bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of lipid and protein components of ejaculated bull sperm surface and seminal plasma
Authors: Young Leona G. , Goodman S. A.
Published: February 2005

Surface components of ejaculated bull sperm were radiolabeled by enzymatic iodination with lactoperoxidase and Na125I. The sperm were lysed and the labeled components analyzed on SDS‐7.5% polyacrylamide gels. Electrophoresis of solubilized radioactivity resolved six components with approximate molecular weights of 77, 61, 44, 36, 24, and 15 kilodaltons. To identify components that might be adsorbed to the bull sperm surface from seminal secretions, seminal plasma was labeled. Electrophoresis of labeled seminal plasma resolved four components with approximate molecular weights of 74, 33, 24, and 15 kilodaltons, each of which comigrated with a labeled sperm surface component. To identify the chemical composition of the radiolabeled components, labeled sperm surface and labeled seminal plasma were submitted to isopycnic density gradient centrifugation in cesium chloride. The 125I incorporated into bull sperm surface separated into two discrete areas of radioactivity, one having a density characteristic of protein and the other, of lipid. Iodinated seminal plasma banded in one discrete area that had a density characteristic of protein. Electrophoretic analysis of each area of radioactivity recovered from the gradients demonstrated that five of the six sperm surface and all of the seminal plasma components were in the protein fractions. The 15‐kilodalton sperm surface component banded as a lipid, whereas the 15‐kilodalton seminal plasma componènt banded as a protein.
Light microscopic observations of alterations in staining of the zona pellucida of porcine follicular oocytes: Possible early indication of atresia
Authors: Centola Grace M.
Published: February 2005

Serial sections of porcine ovaries were examined in an attempt to detect early signs of oocyte degeneration/atresia using special staining. Porcine ovaries were fixed in Bouin's fixative and embedded in paraffin using routine techniques. Serial sections (8 μm) were mounted on glass slides and stained with Shorr's S3 and hematoxylin stain. Several criteria were used for examining general histology of the antral follicles: condition of the granulosa layer, antral cavity, the oocyte and its surrounding zona pellucida, and the cumulus layers. A change in the staining characteristic of the zona pellucida was the single most striking observation in all ovaries examined. In presumably healthy follicles, the zona pellucida was uniformly stained green, the granulosa layer was intact with fewer than three pyknotic cells per section, and a uniform basement membrane (stained green) separated the intact theca layers from the remainder of the follicle. In those follicles showing some degree of degenerative changes in the follicular wall, the zona pellucida was stained a bright orange. In the last stages of degeneration, follicles exhibited many pyknotic nuclei throughout the granulosa layers, the layer of granulosa cells was in many cases separated from the basement membrane, and the antrum was infiltrated with lymphocytes. In these follicles, the zona pellucida was always stained orange. Frequently, the zona pellucida was partially stained orange before any detectable changes could be seen in other elements of the follicular wall. Additionally, many non‐antral (primary) follicles exhibited oocytes with orange‐stained zonae pellucidae. In terminal stages of follicular degeneration, collapsed follicles were infiltrated by connective tissue elements stained bright orange and green. These structures very often contained dying oocytes always with bright orange‐stained zonae pellucidae. Scattered throughout the ovarian stroma were many orange‐stained remnants of zonae pellucidae. It is thought that perhaps the characteristic staining of the zona pellucida with Shorr's S3 stain may give an early, previously undetectable indication of follicular atresia.
Effect of albumin on fertilization of mouse ova in vitro
Authors: Quinn P. , Stanger J. D. , Whittingham D. G.
Published: February 2005

Serum albumin is an obligatory component of the incubation medium for the fertilization of mouse ova. Normal, untreated bovine serum albumin supports high rates of fertilization of cumulus‐free ova both with and without their zonae pellucidae. Heat‐treated or trichloroacetic acid‐extracted bovine serum albumin is unable to support the fertilization of a majority of zona‐intact ova but fertilization of zona‐free ova is unimpaired. Spermatozoa incubated in medium containing heated bovine serum albumin fertilize zona‐intact ova when 2 mM caffeine is present but the progress of sperm head decondensation is delayed when compared to normal controls. Trichloroacetic acid extracted BSA preferentially and irreversibly inhibits zona penetration by spermatozoa, but this effect is not mediated by an inhibition of spermatozoal motility or zona‐binding ability. This effect occurs after only a 10‐min preincubation of the spermatozoa in the extracted BSA or when the medium contains only a 10% (v/v) proportion of this albumin. It is estimated that mouse spermatozoa under the conditions used take 2 hr to penetrate the zonae pellucidae of 50% of ova and effect fertilization.
Acrosome reaction in sperm of the toad, bufo bufo japonicus
Authors: Yoshizaki Norio , Katagiri Chiaki
Published: February 2005

The acrosome in the sperm of the toad, Bufo bufo japonicus, consists of a membrane‐limited acrosomal cap and a fibrous perforatorium. When sperm are incubated with the oviducal pars recta extract (PRE) for 30–60 min, the outer acrosomal membrane fuses with the overlying plasma membrane at several points with concomitant loss of the contents of the acrosomal cap. The inner acrosomal membrane thus exposed fuses with the plasma membrane at the caudal end of the acrosomal region. This PRE‐induced acrosome reaction is completely inhibited by soybean trypsin inhibitor. Sperm found in the innermost jelly layer of inseminated eggs possess an intact acrosome, but those either passing through the vitelline coat or localizing in the perivitelline space are acrosome‐reacted in the same manner as when treated with PRE. These observations, combined with recent evidence showing involvement of the pars recta substance in fertilization, indicate that the acrosome reaction occurring in a fertilizing sperm at or near the surface of the vitelline coat is a response to a substance that is derived from the pars recta and deposited in the vitelline coat.
The effect of catecholamines and their antagonists on the fertilization of cumulus‐free mouse ova in vitro at a suboptimal spermatozoal density
Authors: Stanger J. D.
Published: February 2005

A suboptimal sperm concentration was used to assess the capacity of catecholamines to stimulate the fertilization of cumulus free F1,(C57BL × CBA) mouse ova in vitro. At a concentration of 50 μM, (L) epinephrine significantly increased the proportion of ova fertilized at 2 × l05 spermatozoa/ml. However, when (D, L) propranolol at an equimolar concentration was tested for inhibition of the (L) epinephrine effect, fertilization was inhibited in both the test and control dishes. At l0μM, propanolol by itself or in the presence of 50μM (L) epinephrine significantly increased the number of ova fertilized at 2 × l05 sperm/ml. Norepinephrine (50 μM) and phentolamine (50 μM), either alone or together, were also slightly stimulatory. Some data are presented to suggest that propranolol may act in a nonadrenergic manner to precipitate the acrosome reaction and that the stimulatory effect is maximised when it is added to spermatozoa at the same time as ova addition. It was suggested that propranolol may act to trigger calcium influx by a nonspecific alteration in membrane function for example in (Ca + Mg) ATPase activity. It was concluded that spermatozoa at suboptimal densities are capable of achieving fertilization and that sperm concentration dependency in fertilization in vitro may be a reflection of the proportion of spermatozoa achieving capacitation.
Reduced survival in utero from transferred mouse blastocysts compared with morulae
Authors: Hoppe Peter C. , Coman Dale Rex
Published: February 2005

Studies on the survival of mouse embryos revealed that fewer offspring were produced when blastocysts, rather than morulae, were transferred to foster mothers. Approximately 8–10 h after fertilization F1 hybrid eggs (C57BL/6J × LT/Sv) were collected and cultured to morulae (day 4) or blastocysts (day 5 ) before transfer into uteri of day 3 foster mothers. A few recipients were killed on day 8 of gestation and deciduae were examined histologically. Embryos developing from transferred morulae were found to lie deep within the deciduae and were surrounded by numerous, large blood islands. Conversely, embryos developing from transferred blastocysts implanted more distally to the maternal blood vessels with only a few blood islands surrounding the embryos. These observations, suggesting abnormal implantation with insufficient embyro nourishment, were confirmed when uteri of foster mothers were examined on day 19 of gestation. Although the proportion of implantations from transferred morulae or blastocysts was similar (42 % and 47%, respectively), significantly more of the implantations were resorbed after transfer of blastocysts (78%) as compared with morulae (15%). These results demonstrate that transfer of day 5 cultured blastocysts into uteri of foster mothers increases embryonic mortality as a consequence of improper implantation.
Selective binding of specific rat epididymal secretory proteins to spermatozoa and erythrocytes
Authors: Brooks D. E.
Published: February 2005

Tissue pieces from the caput epididymidis of the rat were incubated in vitro with (35S) methionine to produce radioactive secretory proteins. The radioactive secretory proteins so formed were tested for their ability to bind to washed rat spermatozoa collected from the rete testis and cauda epididymidis, and to rat erythrocytes. The sperm and erythrocytes bound approximately 5% of the total radioactive protein. Binding was protein‐specific in that only selected proteins became associated with the cells. Binding was not cell‐specific, however, since testicular spermatozoa, caudal spermatozoa, and erythrocytes all bound the same proteins to a similar degree.
Parameters influencing ovum pickup by oviductal fimbria in the golden hamster
Authors: Mahi‐Brown Cherrie A. , Yanagimachi Ryuzo
Published: February 2005

The process whereby hamster oviductal fimbria transport ova to the ampulla was investigated in vitro and in vivo with the intent of determining whether cumulus‐free ova were transported and resolving some of the parameters involved in cumulus‐isolated from ovaries could not be picked up until 16–17 h before it would be ovulated. This was apparently related to the time at which the cumulus matrix begins to expand. The fimbria were not species‐specific, as they readily picked up rat, mouse, and rabbit ovulated cumul, but they were partially tissue‐specific as they would only pick up tissues such as the vitreous humor and loose connective tissue, which contain considerable extracellular glycosaminoglycan. Interaction of cumulus and fimbria could be prevented b treating either structure with polycatinic macromolecules such as poly‐l‐lysine, cationic ferritin, and protamine. Treatment of fimbria with proteolytic enzymes or hyaluronidase did not prevent pickup, but neuraminidase did. The only “artificial cumulus” the fimbria would pick up was chicken egg‐white. Cumulus‐free ova were not picked up in vitro, but part of them placed into the ovarian bursa were transported to the ampulla in vivo. Coating the ova with chicken egg‐white allowed their pickup in vitro, but had little effect on the success of transport of ova transplanted into the ovarian bursa in vivo. The transport of ova from the ovarian bursa to the oviductal ampulla is apparently a rather complex process involving interaction of some component of the cilia surfaces with the cumulus as well as other factors not necessarily related to this interaction.
Immunocytochemical localization of protamine in the spermatids of the ram
Authors: Courtens J. L. , Delaleu B. , Dubois M. P. , Lanneau M. , Loir M. , Rozinek J.
Published: February 2005

Pure ram protamine isolated from epididymal spermatozoa was used to raise antisera in castrated rabbits. The antibodies were visualized in the electron microscope using the method of Moriarty and Halmi [1972] with either peroxidase or coupling with colloidal gold. The gold gave better contrast but lower afinity than the peroxidase method. With the use of fixation according to Thiery and Rambourg [1976] and thick sections treated with hydrogen peroxide, it was possible to detect the protamine in the cytoplasm near the flagellum and the chromatoid body of step 12 spermatids. It was concluded that protamine enters the nucleus and concentrates at that step in the ram.
The localization of Ca2+‐ATPase and Ca2+ binding proteins in the flagellum of guinea pig sperm
Authors: Gordon Mildred , Morris Eugene G. , Young Ronald J.
Published: February 2005

Ferritin‐labeled antibody to calmodulin was localized in flagella of guinea pig sperm. Ferritin granules were present on the surface of the medial portion of the coarse fibers, and in the matrix immediately surrounding this segment of the fibers. The results demonstrate that calmodulin is confined to regions close to the tubulin‐dynein systems os the axoneme. A calcium‐dependent ATPase was localized in the same region by trapping the reaction product with cadmium. The presence together of calmodulin and the Ca2+‐ATPase close to the axonemal complex suggests that they function to control the concentration of Ca2+, which in turn may regulate the action of the dynein ATPase, and sperm motility. The remainder of the coarse fiber surface also contains specialized material as evidenced by intense staining with ethanolic phosphotungstic acid. These data suggest that the role of the coarse fibers is complex and may not be solely structural.
Nuclear maturation of follicle‐enclosed rat oocytes during inhibition of steriodogenesis
Authors: Billig Håkan , Hillensjö Torbjörn , Tsafriri Alexander , Mangusson Claes , Brodie Angela
Published: February 2005

This study examined the possible role of steriods in meiotic maturation of preovulatory oocytes. Follicles were isolated from PMSG‐treated immature rats and incubated with or withour LH in the presence of one of four inhibitors of steroidogenesis. The inhibitors employed had different sites of attack in the steriodogenic pathway and were aminoglutethimide, cyanoketone, SU 10603 (17β‐hydroxylase inhibitor), and 4‐OH‐androstenedione (aromatase inhibitor). As predicted, the inhibitors drastically altered the pattern of steroid production. None of the inhibitors, however, changed the proportion of oocytes resuming or completing meiosis in response to LH, and there was also no effect of the inhibitors on the oocytes in the absence of LH. It was concluded that steriods are not required for preovulatory nuclear maturation of oocytes in the rat.
Gametes and fertilization in the Chinese hamster
Authors: Yanagimachi R. , Kamiguchi Y. , Sugawara S. , Mikamo K.
Published: February 2005

Freshly ovulated eggs are each surrounded by a compact cumulus oophorus. The overall diameter of the normal egg (including the zona pellucida) is about 100 μm. Cumulus cells, particularly those near the egg, are arranged redially in a viscous noncellular matrix. The spermatozoon is about 250 μm in length. The head a large acrosome, changes in which can be readily examined with the light (phase‐ contrast) microsope. When exposed to physiological salt solutions, testicular spermatozoa either were motionless or flexed the posterior half of their tails slowly. Spermatozoa from the caput epididymis were highly motile, flexing the entire tail. A few of them moved progressively. Mature spermatozoa from the vas deferens were highly motile and moved either straightforward or in a circle. They vibrated their tails stiffly without flexing them.

In normally mated females, fertilization began sometime between 2 and 3 h after ovulation and was completed within the next 4 to 5 h. Spermatozoa swimming in the ampullary fluid or within the cumulus oophorus about the time of fertilization flexed the anterior half (which roughly corresponds to the midpieac region) of their tails. This peculiar movement may be homologous to the so‐called “hyperactivation” of spermatozoa as reported in several other mammalian species. Actively motile spermatozoa within the cumulus or no the zona pellucida had either modified (“collapsed”) or no acrosomal caps. The sperm head usually passed verticually or nearly through the zona, but the path was oblique in some instances. In 54% of the recently fertilized eggs examined, the entire length of the sperm tail was within the perivitelline space; in the other 46% of the eggs varying lenghts of the tail remined the perivitelline space, the tails were extruded from the vitellus of many eggs even before the eggs began their first cleavage.

When unfertilized eggs in the cumulus oophorus were inseminated with vas deferens spermatozoa in a modified Tyrode's solution (m‐TALP), about 80% of them were ferrtilized by 4–6 h after insemination. The vast majority were monospermic. When eggs were freed from the cumulus prior to insemination, none were fertilized, suggesting that the cumulus cells or their matrix assisted capacitation and/or the acrosome reaction of the spermatozoa under the in vitro conditions employed. No eggs were fertilized by the testicular or caput epididymal spermatozoa regardless of the presence or absence of cumulus oophorus around the eggs at the time of insemination.
p15, A nuclear‐associated protein of human sperm: Identification of four variants and their occurrence in normal and abnormal seminal cells
Authors: Rodman Tody C. , Pruslin Fred H. , Chiorazzi Nicholas , Michelis Mary Ann , Winston Ronald
Published: February 2005

A basic protein of apparent molecular weight 15,000 d (p15) has been identified as a tissue‐specific species‐unique component of spermatogenic cells of human semen. Cytoimmunochemical study with a monoclonal antibody indicates that p15 resides in a perinuclear space in morphologically normal spermatozoa and differs in distribution and stat in abnormal seminal cell and nonnucleated bodeis. Biochemical analysis indicateds that p15 occurs as four variiants, differentially migratory on acetic acid/urea gel and differnetially extractable by NaCl in reducing solution. By correlation of the cytologic and biochemical data, we propose that variant 1 is the unmodified form of p15; in the normal progression of spermiogenesis p15 is modified to variants 2 and 3 and in the absence of the normal progression is unmodified or aberrantly modified to variant 4. The association of molecular abnormality in p15 with morphologically abnormal sperm suggests that p15 may play a role in sperm‐head shaping.
Surface and internal antigens of rat spermatozoa distinguished using monoclonal antibodies
Authors: Jones Roy , Brown Colin R. , Cran David G. , Gaunt Stephen J.
Published: February 2005

Fluorescent antibody labeling techniques are frequently used to investigate the topography of antigens on spermatozoa. It is generally assumed that these procedures detect molecules only on the sperm surface but we now show that this assumption is not always valid. Using monoclonal antibodies that recognize either surface or internal antigens we demonstrate how spurious conclusions can be made, and we suggest simple procedures for assigning the position of an antigen to the cell surface or to an intracellular organelle. Antibodies against plasma membrane antigens should stain 100% of normal intact spermatozoa, but this proportion should be greatly reduced if the spermatozoa have previously been demembranated. If ≪ 100% of spermatozoa are stained but the proportion increases following permeabilization, then the possibility should be considered that the antigens are intracellular. We conclude that assignment of an antigen to a regional domain on the sperm surface using fluorescent antibody techniques should be validated by a demonstration that the antigen is actually located on the cell surface.
The status of acrosomal caps of hamster spermatozoa immediately before fertilization in vivo
Authors: Yanagimachi Ryuzo , Phillips David M.
Published: February 2005

Adult female golden hamsters were induced to superovulate. When they were mated several hours prior to ovulation or artificially inseminated about the time of ovulation, nearly 100% of their eggs were subsequently fertilized monospermically. During the progression of fertilization when the eggs were still surrounded by compact cumulus oophorus, the contents of the ampullary region of the oviducts were collected and spermatozoa moving in the ampullary fluid, within the cumulus and on/in the zonae pellucidae of unfertilized eggs, were examined by light and electron microscopy to evaluate the status of their acrosomal caps.

Most spermatozoa swimming in the ampullary fluid had apparently intact acrosomal caps, while the vast majority moving within the cumulus had distinctly modified acrosomal caps. Most spermatozoa that had passed through the cumulus and reached the zona surfaces had remnants of their acrosomal caps (“acrosomal ghosts”). When the ghosts were present around the sperm heads on the zona, the heads pivoted about a point roughly corresponding to the places where the ghosts were located. The ghosts seemed to firmly attach to the zona surfaces, then were split open by the sperm heads and left behind as the sperm heads advanced into the zona. A few spermatozoa on the zona surfaces had no acrosomal ghosts (at least not detectable by light microscopy). In this case, the sperm head pivoted about either the inner acrosomal membrane or the equatorial segment of the acrosome. In no instance were spermatozoa with intact acrosomal caps found on zona surfaces.

We infer from these observations that most spermatozoa in vivo initiate their acrosome reactions while they are advancing through the cumulus. When they arrive at the zona surfaces, acrosomal ghosts are generally present on the sperm heads. These ghosts appear to hold sperm heads to zona surfaces as well as to restrict the direction of advancement of sperm head through the zona. In a minority of cases, ghostless spermatozoa reach the zona surfaces. As these spermatozoa appear to be able to penetrate the zona successfully, structures other than the acrosomal ghost (ie, the inner acrosomal membrane and the plasma membrane over the equatorial segment of the acrosome) may also attach to zona surfaces before spermatozoa penetrate into the zona.
Developmental ability of precociously superovulated golden hamster eggs
Authors: Yanagimachi R.
Published: February 2005

Superovulated hamster eggs obtained by injections of PMSG on day 1 (the day of ovulation) and hCG on day 3 (2 days before expected day of next ovulation) have been used widely for assessing the fertilizing capacity of spermatozoa of the homologous as well as heterologous species. The developmental ability of these eggs was assessed by transferring them to host females (recipients) that had ovulated and mated naturally. The proportion of transplanted eggs developing into normal fetuses and young was the same regardless of whether the eggs had been collected from superovulated or naturally ovulating donors. The young originating from superovulated eggs were normal in that they grew at a normal rate and produced normal progeny. It may be concluded that the precociously superovulated eggs were as normal as the control (naturally ovulated) eggs. Thus, the use of precociously superovulated hamster eggs in evaluating the fertilizability of spermatozoa is justified.
Inhibition of mouse embryonic development by calmodulin antagonists
Authors: Lenz R. W. , Hart R. , Ax R. L. , Cormier M. J.
Published: February 2005

Two‐cell mouse embryos were incubated in the presence of calmodulin inhibitors to determine their effect on embryonic development to the blastocyst stage. Calmodulin, a Ca2+‐dependent regulatory protein, has been localized in the cytoplasm and has been implicated in regulation of many cellular events, such as mitosis. Several concentrations of either commercial or synthesized calmodulin inhibitors were tested. Several phenothiazine sulfoxide derivatives were more effective than the three naphthalene sulonic acid derivatives tested; 2‐chloro‐10‐aminopropyl phenothiazine and 10‐aminopropyl phenothiazine were the most potent phenothiazines to inhibit embryonic development at the two‐cell stage. The interesting aspect of this study is that phenothiazine sulfoxide derivatives are not potent inhibitors of calmodulin, however, they were successful in inhibiting embryonic development. Potent inhibitors of calmodulin apparently did not penetrate the embryonic membranes because they had no effect.
Spermatozoa of chromosomally heterozygous mice and their fate in male and female genital tracts
Authors: Redi C. A. , Garagna S. , Pellicciari C. , Romanini M. G. Manfredi , Capanna E. , Winking H. , Gropp A.
Published: February 2005

The fate of morphologically normal but chromosomally abnormal spermatozoa derived from mice with variable degrees and complexity of Robertsonian heterozygosity was studied at different sites along the male and female genital tract by Feulgen‐DNA measurements. In addition, the percentage frequencies of morphologically abnormal spermatozoa in transit along the male and female genital tracts were studied.

It was found that during transit from the epididymis to the vas deferens the distribution of the Feulgen‐DNA contents of morphologically normal spermatozoa changed: Spermatozoa with chromatin with the extremely low or high Feulgen staining intensity disappeared. The percentages of morphologically abnormal sperm cells did not change at these levels. In the female genital tracts, the distribution of Feulgen‐DNA content of morphologically normal spermatozoa did not show significant changes. This indicates that spermatozoa are able to reach the fallopian tube in spite of gross genome unbalance. There is evidence that unbalanced spermatozoa take part in the fertilization process, producing abnormal zygotes subject to postzygotic loss. Conversely morphologically abnormal spermatozoa were preferentially lost before they reached the fallopian tube, suggesting they had been eliminated prezygotically.
Reproduction in mice: Behavioral changes induced in female mice by mating
Authors: Chaykin Sterling
Published: February 2005

Mated C57BL/6J females experienced in food‐motivated maze running were found to make more choices and choose more correctly than virgins. The differences correlated with the receipt of spermatozoa and prior pseudopregnancy. Pregnancy, active pseudopregnancy, and male‐female social interactions did not appear to be required determinants. The biologic ramifications of mating in mice may extend beyond the fusion of sperm and egg and the induction of hormonal changes that regulate the earliest stages of pregnancy.
Association of newly synthesized 3H‐arginine‐labeled proteins with chromatin structures in fertilization
Authors: Kopečný Václav , Pavlok Antonín
Published: February 2005

Zona‐free hamster eggs have been fertilized in vitro with boar spermatozoa in a medium enriched by arginine‐3H and the sites of localization of newly synthesized arginine‐3H–labeled proteins have been investigated using fine‐structure autoradiography. It was confirmed that such proteins are synthesized during fertilization and that they accumulate to a notable degree in decondensing sperm chromatin as well as in the chromatin of the female pronucleus and also of the second polar body. A similar process did evidently take place also in defective pronuclei, characterized by a core of a still condensed chromatin and by remaining nuclear membrane. In such male pronuclei the highest concentration of the label was seen just on the border of the condensed chromatin, on the expected site of nuclear protein exchange. It is supposed that, at least in this experimental system, any morphologically detectable sperm decondensation is accompanied immediately by a shift from sperm basic nuclear proteins to other nuclear proteins.
Ultrastructural localization of proacrosin and acrosin in ram spermatozoa
Authors: Huneau D. , Harrison R. A. P. , Fléchon J.‐E.
Published: February 2005

Proacrosin and acrosin were localized immunocytochemically at the electron microscope level in ram spermatozoa undergoing an ionophore‐induced acrosome reaction. Antigenicity was preserved after fixation with 0.5% w/v ethyl‐(dimethylaminopropyl)‐carbodimide, and an antibody preparation was used that reacted with all major forms of ram acrosin. All stages of the acrosome reaction could be observed in a single preparation. At the earliest stage, labeling was observed throughout the acrosomal contents, which were just beginning to disperse. As dispersal proceeded, labeling diminished, being associated only with visible remnants of the acrosomal matrix. By the time the acrosome had emptied, almost no labeling could be detected on the inner acrosomal membrane.

The relationship between matrix dispersal and proacrosin activation was studied in isolated ram sperm heads. While proacrosin was prevented from activating, the acrosomal matrix remained compact; but as activation proceeded, the matrix decondensed and dispersed in close parallel. By the time proacrosin activation was complete, the acrosomal contents had almost entirely disappeared.

We conclude that proacrosin is distributed throughout the acrosomal contents as an intrinsic constituent of the acrosomal matrix. During the acrosome reaction, proacrosin activation occurs, resulting directly in decondensation of the matrix. All the contents of the acrosome including acrosin disperse and, by the time the acrosome is empty and the acrosomal cap is lost, only occasional traces of acrosin remain on the inner acrosomal membrane. Since the acrosomal cap is normally lost during the earliest stages of zona penetration, acrosin's role in fertilization is unclear: it does not appear to be a zona lysin bound to the inner acrosomal membrane.
The oocyte‐cumulus complex: Ultrastructure of the extracellular components in hamsters and mice
Authors: Talbot P. , Dicarlantonio G.
Published: February 2005

To enhance preservation of the extracellular materials, we have fixed hamster and mouse oocyte cumulus complexes (OCC) for transmission electron microscopy in the presence of ruthenium red. Ruthenium red had four effects on the extracellular components of the freshly ovulated hamster OCC. It interacted with the surface of cumulus and corona radiata cells; it stabilized the extracellular matrix (ECM) that was comprised of granules and filaments; it produced moderate electron density and good structural definition in the zona pellucida, and it revealed occasional smalls granular depsits on the oolemma. The ECM observed between cells of the cumulus and corona radiata layers extended into the outer one third of the zona pellucida. The granule and filament matrix was removed from the cumulus layer, corona radiata, and pores of the zona pellucida by brief treatment with hyaluronidase. The extracellular components of oviducal OCC from hamsters and mice appeared similar to OCC removed from follicles of the hamster shortly before ovulation. However, oviducal OCC did show increased aggregation of granules in the ECM. In most cases where females had been mated and oocytes were fertilized, the extracellular components appeared similar to those seen in fresh OCC. Exceptions were noted in some oocytes that lacked cumulus and corona radiata cells. In these instances, the zona pellucida generally lacked the granule/filament matrix. After fertilization numerous small electrondense granules were noted in the perivitelline space. These were presumed to originate in the cortical granules and formed a new investing layer around the zygote. Our data suggest that the OCC becomes more difficult for a sperm to penetrate as it approaches the oocyte. The significance of these results is discussed with respect to sperm traffic in the OCC and the cortical reaction.
Comparative cytochemistry of the developing ovarian follicles of the dog, rabbit, and mouse: Origin of the zona pellucida
Authors: Tesoriero John V.
Published: February 2005

The reactivity of the glycoproteins of ovarian follicles of the dog, rabbit, and mouse were compared, using the Alcian blue (pH 0.5) and Alcian yellow (pH 2.5) technique at the light‐microscope level and the periodic acid‐chromic acid‐silver methenamine technique at the electron‐microscope level.

In paraffin sections, the rabbit and mouse show the appearance of both zona pellucida and follicular fluid in the earliest growing follicles. In the dog, there is a sequence of development with the follicular fluid appearing late, after much of the zona has been secreted. The zona and follicular fluid are highly sulfated in all animals. Zonae pellucidae of atresia appear to lose all traces of sulfation and become highly acidic. At the electronmicroscope level, oocytes contain little if any reactive glycoprotein material which can be related to zona pellucida formation. The initial appearance of the zona material occurs between follicle cell membranes extending outwards and away from the oocyte. Follicle cells of all species consistently contain a variety of reactive Golgi bodies and granules, with exocytotic vesicles, suggesting active synthesis and secretion of zona material.

Our observations suggest that in the early stages of oogenesis, the follicular epithelium is responsible for at least part of the synthesis of the zona pellucida. It is possible therefore that both the oocyte and its follicle cells participate, probably on the sequential basis, in the synthesis of the mammalian zona pellucida.
Intra‐acrosomally located acrosin‐inhibitors: Evolution and developmental patterns in mammals
Authors: Flörke‐Gerloff S. , Tschesche H. , Müller‐Esterl W. , Engel W.
Published: February 2005

The acrosome of the mammalian spermatozoon contains proacrosin that is autocatalytically activated to become the proteolytic enzyme acrosin during the process of fertilization. Tschesche et al [1982] isolated specific acrosin inhibitors and suggested that they block prematurely activated acrosin. Antibodies against acrosin inhibitors purified from boar spermatozoa were used to demonstrate the evolutionaary relationship and the developmental pattern of the inhibitors in mammals. Using immunofluorescent techniques the following results were obtained: (1) The spermatozoa of man, boar, bull, ram, rat, rabbit, beaver, and mole stained positive in the acrosomal portion. (2) The round‐headed spermatozoa of patients with globozoospermia and those in the ejaculates of fertile men lacked immunostaining for the inhibitors. (3) During spermatogenesis in all species, immunofluorescence for the acrosin inhibitors was first demonstrable in haploid spermatids and increased thereafter during spermatid differentiation. The stained area was found adjacent to the nuclear membrane, the position of the acrosome. (4) During teratogenesis of round‐headed spermatozoa, the immunofluorescent staining for the inhibitors becomes separated from the nuclear membrane of the spermatids and is lost in late spermatids. Since identical results have been described for acrosin and acrosomal membrane proteins both in spermatozoa and spermatids of mammalian species and during spermiogenesis of patients with globozoospermia, our results are consistent with the localisation of the inhibitors in the acrosome. Immunostaining of spermatozoa of species belonging to five different mammalian orders with the antibody against boar acrosin‐inhibitors is indicative of an evolutionary conservation of the inhibitors and their underlying genes.
Effects of caffeine, Ca++, capacitation time, and strain on in vitro fertilization in mice
Authors: Dodds J. W. , Seidel G. E.
Published: February 2005

Oocytes (N=2922) were collected from superovulated female C57B16/J X DBA2/J (B6D2F1) mice and distributed among 48 treatments consisting of a 2×3×2×2×2 factorial design. The factors were strain of spermatozoa, B6D2F1 or SJL/J; caffeine concentration in the fertilization medium, 0,2, or 6 mM; time oocytes were exposed to sperm, 1 or 2 hours; Ca++ concentration in the capacitation medium, 0 or 1.8 mM; and capacitation time, 1 or 2 hr. Ova were observed 400 min after they were initially exposed to 105 spermatozoa per ml. Ova with two or more pronuclei and a second polar body were considered fertilized, In vitro embryonic development was monitored for 5 days.

B6D2F1 spermatozoa resulted in consistently higher rates of fertilization than SJL spermatozoa, 77.5% vs 38.7% when averaged over other treatments. Caffeine concentrations of 0,2, and 6 mM resulted in respective mean fertilization rates of 50.1%, 58.8%, and 65.4% (P<0.005) when averaged over other factors. Fertilization rates of ova exposed 1 and 2 hr to sperm were 53.0% and 63.3% (P<0.005). B6D2F1 spermatozoa capacitated in medium with 1.8 mM Ca++ fertilized more ova (P<0.01), 83.1%, than when no Ca++ was present, 71.9%; this effect was absent with SJL spermatozoa. The effect of capacitation time depended on strain. Fertilization rates with B6D2F1 spermatozoa were higher, 80.1%, with a 2‐hour capacitation time than with a 1‐hour capacitation time, 75.0%. Exactly the opposite was true for the SJL spermatozoa; 43.4% for the 1‐hour and 34.1% for 2‐hour capacitation (P<.01). Development to the blastocyst stage was significantly greater (P<0.025) for ova fertilized by B6D2F1 (26.8%) than by SJL spermatozoa (17.7%).
Enhancement of the acrosome reaction of hamster spermatozoa by the proteolytic enzymes, kallikrein, trypsin, and chymotrypsin
Authors: Shinohara Harumichi , Yanagimachi Ryuzo , Srivastava Prakash N.
Published: February 2005

The involvement of a kallikrein−kinin system in the motility of mammalian spermatozoa has been suggested by several investigators. We found that incorporation of kallikrein (0.1–1.0) unit/ml) in the sperm incubation medium did not enhance the motility of hamster spermatozoa that were already active. However, this enzyme significantly increased the incidence of the acrosome reaction. Trypsin (1.8–18 units/ml) and chymotrypsin (0.34–3.4 units/ml) also increased the incidence of the acrosome reaction, and accelerated its onset. Kinins (bradykinin and kallidin) added to the medium in a wide concentration range (1 ng/ml to 1 mg/ml) had no marked effects on either the motility or the acrosome reaction. A kallikrein−kinin system is apparently not of primary importance at least for the acrosome reaction. The enhancement of the acrosome reaction by exogenous proteinases may be due in part to accelerated removal or alteration of the sperm surface coat (glycoprotein) by the enzyme peior to the acrosome reaction. Exogenous proteinases may also act synergistically with endogenous (acrosomal) proteinases (and other enzymes) in altering membrane proteins and dispersing the acrosome matrix during the course of teh acrosome reaction.
Induction of polyspermic fertilization in sea urchins by the leukotriene antagonist FPL−55712 and the 5−lipoxygenase inhibitor BW755C
Authors: Schuel Herbert , Moss Roberta , Schuel Regina
Published: February 2005

Arachidonic acid can be oxidized via the cyclooxygenase pathway to produce prostaglandins and via the 5−lipoxygenase pathway to produce leukotrienes. These substances are known to be extremely potent regulators of cellular function in somatic tissues, particularly during inflammatory reactions. Recent studies have implicated cyclooxygenase−derived products in preventing polyspermy in sea urchins [Schuel et al, Gamete Res 10:9–19, 1984]. FPL−55712, a receptor antagonist for leukotrienes in somatic tissues, causes a dose−(1–10 μM) and sperm−density−dependent induction of polyspermic fertilization in sea urchins if added before the egg completes the cortical reaction (elevation of the fertilization envelope). Eggs pretreated with FPL−55712 become polyspermic upon subsequent insemination with untreated sperm in sea water. Sperm pretreated with the drug do not cause polyspermy when used to inseminate untreated eggs. The 5−lipoxygenase inhibitor BW775C also promotes polyspermy. FPL−55712 and BW755C do not retard elevation of the fertilization envelope. These findings imply that (1) leukotrienes may be produced via the 5−lipoxygenase pathway during fertilization in sea urchins, and (2) the reaction of leukotrienes with putative receptors on the egg's surface may modulate its receptivity to sperm during the cortical reaction, and thereby help prevent polyspermy.
Electron microscopy of mouse preimplantation embryos and isolated blastomeres immobilized on coverslips
Authors: Wiley Lynn M. , Takaki Kathy K. , Yamagata Maya
Published: February 2005

In this report we describe a new method of immobilizing intact mouse preimplantation embryos and their dissociated blastomeres on a flat substrate so that they may be processed en masse for electron microscopy. The embryos can be arranged so that they will be coplanar and grouped together closely enough to be included within the same plane of section. The substrate consists of Thermonex plastic coverslips that have been coated with a layer of fibroblasts or with fibronectin. The plant lectin phytohemaggiutinin (PHA) is used as a ligand to adhere the embryos onto the coverslips. This method is compatible with other ligands and with a variety of immunocytochemical procedures and can be used with live or fixed embryos.
Partial purification and characterization of an alkaline proteinase from the rabbit testis
Authors: Ninjoor Vyas , Srivastava Prakash N.
Published: February 2005

A proteolytic enzyme capable of cleaving intact proteins and synthetic substrates α−N−benzoyl−DL−arginine β−naphthylamide (Bz‐Arg‐NNap), α‐N‐benzoyl‐L‐arginine p‐nitroanilde (Bz‐Arg‐NPhNO2), and α‐N‐benzoyl‐L‐arginine ethyl ester (Bz‐Arg‐OEt) was purified 92– fold from the rabbit testes. The enzyme exhibited optimal activity at pH 9.0 and 50°C. The polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis of the purified enzyme demonstrated multiple forms; the major band in the SDS‐polyacrylamide gel electrophoresis corresponded to a Mt 48,000. The same value was established by the gel filtration over Sephadex G‐75. The rabbit testicular alkaline proteinase (TAP) resembled acrosin in the hydrolysis of Bz‐Arg‐OEt. However, CaCl2, a potential stimulator of acrosin activity, inhibited the alkaline proteinase. The strong inhibitors of acrosin, eg pheny methyl sulphonyl fluoride (PMSF), tosyl lysine chloromethyl ketone (TLCK), and benzamidine did not inhibit the alkaline proteinase. TAP was activated by an acrosin inhibitor isolated from the rabbit testes. Since 0.5 M KCl was necessary for complete extraction of the enzyme and the bulk of the activity was present in 9,000g pellet of the testicular homogenate. The alkaline proteinase appeared to be associated with the membranous structures.
A follicular fluid component prevents gonadotropin reversal of cyclic adenosine monophosphate‐dependent meiotic arrest in murine oocytes
Authors: Downs Stephen M. , Eppig John J.
Published: February 2005

The effects of the putative maturation inhibitor in porcine follicular fluid on gonadotropinstimulated reversal of cyclic adenosine monophosphate (cAMP)‐maintained meiotic arrest in mouse oocytes in vitro were assessed in this study. When cumulus cell‐enclosed oocytes were cultured in a suboptimal inhibitory concentration of dibutyryl cAMP (dbcAMP), the effect of follicle‐stimulating hormone (FSH) on oocyte maturation was initially inhibitory at 3 hr, but stimulatory at 6 hr. Supplementation of the medium with an ultrafiltrate of porcine follicuiar fluid (PM10‐filtrate) completely suppressed FSH‐promoted reversal of inhibition at 6 hr. Charcoal extraction eliminated this effect of the PM10‐filtrate. FSH reversed the inhibition of maturation of cumulus cell‐enclosed oocytes maintained by a high concentration of dbcAMP and suboptimal concentrations of the phosphodiesterase inhibitor, 3‐isobutyl‐1‐methyl xanthine (IBMX), during a 21–22‐hr culture period. However, the effect of a completely inhibitory concentration of IBMX was not reversed by gonadotropin. A component of serum was also found to inhibit FSH reversal of dbcAMP‐maintained meiotic arrest, and this activity was removed by charcoal extraction. In addition, when oocytes were cultured in medium containing a suboptimal concentration of dbcAMP plus a low molecular weight fraction (< 1,000) of porcine follicular fluid, porcine serum, or fetal bovine serum, a synergistic inhibition of maturation was observed. Experiments with highly purified gonadotropins revealed that reversal of dbcAMP‐maintained meiotic arrest occurred only in response to FSH; neither highly purified luteinizing hormone nor human chorionic gonadotropin could mimic this action of FSH. Also, this effect was mediated by the cumulus cells, since FSH could not reverse dbcAMP‐maintained meiotic arrest in denuded oocytes. Furthermore, elevating cAMP levels in denuded oocytes augmented, rather than reversed, the inhibitory action of dbcAMP on oocyte maturation. These data therefore suggest that dbcAMP‐ or IBMX‐maintained meiotic arrest in vitro is reversed by an FSH‐stimulated, cAMP‐dependent process mediated by the cumulus cells and demonstrate that a factor present both in follicular fluid and serum prevents this action of the gonadotropin.
Characterization of a spectrophotometric technique for the estimation of fowl and turkey sperm motility
Authors: Wishart Graham J , Ross Frances H.
Published: February 2005

An objective method of estimating the motility of fowl and turkey spermatozoa, depending on their rheotactic and light‐scattering properties, has been developed. From a 1‐ to 2‐minute recorder trace of the optical density of diluted semen before and after stopping its passage through a flow cuvette, three independent constants may be simply and graphically determined. These are: ODm, the maximum optical density of semen flowing through the cuvette, shown to be dependent on the concentration of spermatozoa; % (ΔOD)m, the maximal change of optical density following cessation of flow, which has been correlated with the percentage of motile spermatozoa in the sample; and t½, the time taken for the change of optical density to reach % (ΔOD)m. This latter parameter has been correlated with forward motility of both fowl and turkey spermatozoa.
Transglutaminase inhibitors, calmodulin antagonists, and calcium channel blockers: Influence on Arbacia sperm motility
Authors: Cariello Lucio , Nelson Leonard
Published: February 2005

Sea urchin spermatozoa react to transglutaminase inhibitors, which may antagonize the function of calmodulin, in dose‐dependent fashion. The optimum concentration of diallyl‐amino propionyl benzothiophenc (DAPBT), a potent, noncompetitive inhibitor of transglutaminase, 0.01 mM, causes a 3 1/2‐fold increase in the forward swimming speed of Arbacia sperm. This effect apparently involves calcium‐dependent enzymes since manipulation of both extracellular and intracellular calcium by means of chelating agents, calcium‐channel blockers, and calmodulin antagonists depresses the stimulatory effect of DAPBT. These results suggest that in the spermatozoa the interaction of flagellar sliding filaments may be mediated by reversible cross‐linking of the contractile proteins catalyzed by a calcium‐ dependent transglutaminase.