bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of hamster two‐cell embryos in the isolated mouse oviduct in organ culture system
Authors: Minami N. , Bavister B. D. , Iritani A.
Published: February 2005

Hamster early two‐cell embryos developed to the expanded blastocyst stage within the isolated mouse ampulla maintained in organ culture system. Mouse ampullae isolated at different times after treating the mice with human chorionic gonadotropin (hCG) (0–72 h) or pregnant mare's serum gonadotropin (PMSG) (30–32 h) were flushed with culture medium, and hamster early two‐cell embryos were introduced into these ampullae. Mouse ampullae isolated at 14–32 h after hCG injection were more favorable for the development of the embryos than those isolated at 70–72 h. When mouse ampullae were isolated 30–32 h after hCG or PMSG treatment, 39% of the cultured eggs developed, some of them to the expanded blastocyst stage after additional culture for 65–70 h. These results indicate that unknown oviductal factors stimulate the development of hamster early two‐cell embryos, and these factors are under the control of hCG or PMSG. In addition, these factors are common to the mouse and hamster.
Effect of defolliculation and 17α‐hydroxy, 20β‐dihydroprogesterone on cyclic AMP level in full‐grown oocytes of the rainbow trout, Salmo gairdneri
Authors: Finet B. , Jalabert B. , Garg S. K.
Published: February 2005

The changes in cAMP were followed in trout oocytes incubated in vitro after defolliculation performed by either enzymatic or manual dissection. Both defolliculation methods induced a highly significant rise in oocyte cAMP level (4.5 times the basal level of control [follicle‐enclosed oocytes], after 6 h). Treatment of defolliculated oocytes with 17α‐hydroxy,20β‐dihydroprogesterone (17α,20β‐OH‐P) (10−6 M), which induced oocyte maturation (germinal vesicle breakdown [GVBD]) was able, first, to interrupt the increase of oocyte cAMP level promoted by defolliculation and then to lower this level significantly down to values that still remained higher than folliculated controls. Very low concentrations of 17α,20β‐OH‐P (1.38–55.6 10−9 M), or physiological doses of testosterone (0.35 10−6 M, in the range found in vivo before ovulation) were able to induce a similar decrease of oocyte cAMP level without inducing GVBD. Under the same experimental conditions estradiol (0.35 10−6 M) exhibited no action. These results suggest that some factor(s) originating in the follicle (FIF), inhibit the oocytes' tendency to accumulate cAMP before the final surge of 17α,20β‐OH‐P. This factor might be a follicular steroid such as testosterone or nonmaturing concentrations of 17α,20β‐OH‐P. Moreover our data favour the hypothesis that the final surge of 17α,20β‐OH‐P could induce distinct intraoocyte mechanisms: the first induces an irreversible blockage of cAMP level before the inhibitory action of the FIF is suppressed by ovulation, and the second mechanism leads to GVBD.
Fine structure of sheep fertilization in vitro
Authors: Crozet N.
Published: February 2005

The fertilization events of the sheep oocyte have been investigated by electron microscopy. Thirty‐two oocytes recovered from the oviducts of superovulated ewes were fertilized in vitro by a procedure that has given rise to full embryonic development (Crozet et al.: Gamete Res 16:159–170, 1987) and were analyzed at different stages of fertilization.

Sperm incorporation in the ooplasm occurred as early as 2 h postinsemination (PI). One hour later, a fertilization cone containing very few cytoplasmic organelles developed above the decondensing sperm chromatin. It was a transitory structure which disappeared when the male pronucleus (PN) was formed. The second meiotic division of the oocyte resumed soon after sperm incorporation; abstriction of the second polar body occurred by 3–4 h PI; the polar body constriction was associated with microfilaments.

Both the male and female PN were formed at 5 h PI and were surrounded by nuclear envelopes containing pore complexes. The female PN was characterized by patches of condensed chromatin; the male PN was closely associated with the sperm neck and proximal midpiece. Thereafter the PN migrated toward the center of the egg, where the spindle of the first cleavage division was observed at 21 h PI.

Dispermic ova analyzed at 2–4 h PI contained 2 decondensing sperm heads and 2 fertilization cones which have reached the same stage, thus suggesting that a few sperm may penetrate quite simultaneously. Multipronucleate eggs were characterized by disturbance of the PN migration.
Human sperm nuclei can transform into condensed chromosomes in Xenopus egg extracts
Authors: Ohsumi Keita , Katagiri Chiaki , Yanagimachi Ryuzo
Published: February 2005

The conditions necessary for inducing pronucleus formation and chromosome condensation of human sperm nuclei were studied using Xenopus egg extracts. When lysolecithin (LC)‐and dithiothreitol (DTT)‐treated spermatozoa were incubated with egg extract prepared with an extraction medium containing 10 mM EGTA, sperm nuclei transformed into chromosomes, bypassing the pronuclear stage. On the contrary, LC‐DTT‐treated spermatozoa incubated in egg extract without EGTA mimicked LC‐permeabilized Xenopus spermatozoa in that they underwent pronucleus formation accompanied by DNA synthesis and subsequent chromosome condensation in the correct chronological order. These observations suggest the importance of intracellular calcium for regulating the states of nuclear chromatin. LC‐permeabilized human spermatozoa were not responsive to the egg extract without any additives, but formed pronuclei when incubated with the egg extract supplemented with 5–10 mM DTT. However, subsequent chromosome condensation of sperm nucleus did not occur in the DTT extract. This suggests that disulfide‐reducing agent blocks transformation of sperm pronuclei into condensed chromosomes.
Protein‐carbohydrate complementarity in mammalian gamete recognition
Authors: Macek Mary Beth , Shur Barry D.
Published: February 2005

Recent studies suggest that gamete recognition in a number of species is mediated by complementary proteins and carbohydrates on opposing gamete surfaces. Studies in invertebrates and vertebrates have shown that carbohydrate‐binding proteins on the sperm surface recognize and bind to complementary glycoconjugates on the egg's extracellular coat. This chapter reviews our current knowledge of gamete recognition in the mouse. The complementary receptors for both mouse sperm and egg have been identified, purified, and characterized. Their synthesis during gametogenesis has been defined, as have the effects of sperm capacitation and of the acrosome reaction on their expression and distribution. Their relationship to gamete receptors that function in other species is discussed. Finally, evidence is presented that suggests that one of the receptors that mediate mouse gamete recognition belongs to a family of cell surface receptors that function during multiple cellular interactions in development.
Gonadotropin‐induced murine oocyte maturation in vivo is not associated with decreased cyclic adenosine monophosphate in the oocyte‐cumulus cell complex
Authors: Eppig John J. , Downs Stephen M.
Published: February 2005

The cyclic adenosine monophosphate (cAMP) content of intact oocyte‐cumulus cell complexes at various times after the induction of oocyte maturation in mice in vivo was correlated with the time of commitment by the oocytes to undergo germinal vesicle breakdown (GVB) and metabolic coupling between the oocyte and cumulus cells. Seventy‐nine percent of the oocytes either underwent GVB or were committed to do so by 2 h after injection of human chorionic gonadotropin (hCG). This occurred without a decrease in the coupling between cumulus cells and the oocyte and with increasing cAMP levels in the oocyte‐cumulus cell complex. Maintenance of threshold levels of cAMP within mammalian oocytes appears essential for the maintenance of meiotic arrest, but data presented here suggest that oocyte maturation in mice is induced by gonadotropins in nonatretic follicles in vivo by some mechanism other than one which decreases the cAMP content of the intact oocyte‐cumulus cell complex.
Cholesterol efflux from bovine sperm. I. Induction of the acrosome reaction with lysophosphatidylcholine after reducing sperm cholesterol
Authors: Ehrenwald Eduardo , Parks John E. , Foote Robert H.
Published: February 2005

Methods were developed to quantitatively reduce the cholesterol (Chol)/phospholipid (PL) ratio of bovine sperm and to determine the effectiveness of this treatment in capacitating sperm. Washed sperm (2 × 108) were incubated in 1.0 ml of modified Tyrode's solution (TS) containing unilamellar liposomes of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and [14C]‐Chol (35:35:30 molar ratio, 300 nmol total PL). [3H]‐triolein was included as a nonexchangable marker. After 90 min at 39°C, a 13% net exchange of [14C]‐Chol from liposomes to sperm was observed (n = 4), and sperm motility was 80%. Sperm were then washed and 50 × 106 sperm were incubated as before with PC/PE liposomes containing no Chol. After 90 min, sperm were separated from liposomes by centrifugation. Measurement of [14C]‐Chol in the liposomes (supernatant) and parallel gas chromatographic analysis of extracted, saponified liposomes (n = 4) indicated that 30% of sperm Chol was removed by this procedure. Chol efflux decreased percent motile sperm by less than 10% but reduced sperm velocity by more than 50%. Sperm incubated with no liposomes (control), with liposomes containing Chol ( + Chol), and with Chol‐free liposomes (—Chol) were washed and resuspended in TS with 0.2% BSA and 30 μg lysophosphatidylcholine (LPC)/mg bovine serum albumin (BSA). Percent sperm undergoing the acrosome reaction (AR) upon incubation with LPC‐BSA was used as a measure of sperm capacitation. After 60 min of exposure to LPC‐BSA at 39°C, the mean (± SE) percent motile sperm for control, +Chol, and —Chol treatments was 57.0 ± 4.9, 60.0 ± 4.7, and 57.0 ± 6.8, respectively. Corresponding values for percent AR were 14.0 ± 3.4, 20.3 ± 4.4, and 39.7 ± 1.2. These results suggest that loss of Chol from bovine sperm may be an early step in sperm capacitation in this species.
Influence of image sampling frequency on the perceived movement characteristics of progressively motile human spermatozoa
Authors: Mortimer D. , Serres C. , Mortimer S. T. , Jouannet P.
Published: February 2005

Tracks of 30 progressively motile washed human spermatozoa were plotted manually from 200‐Hz frame rate video recordings. Tracks at 100, 66.7, 50, 40, 33.3., 25, 20, 10, and 5 Hz were then constructed using every 2nd, 3rd, 4th, 5th, 6th, 8th, 10th, 20th, or 40th point. The 200‐Hz tracks were analyzed by traditional manual methods, and all ten sets of tracks analyzed using a computer‐assisted method (“Videomot,” developed originally to analyze 30‐Hz tracks) to eliminate observer bias. Progression velocity (VSL) remained constant under all analysis conditions. Average path velocity (VAP) also remained essentially constant, although Videomot was less reliable at high frame rates due to problems in determining the average path. Curvilinear velocity (VCL) was very frame rate dependent (the 25‐Hz mean value was only 56.5% of that at 200 Hz), and Videomot was more accurate than manual analysis at 200 Hz. Values of the amplitude of lateral head displacement (ALH) were acceptable at most frame rates. At < 66.7 Hz the inclusion of spurious curvilinear track deviations caused lower mean ALH values, and at 5 Hz ALH could not be measured since the track was essentially the average path. Beat/cross frequency (BCF) was also frame rate dependent; at high rates there was the same problem as with ALH measurements, while at ⩽ 25 Hz the maximum BCF was restricted by the frame rate. We conclude that human sperm movement characteristics can be measured at frame rates ca. 30 Hz but only if the constraints affecting VCL and BCF values are understood and accepted. Finally, < 10 Hz can only give values for VSL and, perhaps, VAP.
Incubation of mouse sperm with lactate delays capacitation and hyperactivation and lowers fertilization levels in vitro
Authors: Neill James M. , Olds‐Clarke Patricia
Published: February 2005

Mouse sperm were incubated in medium with or without 24 mM lactate and assessed for (1) motility characteristics including hyperactivation—a computer‐assisted motion analysis system was used; (2) capacitation—a chlortetracycline fluorescent dye binding assay was used; and (3) ability to penetrate oocytes. Lactate affected all aspects of motility and delayed the rates of both hyperactivation and capacitation. When a concentration of 8 × 103 sperm/ml was used for insemination in vitro, sperm preincubated 60–90 minutes in medium with lactate prior to insemination in lactate‐free medium fertilized fewer oocytes than did sperm preincubated in lactate‐free medium. Use of a calcium‐sensitive electrode demonstrated that lactate chelated appreciable amounts of calcium in the medium. Capacitation was assayed in sperm incubated 60 minutes in medium with various concentrations of lactate or CaCl2. When medium containing lactate was compared to medium without lactate but having a similar level of free calcium, the level of capacitation of sperm incubated with lactate was less than half that of sperm incubated without lactate. These results demonstrate that including 24 mM lactate in the medium can have detrimental effects on mouse sperm hyperactivation and capacitation. The detrimental effects on capacitation are partly but not completely due to the chelation of calcium by lactate.
Fine structure of the neck of epididymal spermatozoa of schreiber's long‐fingered bat (Chiroptera: Mammalia)
Authors: Bernard R. T. F. , Hodgson A. N.
Published: February 2005

The structure of the spermatozoon of the long‐fingered bat (Miniopterus schreibersii) was studied using transmission electron microscopy. The neck is characterized by two asymmetrically arranged features that allow the orientation of sections in different planes and therefore aid the interpretation of the structure. There are six segmented columns, two major and four minor, in the anterior 79% of the neck, and it is only posterior to this that the major columns split. One major column is associated with doublets 9,1, and 2, and the other major column, with doublets 5 and 6. The proximal centriole is surrounded by the capitulum and segmented columns, and the distal centriole persists and is apparent in both longitudinal and transverse sections. The central pair of microtubules pass through the distal centriole and terminate at the proximal centriole. A line drawn through the two central microtubules divides the head into left and right halves. Development of the neck begins during early spermiogenesis at which time the two centrioles are associated with the production of the capitulum, segmented columns, and axoneme complex.
Adenosine potentiates forskolin‐induced delay of meiotic resumption by mouse denuded oocytes: Evidence for an oocyte surface site of adenosine action
Authors: Salustri A. , Petrungaro S. , Conti M. , Siracusa G.
Published: February 2005

Adenosine is present in the mouse follicular fluid and has been shown to interfere with oocyte maturation in vitro. To clarify the mechanism of adenosine action on meiotic arrest, we have characterized the synergistic action of this purine with forskolin on the meiotic resumption of mouse denuded oocytes.

Forskolin delays meiotic resumption by approximately 1 hour; adenosine at concentrations ranging between 30–750 μM has no significant effect. Conversely, adenosine treatment together with forskolin produces a further delay in the resumption of meiosis. This adenosine effect is dose‐dependent and mimicked by adenosine analogs like N6‐phenylisopropyl adenosine (PIA), 2‐chloroadensoine (2‐CLA), 5′‐N‐ethylcarboxamide (NECA). Dipyridamole, which inhibits adenosine transport, does not prevent the meiosis‐arresting synergistic effect of adenosine with forskolin. Adenosine causes a 50% increase of adenosine triphosphate (ATP) content in the oocyte. However, this increase is not directly responsible for the observed delay in the oocyte maturation for the following reasons: (1) the dose response of inhibition of meiotic resumption does not correlate with the doses of adenosine producing an increase in ATP; (2) dipyridamole blocks the increase in intracellular ATP, but it has no effect on the adenosine inhibition of maturation; (3) adenosine analogs inhibit oocyte maturation but do not affect intracellular ATP levels. These results suggest that the synergism of adenosine with forskolin on meiotic arrest does not require uptake of the nucleoside nor its conversion to ATP and that the adenosine effects are exerted at the level of the oocyte plasma membrane.
Wheat germ agglutinin treatment of follicle cell‐free mouse oocytes inhibits fertilization
Authors: Nuzzo Nancy A. , Zaneveld Lourens J. D.
Published: February 2005

Controversy exists whether treatment of follicle cell‐free oocytes with wheat germ agglutinin (WGA) prevents fertilization. Lack of inhibition in one case has led to the suggestion that acrosin may not be a zona lysin. To re‐examine the effect of the WGA, the zona pellucida of follicle cell‐free mouse oocytes was made more resistant to proteinase digestion by treatment with 10 or 50 μg/ml WGA. Such WGA‐treated oocytes showed decreased fertilizability when washed to remove excess WGA and incubated with capacitated spermatozoa. Oocyte cleavage was used as an end point, because a large number of spermatozoa adhered to the eggs after WGA treatment, making observation of sperm penetration and pronucleus formation unreliable. Resistance to proteinase digestion increased, and the fertilizability decreased with the higher amount of WGA. The action of WGA was most likely not mediated by a direct effect on sperm motility, sperm acrosin activity, sperm binding to the zona pellucida, or oocyte cleavage. WGA did not affect the acrosome reaction of guinea pig spermatozoa. These data show that WGA treatment of follicle cell‐free mouse oocytes results in decreased fertilizability, possibly by rendering the zona pellucida more resistant to sperm proteinase digestion.
Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion sperm
Authors: Blach E. L. , Amann R. P. , Bowen R. A. , Sawyer H. R. , Hermenet M. J.
Published: February 2005

Transmission electron microscopy was used to confirm that a monoclonal antibody (F79.3E2; class IgGlK) was specifically localized to an antigen in the acrosomal ground substance of stallion sperm. This antibody was used to develop and validate an indirect immunofluorescent procedure to evaluate integrity of the plasma‐acrosomal membranes of stallion sperm. The concept was that primary monoclonal antibody would be “shielded” from its acrosomal antigen by an intact plasma membrane. Conversely, sperm with damaged plasma‐acrosomal membranes would exhibit green acrosomal fluorescence when viewed with an epifluorescence microscope. A lipophilic counterstain (red fluorescence) was used to insure that all sperm were visualized. Sperm in fresh‐extended or frozen‐thawed semen were incubated with hybridoma supernatant containing monoclonal antibody for 30 min at 37°C, then a second antibody (rabbit anti‐mouse IgG‐FITC) was added for 30 min at 37°C. Unbound antibody was removed by dilution and centrifugation. Sperm were resuspended in phosphate‐buffered saline containing Evan's blue as a counterstain. All sperm fluoresced bright red, regardless of the status of cell membranes, except that in cells with damaged plasma‐acrosomal membranes, the green fluorescence associated with antibody was overriding for the rostral portion. By counting fluorescent and nonflourescent “acrosomes”, the percentage of sperm with intact plasma‐acrosomal membranes was easily determined. Evaluation of five mixtures of undamaged and damaged sperm by this procedure gave a correlation of 0.91 between the percentage of damaged sperm in a mixture and the percentage of sperm with a fluorescent acrosome. Intra‐ and interassay coefficients of variability were < 6%.
Phospholipid composition of isolated guinea pig sperm outer acrosomal membrane and plasma membrane during capacitation in vitro
Authors: Stojanoff A. , Bourne H. , Andrews A. G. , Hyne R. V.
Published: February 2005

After capacitation of guinea pig spermatozoa in vitro, the plasma membrane was mechanically separated from the spermatozoa in the presence or absence of HgCl2 and subsequently isolated by density gradient centrifugation. Examination of the spermatozoa by electron microscopy after homogenization in the presence of HgCl2 revealed that plasma membrane was removed only from the acrosomal region and remained predominately intact posterior to the equatorial segment of the sperm head, as well as the midpiece and tail. In comparison, spermatozoa homogenized under similar buffer conditions but in the absence of HgCl2 lose the large apical segment of the acrosome and the plasma membrane is removed essentially from the entire cell. If spermatozoa were homogenized in the absence of Hg2+, analysis of plasma membrane phospholipid composition revealed a complete loss of lysophosphatidylcholine (LPC) from the plasma membrane after incubation of spermatozoa in minimal capacitating medium (MCM‐PL) for 2 hours. Under these culture conditions the addition of Ca2+ (5 mM) to the capacitated spermatozoa induced approximately 78 ± 5% (n = 3) of the motile spermatozoa to undergo acrosome reactions while still maintaining sperm motility (80 ± 5%) (n = 3). If the spermatozoa were homogenized in the presence of Hg2+, a time course study revealed that plasma membrane LPC loss occurred between 60 and 90 minutes of incubation. This complete loss of LPC was evident when approximately half of the capacitated spermatozoa had undergone acrosome reactions. Incubation of the spermatozoa with the metabolic and acrosome reaction inhibitor, 2‐deoxyglucose (10 mM) for 2 hours, maintained the plasma membrane phospholipid composition similar to that in the noncapacitated state. These data provide evidence that changes in the plasma membrane phospholipid composition may be associated with guinea pig sperm capacitation.
Role of protein kinase C activation in oocyte maturation and steroidogenesis in ovarian follicles of Rana pipiens: Studies with phorbol 12‐myristate 13‐acetate
Authors: Francisco Susan Kleis‐San , Schuetz Allen W.
Published: February 2005

In ovarian follicles of Rana pipiens, frog pituitary homogenates (FPH) elevate intrafollicular progesterone levels which in turn is thought to induce meiotic resumption in the prophase I arrested oocytes. Calcium plays a role in FPH and steroid‐provoked responses in the somatic and gametic components of the follicle, presumably via effects exerted at the plasma membrane of their respective target cells. Many membrane active hormones which utilize Ca2+ in their intracellular transduction also provoke membrane phosphoinositide hydrolysis yielding inositol triphosphate (IP3) and diacyl glycerol (DAG), an activator of the CA2+‐dependent protein kinase C (PKC). The actions of phorbol 12‐myristate 13‐acetate (TPA), a potent synthetic activator of PKC, on progesterone production and oocyte maturation was examined in in vitro cultured ovarian follicles. TPA induced germinal vesicle breakdown (GVBD) in intact follicles and in oocytes denuded of somatic components, while the inactive compound phorbol 13‐monoacetate was ineffective. Further, TPA induction of GVBD exhibited similarities to progesterone‐induced GVBD, being inhibited by treatments which elevate cAMP or inhibit protein synthesis. TPA alone did not elevate intrafollicular or medium progesterone levels, as occurred in FPH‐treated follicles. TPA partially inhibited intrafollicular progesterone accumulation induced by FPH or treatments which elevate cAMP levels. These data suggest that activation of PKC plays a role in oocyte maturation independent of follicular progesterone production as occurs in response to FPH. Further, it appears that the somatic cells of the amphibian follicle also possess PKC which when activated, antagonizes cAMP generating pathway in these cells. Results indicate that protein kinase can influence oocyte maturation in Rana follicular oocytes by several mechanisms.
Kinematics of hamster sperm during penetration of the cumulus cell matrix
Authors: Drobnis E. Z. , Yudin A. I. , Cherr G. N. , Katz D. F.
Published: February 2005

During capacitation, mammalian spermatozoa gain the ability to penetrate the cumulus cell matrix (CCM). The role of hyperactivated motility for this capacity is uncertain. In the present study, hamster sperm were observed during penetration and progression through the CCM, and flagellar beat patterns were quantitated by characterization of the underlying flagellar bends. Small numbers of sperm were added to cumulus masses slightly compressed on a slide (150 μm depth), and penetration was videorecorded using interference contrast optics. During penetration of the cumulus surface, sperm did not generate the large flagellar bends and asymmetric beats that are hallmarks of hyperactivation in low viscosity media. Instead, they entered slowly using high‐frequency, low‐amplitude sinusoidal flagellar motions. Within the CCM, sperm continued to move slowly, and they exhibited three distinct patterns of motility. The first was sinusoidal, produced by alternating, propagated bends: principal bends (PB) moved the head away from the beat midline, with the convex edge of the head leading, and reverse bends (RB) had the opposite curvature. The second pattern was asymmetric and sinusoidal: an extreme RB developed in the distal flagellum, was propagated distally, and was followed by a PB of less curvature. The third motility pattern was a hatchet‐like stroke of the sperm head which resulted when an extreme, nonpropagated PB developed slowly in the proximal midpiece, and was released rapidly. In this mode there were no reverse bends, and sperm did not progress. There were subpopulations of capacitating sperm in free‐swimming medium which had these same bend types and motility patterns, suggesting that qualitative flagellar movement may not change during CCM penetration. Sperm velocity in the CCM was not strongly correlated with flagellar beat kinematics, suggesting local heterogeneity in cumulus mechanical resistance and/or differences in interaction of the matrix with the surfaces of individual sperm. An effective viscosity of the cumulus near its border was estimated to be of the order of 1–4 P.
The major component of the rat sperm fibrous sheath is a phosphoprotein
Authors: Brito Monica , Figueroa Jaime , Maldonado Enrique U. , Vera Juan C. , Burzio Luis O.
Published: February 2005

The fibrous sheath from rat epididymal sperm was isolated by sequential extraction, first with Triton X‐100 and dithiothreitol, and then with 6 M urea and dithiothreitol. The latter extraction procedure solubilized most of the sperm components, leaving the head and the fibrous sheath as the only intact structures. This material was purified by sucrose gradient centrifugation. Electron microscopy confirmed the purity of the isolated material and revealed the characteristic structural features of the fibrous sheath.

Polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulfate) of the fibrillar material, showed a complex polypeptide composition. The polypeptides with molecular weights of 80,000, 24,000, and 11,500 accounted for about 65% of the total protein of the fibrous sheath. Peptide map analyses indicated that the components of molecular weights of 80,000 and 24,000 are unrelated to the polypeptides of similar size of the outer dense fibers. On the other hand, it appears that the fibrous sheath and the outer dense fibers share the polypeptide of 11,500 daltons. The component of 80,000 daltons contains on the average about 3 mol of phosphoserine per mol of polypeptide, indicating that the most abundant polypeptide of the fibrous sheath is a phosphoprotein.
Parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro
Authors: Onodera M. , Tsunoda Y.
Published: February 2005

The present study was undertaken to determine conditions for parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro. The cumulus‐free eggs were submitted to square direct current pulses at output voltage of 1.0 to 2.5 kV/cm for 25 to 200 μsec. The best conditions for the activation of mouse eggs were 1.5 kV/cm for 100 μsec, in which 78% of eggs were activated, 32% of which developed to blastocysts in vitro. When the nonelectric solution (0.3M mannitol) was used for electric stimulation, the activation rate was quite low (16%). Optimal conditions for activation of rabbit eggs were 1.5 kV/cm for 200 μsec, in which 77% of eggs were activated, 25% of which developed to blastocysts. Unlike mouse eggs, rabbit eggs frequently had three pronuclei after electric stimulation.

It is clearly shown that electric stimulus can induce parthenogenetic activation of the mouse and rabbit eggs in vitro.
Assessment of the viability and fertilizing potential of cryopreserved bovine spermatozoa using dual fluorescent staining and two‐flow cytometric systems
Authors: Ericsson S. A. , Garner D. L. , Redelman D. , Ahmad K.
Published: February 2005

A dual fluorescent staining system utilizing 5 (and‐6)‐carboxy‐4′,5′‐dimethyl fluorescein diacetate (CDMFDA) and Hydroethidine (HED) was developed to provide quantifiable information reflective of spermatozoal viability and fertilizing potential. Cryopreserved spermatozoa from ten bulls on which there was fertilizing capacity information were incubated for 1.5, and 3 hr at 39°C prior to fluorogenic staining. Spermatozoa were analyzed using both a FACS Analyzer and an EPICS V flow cytometer to determine if a particular fluorescence pattern was due to an instrumental artifact or cellular processes. Five fluorescent cellular populations were identified by the FACS Analyzer and three populations by the EPICS V. Spermatozoa were quantified after each incubation time for red (HED) and green (CDMFDA) fluorescence. Viable spermatozoa retained the greatest amount of both green and red fluorescence. Dead or moribund spermatozoa had a decrease in over‐all fluorescence. The number of viable cells at 0 hr plus the number of dead or morbid cells at any time period were identified by the FACS Analyzer as important in estimating the potential fertility of a bull. The EPICS V identified the number of dead or moribund cells as being related to nonreturn rates. Incubation of samples decreased cellular viability, which resulted in reduced levels of both green and red fluorescence. Similarities between data obtained with both flow cytometers illustrated that cellular processes, not instrumental artifacts, were responsible for the decrease in over‐all fluorescence when viability declined, the relationship between the number of cells with specific fluorescence levels and nonreturn rates, and the incubative‐induced changes in fluorescence patterns.
Differentiation of binuclear spermatozoa in mice
Authors: Burkhart J. G. , Malling H. V.
Published: February 2005

In an electron microscopy study of abnormal spermatogenesis in mice, we have found that two discrete haploid nuclei may be located in a single spermatid cytoplasm after the second meiotic division. The spermatid continues to differentiate and forms a binucleate spermatozoon with both nuclei separately packaged within the sperm head. The Golgi apparatus of the double spermatid forms a single proacrosome that attaches to both nuclei. Apparently, one acrosomal structure differentiates to cover and compartmentalize the two haploid nuclei within the sperm head. Chromatin condensation appears normal. The head morphology and number of flagella vary in mature spermatozoa produced by this process. This work demonstrates one pathway by which polyploid spermatids continue to differentiate to spermatozoa after failure of cytoplasmic division or possibly cellular fusion.
Distribution and role of microfilaments during early events of sheep fertilization
Authors: Le Guen P. , Crozet N. , Huneau D. , Gall L.
Published: February 2005

The distribution of actin was studied during early events of sheep fertilization by fluorescence microscopy after staining with 7‐nitrobenz‐2‐oxal‐1.3 diazole (NBD)‐phallacidin and anti‐actin antibody and by electron microscopy after heavy meromyosin labelling. Unfertilized and fertilized eggs exhibited a continuous band of fluorescence with both NBD‐phallacidin and anti‐actin antibody. Unlike in mice, no high concentration of actin overlying the spindle was detected in ovulated sheep oocytes. At the site of sperm head incorporation, the fertilization cone developed above the decondensing male chromatin and was underlined by a submembranous area rich in microfilaments. A similar actin network was observed in the cortex of the second polar body.

Cytochalasin D was used to investigate the role of actin during the fertilization process. This drug did not prevent sperm fusion and incorporation but inhibited polar body abstriction and fertilization cone development and retarded sperm tail incorporation. Moreover, in the presence of the drug, the anchorage of the metaphase II spindle at the surface of the egg was destroyed. The role of microfilaments in these early events is discussed.
Effects of non‐24‐hour days on reproductive efficacy and embryonic development in mice
Authors: Endo Akira , Watanabe Toshiaki
Published: February 2005

ICR female mice were exposed to either 22 (L11, D11) or 26 hour day (L13, D13) light/dark cycles for at least 2 weeks before mating and/or during pregnancy. The mating rates of these animals decreased considerably. When pregnant females were examined at gestation days 12.0 or 17.5, resorption rates were increased, the embryos weighed less, and development was retarded in the experimental groups with preconceptional exposure to non‐24‐hour days. We speculate that in mice maternal and paternal pre‐ and periconceptional environment of daily light/dark cycles is important for normal reproductive efficacy and normal embryonic development during pregnancy.
Factors regulating mammalian sperm migration through the female reproductive tract and oocyte vestments
Authors: Katz David F. , Drobnis Erma Z. , Overstreet James W.
Published: February 2005

Mechanisms of mammalian sperm migration through the female reproductive tract and ovum vestments are described. The perspective is biophysical as well as biochemical and morphological, and the focus is upon the role of sperm motility in these processes. Sperm forward progression is characterized as an interactive process between the the cell and its environment, and the mediation of flagellar bend propagation by the physical properties of its surroundings is described. These properties, together with flagellar beat kinematics, sperm morphology, and surface properties, determine the magnitude of the forces generated by sperm and their consequent rate of progression. Sperm interactions with the cervical mucus, the cumulus oophorus, and the zona pellucida are described. The poorly understood affinity of the sperm surface for the macromolecules of the mucus, cumulus, and zona is stressed, as is the viscoelastic structural mechanical resistance of these biopolymers to sperm motion. The kinematics and consequences of hyperactivated sperm motion are presented, with emphasis on objective characterization of such motion (as a biomarker), along with analysis of the mechanical advantage that such motion may confer on spermatozoa during egg–vestment interaction.
Preimplantation embryo development and serum steroid levels in immature rats induced to ovulate or superovulate with pregnant mares' serum gonadotropin injection or follicle‐stimulating hormone infusions
Authors: Leveille M.‐C. , Armstrong D. T.
Published: February 2005

Follicular stimulation protocols using pregnant mares' serum gonadotropin (PMSG) or a follicle‐stimulating hormone (FSH) preparation were compared to evaluate the yield and quality of embryos obtained from immature rats. Rats received a superovulatory dose of PMSG (401U), a nonsuperovulatory dose of the same gonadotrophin (4 IU), or a continu ous s.c. infusion over a 72‐h period with a purified FSH preparation containing an opti mum ratio of luteinizing hormone (LH): FSH (FSH‐hCG). The females were caged with fertile males on the evening of the 3rd day of gonadotropin treatment and scored for the occurrence of mating on the next morning; subgroups were killed on days 1–4 of preg‐nancy. High fertilization rates were observed in rats treated with 4 IU PMSG (84.1%) and in rats infused with FSH‐hCG (91.0%); however, a much lower fertilization rate was observed following treatment with 40 IU PMSG (41.5%). From median ovulation rates of 9 and 79 in rats treated with 4 IU PMSG and in rats infused with FSH‐hCG, medians of 8 and 69 embryos, respectively, were recovered from reproductive tracts flushed on day 4 of pregnancy, from which 75% were morulae or blastocysts; in contrast, from a median ovu lation rate of 42.5, a median of only 12 embryos was recovered on day 3 of pregnancy following superovulation with 40 IU PMSG of which 80% were degenerate ova. Serum steroid profiles during the first 4 days of pregnancy differed significantly among treatment groups, the major differences being in substantially elevated levels of estradiol and andro‐gens on days 1–3 in rats receiving the high (40 IU) dose of PMSG. Levels of these steroids in rats superovulated with the FSH‐hCG infusion regimen were only marginally elevated above levels observed in rats treated with the low (4 IU) nonsuperovulatory dose of PMSG. Consistent with high ovulation rates, serum progesterone levels rose to considera bly higher levels during the period in both superovulated groups than in animals receiving the low, nonsuperovulatory dose of PMSG. This work describes a novel method to superovulate rate (FSH‐hCG) leading to high yields of normally developing embryos at all preimplantation stages and illustrates the close association between high yield of emyryos and low levels of circulating andorgens and estradiol‐17β during the preimplantation period.
Effects of a superovulatory dose of pregnant mare serum gonadotropin on follicular steroid contents and oocyte maturation in rats
Authors: Yun Young W. , Yu Frank H. , Yuen Basil Ho , Moon Young S.
Published: February 2005

Our previous study has shown that superovulatory treatment with pregnant mare serum gonadotropin (PMSG) in rats caused marked alterations in ovarian and serum steroid responses and coincidental increase in degenerate oocytes (Yun et al., 1987). This study examined the effects of superovulatory treatment (20 IU PMSG) on follicular steroid contents and oocyte maturation. Immature female rats aged 28‐30 days were injected with 4 or 20 IU PMSG and sacrificed at 24, 48, 60, and 72 hr. Compared to control regimen, follicular content of progesterone (P) in superovulated rats significantly (P < .05) increased at 48 hr. Androgen (A) content significantly (P < .01) decreased below control level at 24 hr but significantly (P < .05) increased above control level at 48 hr and 60 hr. There was no significant change in 17β‐estradiol (E) content between the two groups. In control regimen, the ratio of A/E sharply decreased and the ratios of P/Eand P/A increased steadily from 24 hr. However, superovulatory regimen showed a consistently steady state in the overall ratios of follicular steroids after 24 hr. Nuclear maturation of the majority of control oocytes recovered from oviducts at 72 hr was synchronized at metaphase II, whereas superovulated oocytes displayed different stages varying from prophase I to metaphase II at 24, 48, and 72 hr. The results provide direct evidence of atypical ovulations in superovulated oocytes with premature or asynchronous nuclear maturation and demonstrate a close relationship between meiotically aberrant oocytes and abnormal follicular steroidogenesis following superovulation with PMSG in rats.
Analysis of the first cell cycle in the cross between hamster eggs and human sperm
Authors: Brandriff B. F. , Gordon L. A.
Published: February 2005

Golden hamster eggs fused with human sperm were pulsed with bromodeoxyuridine to determine the timing of S‐phase and the length of the first ceil cycie in this hybrid cross. Fused eggs were fixed and pronuclei scored for incorporation of the thymidine analogue detected by indirect immunofiuorescence. Although S‐phase started synchronously 3–3.5 hr after coincuba‐tion of sperm and eggs, its duration was variable such that two‐cell stages appeared at 16 hr while a proportion of pronuclei was still engaged in DNA synthesis. Unlike rodent sperm chromatin, human sperrn chromatin was able to participate in DNA synthesis well before its maturation into a fully developed pronucleus. Human sperm chromatin appears able to function under conditions different in several respects from those in human eggs.
Modulation of phagocytic activity in cultured sertoli cells
Authors: Filippini A. , Russo M. A. , Palombi F. , Bertalot G. , De Cesaris P. , Stefanini M. , Ziparo E.
Published: February 2005

Phagocytic activity of rat Sertoli cells that were cultured in vitro has been evaluated as the ability to internalize polystyrene beads. Our data demonstrate that these ceils are active phagocytes and that such phagocytic activity is under negative control by follicle‐stimulating hormone (FSH). The hormonal control is mediated by increased intracellular levels of cAMP. Moreover Sertoli cdl responds to tuftsin, an oligopeptide known to act only on macrophages and granulocytes, by increasing up to five times its phagocytic activity. Phagocytic uptake of polystyrene beads is associated with dramatic changes of the cellular shape. Such morphological modifications are significantly reduced under FSH stimulation. The physiological implications of the data are discussed.
Membrane specializations associated with the acrosomal complex of sea urchin sperm as revealed by immunocytochemistry and freeze fracture replication
Authors: Longo Frank J. , Georgiou Christos , Cook Susan
Published: February 2005

Observations, employing freeze fracture replication and electron microscopic imrnunochemis‐try, have been carried out to determine structural correlations of the plasma membrane domain occupied by a 210 kDa protein involved in the acrosomal reaction of sea urchin sperm and recognized by the monoclonal antibody, J10/14 (Trimmer et al: Cell 40:697–703, 1985; Proceedings of the National Academy of Sciences of the United States of America 83: 9055–9059, 1986). Immunogold‐J10/14 staining of acrosorne‐intact sperm was intense along the flagellum and a narrow collar just posterior to the sperm apex that surrounded the acrosomal complex (acrosomal vesicle and subjacent anterior nuclear fossa containing g‐actin). Counts of gold particles revealed a density (average number of particles/μm2 of surface area) eightfold greater along the plasma membrane associated with the acrosomal complex than membrane delimiting the remainder of the sperm head. The collar of J10/14 staining was isomorphic with a dense aggregation of intramembranous particles in the P‐face of the plasma membrane and a thin cytoplasmic region that surrounded the acrosomal complex. In acrosomc‐reacted sperm, intense J10/14 staining was distributed along the flagellum and sperm head; prominent anterior staining was not apparent in all specimens. The density of gold particles associated with plasma membrane delimiting components of the former acrosomal complex, nucleus and mitochondrion, as well as the total average number of particles along the entire sperm surface, were increased in sperm acrosome‐reacted with A‐23187. Concomitant with this change in staining was the disappearance/reduction of the collars of intramembranous particles and cytoplasm. These observations indicate that plasma membrane components (210 kDa protein and intramembranous particles) and the collar of cytoplasm which are associated with the acrosomal complex are functionally, as well as structurally related. Analyses of particle density distributions along acrosome‐ and non‐acrosome‐reacted sperm suggest that the different staining patterns observed may be brought about by the recognition of cryptic sites at the time of the acrosomal reaction.
On the number and rate of formation of sperm‐zona bonds in the mouse
Authors: Baltz Jay M. , Cardullo Richard A.
Published: February 2005

In mammalian fertilization, sperm bind to the zona pellucida, a glycoprotein matrix forming a shell surrounding the oocyte. Subsequently, one of the bound sperm penetrates the zona and fertilizes the egg. The adhesion between sperm and zona is mediated by complementary receptor‐ligand pairs. Recent biochemical evidence has identified likely candidates for these molecules in the mouse. Biophysical studies have predicted that very few (possibly as few as one) bonds are needed to tether a motile sperm to the zona. We have used the data characterizing the putative receptors of the mouse sperm to predict the number of bonds they can form with the zona ligands. Our calculations indicate that few bonds probably form between the sperm and zona during the initial contact when the sperm is captured, supporting the hypothesis that fertilization depends on the action of a very few sperm‐zona bonds.
Hamster oocyte penetration tests with oocytes frozen in propanediol: Comparison with non‐frozen oocytes
Authors: Sachs H. Hyatt , Pink M. J. , Gwatkin R. B. L.
Published: February 2005

Hamster oocytes were frozen using a 1,2‐propanediol‐sucrose procedure, which resulted in over 90% survival. After thawing and zona removal the oocytes were compared with non‐frozen oocytes in a zona‐free hamster egg test employing spermatozoa from human semen donors and suspected infertility patients. Similar data were obtained, indicating that propancdiol‐sucrose frozen hamster eggs may be used in place of fresh eggs for convenience and to a void scheduling problems.
Developmental capacity of mouse oocytes that undergo maturation in vitro: Effect of the hormonal state of the oocyte donor
Authors: Schroeder Allen C. , Eppig John J.
Published: February 2005

In a previous study, it was shown that cumulus cell‐enclosed germinal vesicle (GV)‐stage oocytes, isolated from pregnant mares' serum gonadotropin (PMSG)‐primed immature (22–24 day old) mice and that underwent spontaneous maturation in vitro, exhibited frequencies of embryonic development similar to oocytes stimulated to mature and ovulate in vivo by administration of gonadotropins [Schroeder AC, Eppig JJ, (1984) Dev Biol 102:493–497]. In the present study, the effect of the hormonal state of the oocyte donor on the capacity of in vitro matured oocytes to be fertilized and undergo pre‐ and post‐implantation development was explored further. Oocytes were isolated at the GV‐stage from the following groups of mice: 1) unprimed immature mice; 2) adult cycling mice; 3) unprimed Snell dwarf (dw) mice that have undetectable levels of growth hormone (GH), prolactin, and thyroid‐stimulating hormone (TSH); and 4) primed and unprimed hypogonadal (hpg) mice that have undetectable levels of circulating gonadotropins. Oocytes maturing in vitro after isolation from normal unprimed immature or adult mice at all stages of the estrous cycle acquired full developmental capacity. GV‐stage oocytes isolated from dwarf mice showed embryonic development equivalent to normal ( + /?) littermate controls. Therefore, GH, TSH, or prolactin are not required during oogencsis in vivo to promote the acquisition of competence to complete embryogenesis after maturation in vitro. Oocytes from hypogonadal mice had a much reduced capacity for preimplantation development when compared with normal littermates. Administration of PMSG to the hypogonadal mice significantly increased the developmental capacity of oocytes that underwent maturation in vitro. Gonadotropins, therefore, have a beneficial effect on the oocytc's capacity for embryonic development.