2 =0.853, p &lt;0.01). Correlation between 
follicular fluid progesterone concentrations and FRP activity for 
untreated patients (r=0.622, p &lt;0.05) and hMG treated patients r=0.756, p 
&lt;0.005) was significant. For clomiphene treated patients, correlation 
between follicular fluid progesterone and FRP activity were not 
significant. The correlation between follicular fluid 
17-hydroxyprogesterone values and FRP activity for the untreated (r=0.833, 
P&lt;0.001), as well as hMG (r.sub.2 =0.853, p &lt;0.0025) and clomiphene 
(r.sub.2 =0.637, p &lt;0.025) treated patients was significant by second 
order regression analysis. The correlation between FRP and androstenedione 
concentration from untreated (r=0.241), hMG (r=0.357), and clomiphene 
(r=0.219) treated patients was not significant, nor was the correlation 
between testosterone and FRP activity significant (r=0.477, 0.409, 0.480, 
respectively). The average inhibin activity for untreated patients was 
50.+-.1.9 CU. In patients receiving treatment, inhibin activity was 
8.2.+-.2.3 and 35.4.+-.3.7 CU for clomiphene and hMG treatment, 
respectively. Differences between hMG and either clomiphene or untreated 
patients were highly significant (p &lt;0.001). Correlation between inhibin 
and FRP activities for untreated patients was significant (r=0.654; p 
=0.05; FIG. 26). However, there was no statistically significant 
correlation between inhibin and FRP activities in patients receiving 
either hMG (r=0.270) or clomiphene (r=0.262). 
These observations report the presence of an aromatase inhibitor (FRP) in a 
purified fraction of human follicular fluid. This follicular fluid protein 
fraction (FRP) has previously been shown to inhibit hMG-mediated increases 
in rat ovarian weight and serum estradiol concentrations. As is shown 
hereinafter in Example Thirteen, when this protein fraction is injected 
into regularly menstruating monkeys, it disrupts folliculogenesis 
resulting in either anovulatory cycles or luteal phase insufficiencies 
accompanied by low serum estradiol and progesterone concentrations without 
markedly altered peripheral serum gonadotropin levels. This data, taken 
together with those presented previously, suggest that the developing 
granulosa cell, through production of an aromatase inhibitor, is capable 
of autoregulating the estrogen production of its own and other follicles. 
EXAMPLE EIGHT 
Gonadal Regulatory Protein Inhibition of Microsomal Aromatase Activity 
In this example, the effect of the gonadal-regulating protein on aromatase 
activity was studied in cell-free placental microsome preparations which 
were prepared in accordance with the techniques described in Mol. Cell. 
Endocrinol., 6 (1976) pp.10 5-115 and J. Clin. Endocrinol. Metab., 39 
(1974) 754-760. 
Aromatase incubations were carried out in a total volume of 0.6 ml in 
12.times.75 ml glass tubes as described in the above-identified 
references. Incubations contained 0.3 ml of placental cell-free 
preparation in buffer A, 0.10 ml of .sup.14 C-androstenedione (A,10.sup.-6 
molar), NADPH (10.sup.-6 molar) and nicotinamide (0.4 molar) in buffer A. 
Gonadal regulatory protein test fractions (0.2ml, 12 to 18 kd, pI 
4.0-6.5), were preincubated with placental extracts (20 minutes) and then 
the .sup.14 C-androstenedione-NADPH mixture was added. Reaction at 
termination/quenching was performed by addition of 100-fold excess 
unlabeled A. Estrogen concentrations were determined by radioimmunoassay 
as described in J. Clin. Endocrinol. Metab., 39, 754-760. 
FRP-dose response determinations were performed using a three-minute 
reaction time. The velocities determined over a range of substrate 
concentrations (0.5-2 mM) demonstrated Michaelis-Menton type kinetics with 
a Km of 0.8 mM as shown in FIG. 27. The aromatase velocities were 
determined over a range of FRP concentrations (0, 62.5, 125, 250, 500 and 
1,000 .mu.g/ml). The use of Dixon kinetic plotting techniques demonstrated 
that FRP inhibited microsomal aromatase with an App Ki=3.times.10.sup.-5 M 
(FIG. 28). 
These data demonstrate the non-competitive inhibition of FRP on aromatase 
activity. Enzyme inhibition may be of three types. First, competitive 
inhibition describes the competition of the inhibitor for the 
substrate-specific site on the enzyme. Second, non-competitive inhibition 
describes a direct inhibition of the enzyme without competition for the 
substrate-specific sites. Third, uncompetitive inhibition describes an 
indirect inhibition of the enzyme by additional mechanisms. In FIG. 28, 
the intersection of the lines at the abscissa, as opposed to ordinal 
intersection (competitive) or non-intersection (uncompetitive) show that 
FRP is a non-competitive inhibitor of aromatase activity. FRP directly 
interacts with the aromatase molecule to inhibit its activity. 
EXAMPLE NINE 
Modulation of Beta-ol-Dehydrogenase (3.beta.ol) Activity By Follicular 
Protein 
Porcine and human granulosa cells from medium sized follicles (2-5 mm in 
diameter) were cultured (100,000 cells per culture) with various 
concentrations of FRP (12-18 kd. pI 4.0-6.5) isolated from follicular 
fluid. To these cultures pregnenolone (10.sup.-5 M) and either hCG or pFSH 
were added. The conversion of pregnenolone to progesterone was used to 
determine 3-beta-ol-dehydrogenase activity in the granulosa cells. FRP 
caused a biphasic response in progesterone production. Gonadal regulatory 
protein in the concentration of 167 ug/ml caused a 10 fold increase in 
progesterone production, while the 500 ug/ml concentration caused a return 
to baseline levels. These results are shown in FIGS. 29 and 30. Although 
pFSH induced a dose response increase in progesterone production, hCG 
produced no change in progesterone levels. Low doses of the gonadal 
regulatory protein acted synergistically with low doses of pFSH to 
increase 3-beta-ol-dehydrogenase activity. However, high doses of FRP 
inhibited the low dose pFSH stimulation of 3-beta-ol-dehydrogenase 
activity. High doses of pFSH (10 ug/culture) overcame both the low dose 
enhancement and the high dose inhibition of FRP on 3-beta-ol-dehydrogenase 
activity. Kinetic analysis of FRP modulation of 3-beta-ol-dehydrogenase 
activity was performed substantially as described in Example Eight. The 
effects of FRP on 3-beta-ol-dehydrogenase activity in human placental 
microsomes are of a non-competitive nature. 
EXAMPLE TEN 
Inhibition of LH/hCG Receptors In Granulosa Cells by FRP 
FRP (12-18 kd., pI about 4.0-6.5) was isolated from porcine follicular 
fluid substantially as described in Example Three. 
Granulosa Cell Cultures 
The granulosa cells were counted using a hemocytometer and viability was 
determined by 1% trypan blue exclusion. Cells were cultured 
(2.times.10.sup.5) in 2 ml of Medium 100 containing 10% fetal calf serum 
with penicillin and streptomycin (100 g/ml and 100 U/ml respectively) in 
12.times.75 mm Falcon plastic test tubes. FSH (10 ng), and/or FRP (1 mg) 
were added at the initiation of culture. Media was changed after 
seventy-two hours. 
Binding Analyses 
Porcine granulosa cells were suspended in appropriate volumes of PBS-0.1% 
gelatin (PBS-gel). All assays were run with five concentrations of labeled 
hCG (10 ul), buffer (PBS-0.1% gel, 100 ul), and cells. Reactions were 
initiated by the addition of granulosa cells and were carried out for four 
hours at 25.degree. C. All reaction tubes were precoated with 5% BSA to 
reduce non-specific absorption. Reactions were terminated by adding one ml 
of cold PBS followed by centrifugation at 30,000 x g for ten minutes. The 
supernatant was carefully aspirated and the pellet rewashed with one ml of 
PBS. The final pellet was counted in a gamma counter. Specific binding was 
calculated as the difference between binding in the presence 
(non-specific) and absence (total) of an excess of unlabeled hormone. Data 
were analyzed by Scatchard plots. Duplicate determinations were performed 
in three separate assays at each time interval. 
Follicular protein significantly reduced porcine granulosa cell hCG binding 
by seventy-four hours of culture. This effect was prevented with the 
co-administration of FSH. 
By ninety-six hours of culture, no change in porcine granulosa cell hCG 
binding was apparent with FSH, follicular protein, or both compared to 
control cultures demonstrating cellular recovery after removal of 
follicular protein at seventy-two hours of culture. 
Thus, in addition to regulating key enzymatic steps in the steriodogenic 
pathway (aromatase, 3.beta.-ol dehydrogenase), general granulosa cell 
response to trophic LH stimulation is also mediated by follicular protein 
through inhibition of LH receptor function. 
EXAMPLE ELEVEN 
Adenylate Cyclase Activity 
The effects of FRP on FSH-induced adenylate cyclase activity in porcine 
granulosa cells was evaluated using Gpp (NH)p and forskolin as 
pharmacological probes of adenylate cyclase activity. With the addition of 
100 .mu.g/ml of FRP (12-18,000 pI 4.0-6.5), a significant decrease in this 
activity was found. Maximal inhibition of cAMP formation was achieved with 
1 mg/ml of FRP. Adenylate cyclase activity reached a maximum 20 min after 
incubation with FSH and returned to baseline by 45 minutes. FRP induced a 
parallel reduction in adenylate cyclase activity during this same interval 
of time (FIG. 31). Adenylate cyclase activity in the membranes of FRP+FSH 
and FRP alone treated cells was significantly less than in cells incubated 
with FSH (p&lt;0.05). Adenylate cyclase activity of FRP treated cells was 
unchanged in the presence of methyl-isobutyl-xanthine. Further, when FRP 
was heated (56.degree. C., 45 min.) or precipitated with 10% TCA, it lost 
the capability to inhibit adenylate cyclase. The 50% inhibitory dose 
(ID50) for FRP inhibition of Gpp(NH)p stimulated adenylate cyclase 
activity with preincubation of granulosa cells with FSH was 350 .mu.g/ml 
and 80 .mu.g/ml without FSH. The ID50 for the FRP inhibition of forskolin 
stimulated adenylate cyclase activity was 350 .mu.g/ml (FIG. 32). 
Adenylate cyclase activity was determined after a 10 min. incubation with 
forskolin or Gpp(NH)p. When these responses were compared during a 5-20 
min. interval, the Gpp(NH)p stimulated adenylate cyclase activity was more 
sensitive to inhibition by FRP than forskolin stimulated adenylate cyclase 
activity (p&lt;0.05). Adenylate cyclase activity stimulated by Gpp(NH)p was 
also, on a molar basis, more sensitive to FRP inhibition than forskolin 
stimulated activity. FRP had no apparent effect on LH or hCG responsive 
adenylate cyclase activity in these granulosa cell preparations. No other 
naturally occurring substance has previously been shown to selectively 
inhibit FSH responsive adenylate cyclase activity in granulosa cells 
without also inhibiting LH responsive adenylate cyclase activity. In 
conclusion, these data demonstrate that FRP inhibited FSH responsive 
adenylate cyclase activity in porcine granulosa cells. 
EXAMPLE TWELVE 
Reduction of Follicular Atresia 
Sheep have been injected intramuscularly with FRP (2 mg at 08:00 and 16:00 
hours) for fourteen days. During this interval, there was a destruction of 
the estrous cycle such that they did not ovulate. On Day 14 of treatment 
the ovaries were removed, fixed in formalin, serially sectioned (4 
microns) and the sections were mounted on glass slides for microscopic 
evaluation after staining with hematoxylin and eosin. It was evident that 
FRP had inhibited ovulation by blocking the normal development process 
whereby a developing follicle either ovulates or degenerates by becoming 
atretic. This was evident in development of the follicles. These findings 
demonstrate that the normal life span of the total follicular pool could 
be significantly prolonged by the therapeutic administration of FRP. With 
prolongation of the life span of the follicular pool, a prolongation of 
the reproductive capacity of an individual would naturally follow. Since 
the alteration in function and composition of sex steroid-dependent 
structures, which is commonly referred to as menopause in females, would 
be prevented or forestalled totally, or in part, if the sex steroid 
secretion was maintained, and since the ovarian follicles are the source 
of such sex steroids, it follows that prolongation of the life span of 
ovarian follicles by FRP therapy will lead to prevention or reduction of 
the clinical manifestations of the menopause. 
III. Whole Animal Studies 
EXAMPLE THIRTEEN 
Inhibition of the Primate Ovarian Cycle by FRP 
The Examples heretofore presented report the identification of a heat and 
trypsin labile protein extracted from porcine, bovine and human gonadal 
fluid which inhibited ovarian response to gonadotropins. The activity of 
this protein, secreted by human granulosa cells, increased with increasing 
follicular fluid estradiol levels and decreased with increasing follicular 
fluid progesterone levels (as shown in Example Four) both in vivo and 
during granulosa cells'luteinization in vitro (Example Four). This 
material has also been found to inhibit granulosa cell aromatase activity 
in both porcine (Example Three) and human (Example Four) granulosa cells 
in vitro. Follicular fluid extracts containing this activity are shown in 
said Examples to have a molecular weight of 12,000-18,000 and an 
isoelectric point of about pH 4.0-4.5 to 6.0-6.5, owing that the 
biophysical nature of this protein is not inhibin. In the present Example, 
the effects of this follicular protein fraction on the integrated 
hypothalamic-pituitary-ovarian axis of the normally cycling monkey are 
assessed. 
Adult female rhesus monkeys (Macaca mulatta; n=8) were selected because of 
reproductive characteristics indicating normal ovarian function and 
menstrual regularity. The FRP employed in this Example was the inhibitory 
follicular fluid protein fraction which was obtained as described in 
Example Three. 
Five monkeys were treated with FRP extracted from porcine follicular fluid 
and three monkeys served as vehicle controls. The FRP (3 mg in 1 ml of 
.01M PBS, pH 7) was administered (IM) at 07:00 hours and 19:00 hours 
beginning on day 1 of the menstrual cycle for a total of twenty-nine 
injections. Total dose for each monkey was 87 mg. Control monkeys received 
only PBS over the same interval. Iron supplement was administered once 
each week. Daily (09:00-11:00 hours), femoral blood samples (3.5 ml) were 
collected beginning with the onset of menses (day 1) and were continued 
until the onset of next menses. Radioimmunoassays for LH, FSH, 
17.beta.-estradiol, and progesterone in serum were performed. 
Monkeys which received FRP injections had either no serum LH peak nor 
elevation of serum progesterone (n=2, FIG. 33), or a midcycle LH surge 
followed by an inadequate luteal phase as demonstrated by low serum 
progesterone levels and/or early onset (twenty-four days intermenstrual 
interval) of vaginal bleeding (n=2, FIG. 34). One FRP treated monkey had a 
midcycle LH surge followed by depressed follicular phase estradiol and 
luteal phase serum progesterone levels (not shown). The 95% confidence 
limits for the vehicle control and other values obtained for this 
population are depicted by the shaded area in FIGS. 33 and 34. Serum 
estradiol levels were reduced throughout the interval of FRP treatment in 
the monkeys without LH surges in the late follicular phase (FIG. 34) and 
in those with luteal phase defects (FIG. 33). Serum FSH and LH levels were 
within the 95% confidence intervals for FRP treated monkeys (FIG. 33). 
However, in all five FRP treated monkeys, serum FSH levels rose during the 
course of therapy. In the monkeys with inadequate luteal phases (FIG. 33), 
serum estradiol levels were below the 95% confidence intervals in the late 
follicular and mid luteal phases. While the serum levels of both estradiol 
and progesterone were markedly suppressed after the LH surge. In 
subsequent cycles (N=3/monkey) onset of vaginal bleeding occurred in the 
usual twenty-six - thirty day monthly interval and no toxic effect of GRP 
treatments was noted. 
FRP administered to normally cycling monkeys throughout the follicular 
phase of the menstrual cycle reduced peripheral estradiol levels without 
markedly affecting FSH, resulting in either apparent anovulation or 
inadequate luteal phases. The mixed response in serum sex steroid levels 
in the FRP treated monkeys may reflect a variable ovarian sensitivity to 
FRP. That serum FSH levels were not significantly inhibited indicates that 
this material has a biological activity different from that of inhibin 
activity in charcoal-extracted whole porcine follicular fluid. Further, 
when FRP was tested in an inhibin assay, its activity was either at the 
limits of sensitivity or undetectable. In contrast to the inhibition of 
FSH, normal or rising serum FSH levels during FRP treatment were found. 
This indicates that the purification of follicular fluid described herein 
removed the major inhibin activity. The molecular weight of the described 
FRP is less than 20,000 while inhibin activity has been associated with 
molecular weights greater than 45,000. 
The in vivo observations of reduced serum estradiol levels in GRP treated 
monkeys support the previously described Examples of granulosa cell 
aromatase inhibition by both human and porcine derived FRP. Further, these 
observations are in agreement with examples of reduced serum estradiol 
levels in FRP treated rats. 
Luteal phase defects, as evidenced by suppressed serum estradiol and 
progesterone levels and early onset of vaginal bleeding, result from 
inadequate follicular maturation in the preceding follicular phase. Taken 
together with the observations reported in this Example of reduced serum 
estradiol and progesterone levels associated with FRP treatment,these data 
indicate that the FRP described in the present invention has a direct 
intraovarian action which disrupts the normal process of folliculogenesis 
by an action apart from gonadotropin stimulation. 
EXAMPLE FOURTEEN 
Inhibition of Spermatogenesis 
Male mongrel dogs were injected with 2 mg of FRP derived from porcine 
follicular fluid (12-18 kd., pI 4.0-6.5), as described in Example Three, 
at 08:00 and 16:00 hours for 20 days. On Day 20 of therapy the testis were 
obtained, fixed in formalin, sectioned by microtome (four micron 
sections), mounted on slides and stained with hematoxylin and eosin. Upon 
evaluation of the slides, a marked reduction in mature spermatozoa was 
present in the seminiferous tubules in the FRP-treated dogs as compared to 
the controls. Moreover, there was an 87% reduction in pacytene 
spermatocytes and a 44% reduction in mature spermatogonia in the 
FRP-treated dogs. 
IV. Preparation of FRP Antibodies 
EXAMPLE FOURTEEN 
Preparation of FRP Antibodies 
Antibodies (monoclonal and polyclonal) to FRP and its conjuners and analogs 
may be prepared for diagnostic and therapeutic uses including but not 
limited to fertility control. Antibody in this contex refers to a 
synthetic protein which binds FRP and alters FRP biological activity. 
Antibodies to FRP are prepared by either polyclonal or monoclonal 
techniques: 
Polyclonal: 5 adult rabbits are immunized with 0.1 mg of FRP suspended in 
complete Freund's adjuvant (5 ml). One ml of this preparation is injected 
subcutaneously at 20 different sites in the back and neck. This is 
followed by monthly injections thereafter. Ear vein phlebotomies are 
performed after each monthly booster injection and the sera obtained are 
checked for titer, affinity, and specificity. 
In a specific example, 1 .mu.g of FRP (from Example Three) was solubilized 
in 0.5 ml physiological saline and emulsified with an equal volume of 
Freund's adjuvant to prepare an inoculum. 
New Zealand White, female rabbits weighing 21/2-3 kg were bled via the 
median ear artery for pre-immune serum. A 10.times.20 cm area on the back 
was shaved, then each rabbit was intradermally injected at multiple 
points. Approximately 50-75 .mu., of inoculum was injected into 10-25 
sites in the shaved area; rabbits 1 and 3 received 0.6 ml and 1.6 ml, 
respectively. The rabbits were boosted in similar fashion six weeks later 
receiving 0.5 ml and 1.5 ml, respectively. Six and ten day post-boost, the 
rabbits were test bled, again through the median artery. Sera containing 
the polyclonal antibodies thus obtained were titred via RIA against 
.sup.125 I-labelled FRP as follows. 
Rabbit sera were two-fold serially diluted in RIA buffer from 1:1000 to 
1:64,000. 100 .mu.each of the RIA buffer, diluted serum, and .sup.125 
I-GRP and 200.mu.RIA buffer were also prepared. The tubes were incubated 
at room temperature overnight. One-half ml (0.5 ml) precipitating solution 
containing 2% goat-antirabbit gamma globulin and 4% polyethylene glycol in 
RIA buffer was then added to each tube except the total tubes. The tubes 
were vortexed, incubated at room temperature for ten minutes, then 
centrifuged at 3000 rpm for an additional ten minutes. The supernatant was 
aspirated and the precipitin-pellet counted for one minute on a gamma 
counter. The rabbit sera was found to contain antibody which bound 
.sup.125 I-FRP (12 to 18 kd., pI 4.0-6.5) in all dilutions tested, 
including 1:64,000. 
Monoclonal: BALB/c mice are immunized with FRP by intraperitoneal injection 
(250 .mu.g).times.2. Thereafter, the spleens are collected and cell 
suspensions prepared by perfusion with DMEM. The BALB/c spleen cells are 
fused with SP 2/0-Ag 14 mouse myeloma cells by PEG and the resultant 
hybridomas grown in HAT selective tissue culture media+20% fetal calf 
serum. The surviving cells are allowed to grow to confluence. The spent 
culture media is checked for antibody titer, specificity, and affinity. 
Specifically, the mice were immunized with FRP (from Example Three) 
adjuvant emulsion described above. Each mouse first received 0.2 ml of 
this emulsion intraperitoneally, then were reinjected in similar fashion 
with 0.1 ml six weeks later. Mouse serum was obtained ten days after the 
second injection and then tested for anti-FRP activity via ELISA. The 
mouse exhibiting the highest absolute anti-FRP activity was chosen for 
cell fusion. 
Three to four days prior to fusion, the chosen mouse was intravenously 
injected with 0.1 ml FRP solubilized in physiological saline, and 
SP2/0-Ag14 BALB/c myeloma cells maintained in log phase culture. On the 
day of fusion, the mouse was sacrificed and its spleen aseptically 
removed. Spleen cell suspension containing B-lymphocytes and macrophages 
were prepared by prefusion of the spleen. The cell suspension was washed 
and collected by centrifugation. Myeloma cells were also washed in this 
manner. Live cells were counted and the cells were placed in a 37.degree. 
C. water bath and 1 ml of 50% poly-ethylene glycol in DMEM slowly added. 
The cells were incubated in the PEG for one to one-and-a-half minutes at 
37.degree. C., after which the PEG was diluted by the slow addition of 
media. The cells were pelletted and 35 to 40 ml of DMEM containing 10% 
fetal bovine serum was added. The cells were then dispensed into tissue 
culture plates and incubated overnight in a 37.degree. C., 5% CO.sub.2, 
humidified incubator. 
The next day, DMEM-FCS containing hypoxanthine, thymidine, and aminopterin 
(HAT medium) was added to each well. The concentration of HAT in the 
medium to be added was twice the final concentrations 
required; i.e. .sup.H final=1.times.10.sup.-4 M, .sup.A 
final=4.0.times.10.sup.7 M, and .sup.T final=1.6.times.10.sup.-5 M. 
Subsequently, the plates were incubated with 1 x HAT medium every 
three-four days for two weeks. Fused cells were thereafter grown in 
DMEM-FCS containing hypoxanthine and thymidine. As cell growth became 1/2 
to 3/4 confluent on the bottom of the wells, supernatant tissue culture 
fluid was taken and tested for FRP specific antibody by ELISA. Positive 
wells were cloned by limiting dilution over macrophage or thymocyte feeder 
plates, and cultured in DMEM-FCS. Cloned wells were tested and recloned 
three times before a statistically significant monoclonal antibody was 
obtained. Spent culture media from the chosen clone contained antibody 
which bound .sup.125 I-FRP (12-18 kd., pI 4.0-6.5) in all dilutions 
tested, including 1:64,000. 
Each of the above described antibody containing solutions was tested 
against FRP secreted by granulosa cells and found to bind to independently 
produced FRP. Antibodies from both polyclonal and monoclonal preparations 
are screened for affinity by evaluating the ability to inhibit the 
reduction of aromatase activity by FRP in human placental microsomes. 
Those antibodies which block this reaction were titered by incubating 
various dilutions of the antibody with a fixed mass of radioactively 
labelled FRP in 100 ul of assay buffer (TRIS 0.025M, pH 7.4) at 4.degree. 
C. overnight with constant agitation. The dilution of antibody that bound 
50% of the labelled FRP under these conditions was defined as the titer. 
Specificity was determined in the same manner as the above only column 
fractions not containing FRP activity were radiolabelled and screened for 
antibody binding. 
It will be apparent from the above description of FRP antibodies that a 
wide variety of diagnostic tests is possible using the antibodies of the 
invention. The over-production of FRP by the gonads, an indication of the 
presence of a condition such as ovarian cancer, could easily be detected 
in body fluids such as serum through the use of an immunoassay which 
employs the novel antibodies according to methods known in the art. 
Similarly, in attempting to diagnose causes of infertility, an immunoassay 
to detect decreased levels of FRP in the body would be a useful adjunct to 
known hormone assays. Further uses for the antibodies include the 
induction of superovulation in livestock. The direct administration of FRP 
antibodies to sheep has been shown to induce multiple ovulations in ewes. 
It should be noted that other than the novelty of the protein and the 
preparation of FRP antibodies thereto, techniques required for the 
preparation and use of diagnostic test kits and superovulatory antibodies 
are known in the art and will not be described in further detail. 
More specifically, the isolation of FRP from porcine follicular fluid 
(12-18 kd., pI 4.0-6.5) has allowed the production of antisera containing 
antibody to FRP which cross-reacts with human FRP. This antibody enables 
the preparation of an enzyme-linked immunosorbent assay (ELISA) and other 
assays suitable for quantitation of FRP in body fluids. In a particular 
example, an ELISA assay was used to quantitate relative FRP 
immunoreactivity in human serum in postmenopausal, normally menstruating 
and anovulatory patients. Since naturally-occurring FRP plays a central 
role in the ovulatory process, quantification of the level of FRP in body 
fluids provides diagnostic information of significance. 
Polyclonal antibodies were prepared from porcine FRP (12-18 kd. fraction 
having isoelectric points in the range of pH 4.0-4.5 and 6.0-6.5) 
according to the method described above, and titered via RIA against 
labeled porcine FRP (having similar characteristics) in dilutions up to 
1:64,000. 
A parallel-line dilution assay comparing the binding of the antibody to FRP 
in human serum to different antigen-containing samples by ELISA was 
performed with the following samples: porcine granulosa cell culture 
media, human granulosa cell culture media, human urine, bovine serum 
albumin, ribonuclease A, chymotrypsinogen and amniotic fluid. Immuno 
recognition of samples in serially-diluted porcine and human granulosa 
cell culture media and urine showed parallelism, whereas those of bovine 
serum albumin, ribonuclease A, chymotrypsinogen and amniotic fluid showed 
no binding, thus indicating a lack of recognition of the antibody for 
proteins in those materials. 
The levels of FRP in the following patients was determined by ELISA assay. 
Ten patients undergoing ovarian extirpative surgery were all of 
reproductive age, and had regular menses which no clinical or laboratory 
evidence of ovarian dysfunction, and all underwent a total abdominal 
hysterectomy and bilateral salpingo-ophorectomy. Peripheral venous blood 
was obtained before surgery and twenty-four hours postoperative. Ten 
post-menopausal patients were 53-63 years of age and required 
hospitalization for conditions unrelated to ovarian function. Thirteen 
ovulatory patients, all under age 40, had problems unrelated to ovarian 
function including infertility due to tubal, cervical or male factor or 
previous tubal sterilization. These patients had regular menstrual cycles 
which were documented as ovulatory based on serum progersterone levels 
greater than 2 mg/ml. The anovulatory patients each had a history of 
oligomenorrhea with chronic anovulation. The anovulatory patients received 
50-250 mg of chlomiphene citrate on menstrual cycle days 5-9 for ovulation 
induction. 
Using the ELISA-determined levels of FRP in the ovulatory women as a basis, 
post-menopausal women had significantly lower serum levels of FRP, and 
similar levels were found in the serum of reproductive-age women 
twenty-four to forty-eight hours after ophorectomy, versus significantly 
higher pre-operative FRP levels in this group. 
Serum FRP levels at the 3rd to 5th menstrual cycle day differed 
significantly in two groups of anovulatory women. A first group had high 
levels of serum FRP, and a second group very low levels approximating the 
levels of post-menopausal women. Both groups of anovulatory patients had 
similar low peripheral estradiol levels. The first group having low serum 
FRP levels comprised those with whom chlomiphene citrate therapy was 
successful in inducing ovulation. In contrast, the anovulatory patients 
with significantly elevated serum FRP levels failed to ovulate after 
chlomiphene therapy. Significant differences between these two groups were 
also apparent twenty-two to twenty-three days after the beginning of the 
last menstrual period. Elevated FRP levels in serum have thus been shown 
to be useful in predicting which anovulatory patients will or will not 
respond to chlomiphene citrate therapy. 
As detailed above, FRP antibodies provide a wide variety of useful 
diagnostic procedures. For example, FRP has been found to be substantially 
elevated in patients having ovarian cancer. It should be noted that there 
presently exists no reliable diagnostic test for this condition. In 
addition, it has also been found that varying levels of immuno-reactive 
FRP are found in urine during active phases of follicle growth and during 
lutenization, allowing the determination of the activity and timing of 
ovarian functions with the FRP antibodies of the invention. 
The invention in general in certain aspects in particular are broad in 
scope, for example, the proteinaceous substance of the invention may be 
produced by other methods. 
In particular, a c-DNA probe can be prepared against the biologically 
active portion of FRP and used to identify the FRP genome in granulosa 
cells or Sertoli cells from any mammalian species. The identified genome 
can then be synthesized into a plasmid which can then be employed to 
produce recombinant DNA in proliferating bacteria according to methods 
known in the art. 
In addition, granulosa or Sertoli cells may be transformed, e.g. by SV40 
virus, to produce FRP quantity. 
In summary, the intergonadal protein, its identifying characteristics, 
methods of production and uses set forth herein identify a novel and 
unique proteinaceous substance. FRP is described herein to be the only 
proteinaceous substance known which inhibits aromatase activity by 
determination of the extent of the conversion of androgens to estrogens. 
Moreover, such inhibition is unique in that it is non-competitive in 
placental microsomes. Further, FRP produces a biphasic effect on 
3.beta.-ol dehydrogenase activity in cultured granulosa cells as shown in 
FIGS. 29 and 30. 
In addition, FRP inhibits FSH induction of LH receptor formation as 
determined by addition to an in vitro culture of granulosa cells, and 
inhibits FSH responsive adenylate cyclase activity as similarly 
determined. 
In addition to this biological activity, FRP is identified by the elution 
profile through a molecular weight exclusion column under the conditions 
set forth in Example Two, as indicated by the designation "FRP" of the 
elution curve in FIG. 9. The elution profile through a hydrogen ion 
exclusion column, particularly the area designated by the indicated peaks 
numbers 4 and 7 in FIG. 15, further identifies identifying characteristics 
of FRP when conducted under the conditions set forth in the Example Three. 
Moreover, a polyacrylamide gel electrophoretic pattern from a high 
pressure liquid chromatographic column, as shown by the designation "FRP" 
in column 6 of FIG. 11, uniquely identifies FRP when the pattern is 
obtained as set forth in Example Two. 
From the foregoing description, one skilled in the art can easily ascertain 
the essential characteristics of the invention and, without departing from 
the spirit and scope thereof, can adapt the invention to various usages 
and conditions. The regulatory protein described herein is defined 
primarily by its biological effect in a biological system. This phrase is 
meant to define a reversible effect, that is, one that does not involve 
the destruction of endocrine functions such as by the heating or other 
denaturation of proteins. For example, the administration of the protein 
to a mammal inhibits aromatase activity, and the cessation of this 
administration allows the reversal of the inhibitory effect. 
While the biological activities set forth herein define the protein, the 
physical characteristics set forth also provide distinguishing features. 
The protein moiety which provides the described biological activity shows 
a wide range of molecular weights (from 5,500 to 18,000 daltons) and an 
electrophoretic range of from about pH 3.5 to about 7.0. More preferably, 
the physical characteristics of the protein are identified as ranging from 
a molecular weight of about 10,000 up to 18,000 daltons, and having an 
isoelectric point of from about pH 4.0 to about 6.5. Based on the data set 
forth herein, the physical characteristics of the protein moiety which 
produces the above-described antibody has a molecular weight of about 
15,000 daltons and an isoelectric point of about pH 4.5 to 4.75. However, 
changes in form and the substitution of equivalents are contemplated as 
circumstances may suggest or render expedient; and although specific terms 
have been employed herein, they are intended in a descriptive sense and 
not for purposes of limitation, the scope of the invention being 
delineated in the following claims.