Showing papers in "Gamete Research in 1983"

Structural changes in bovine oocytes during final maturation in vivo

Abstract: On the basis of structural observations bovine oocytes were grouped into four successive classed: 0, those before the luteinizing hormone (LH) surge; 1, those up to 8 h following the LH peak level; 2, those between 8 and 19 h after the LH peak level; and 3, those between 19 h after the LH peak level and ovulation. Oocytes in class 0 had mitochondria located in a generally peripheral position. Interior to the mitochondria were elements of rough endoplasmic reticulum (RER) and numerous membrane-bound vesicles which bore ribosome-like particles on their outer surface. The first visible changesater the LH peak level as seen in class 1 were the formation of the periviteline space with loss of contact between the cumulus cells and the oocyte, and ruffing of the nuclear envelope. These changes were followed b the resumption of meiosis as defined by germinal-vesicle breakdown (GVBD), the disappearance of RER, and the formation fo clusters of mitochondria in association with lipid droplets and elementrs of smooth endolasmic reticulum (SER). The period between 8 and 19 h following LH peak level (class 2) was characterized by intensive clustering of mitochoncria in association with lipid droplets and elements of SER, conversion of lipid, fusion of vesicles, and the appearance of ribosomes in the cytoplasm. During the final stage (class 3), the polar body was extruded, the mitochondria dispersed, and the majority of the organelles became located toward the center of the cell. The relatively organelle-free cortical region contained cortical granules immediately adjacent to the plasma membrane together with aggregates of tubular SER. The structural changes are discussed in the context of follicular steroidogenesis and oocyte developmental competence.

Isolation of motile spermatozoa by density gradient centrifugation in Percoll

Abstract: A procedure using centrifugation in density gradients composed of Percoll was developed for isolation of spermatozoa from mammalian semen. To evaluate the technique, rabbit, human, or bovine semen was layered over continuous Percoll gradients ranging in density from 1.02 to 1.13 gm/ml and centrifuged at 1,500g for 45 min. After centrifugation, the seminal plasma remained above the gradient, whereas the spermatozoa and seminal particles were distributed within the gradient according to their buoyant densities. Unlike most washing techniques, no sperm pellet was formed; instead, the spermatozoa were concentrated into a compact band above the most dense layer of Percoll. The spermatozoa recovered from the gradient were easily resuspended by gentle techniques. Thus, the mechanical stress to the spermatozoa was minimized. Osmotic stress to the spermatozoa was also negligible as the Percoll gradients were isotonic throughout. Spermatozoa obtained by this technique possessed motility equivalent to that of spermatozoa in the unfractionated semen. Sperm suspensions recovered from the gradients contained less than 5% of the nonspermatozoal particles present in the original samples of unfractionated semen. Soluble seminal components were also efficiently removed from the spermatozoa. Thirty billion bovine spermatozoa could be fractionated on a single gradient without loss of effectiveness. Recovery of spermatozoa from these preparative separations averaged 80%. These results demonstrated that Percoll was a superior medium for efficient density gradient isolation of motile spermatozoa free of contamination by other seminal constituents.

Ultrastructural studies of spermatozoa in three bivalve species with notes on evolution of elongated sperm nucleus in primitive spermatozoa

Abstract: Sperm ultrastructure and spermiogenesis of the three bivalve species Musculus discors, Nucula sulcata, and Dreissena polymorpha have been studied. During spermatid differentiation in Musculus discors and Nucula sulcata the nucleus attains an elongated rod-like shape. The spermatozoon from Nucula sulcata was found to have a cup-shaped acrosome and five mitochondria surrounding two centrioles in the middle piece. The spermatozoa from Musculus discors has a long complex acrosome. From the distal centriole striated processes extend and attach to the plasma membrane. The spermatozoon of the fresh water species Dreissena polymorpha agrees in all main features with those of other invertebrate groups with external fertilization. It is thus of the primitive type with barrel-shaped nucleus and four to five mitochondria1 spheres in the middle piece. The acrosome is a prominant, complex structure at the apex of the mature spermatozoon. A comparison of sperm ultrastructure among bivalves indicates that there is a certain correlation between the evolution of the elongated sperm nucleus and large, yolk-rich eggs. In species with an elongated sperm nucleus the increased egg size has often led to a lecithotrophic or direct development. The elongated nucleus is a slight modification of the primitive type. There is a great variation in acrosome structure among bivalve spermatozoa, reflecting diverging functional demands at fertilization of the eggs.

Rabbit spermatozoa undergo an acrosome reaction in the presence of glycosaminoglycans

Abstract: Rabbit-ejaculated spermatozoa were incubated in a chemically defined medium containing comercially available glycosaminoglycans (GAGs) at concentrations ranging from 0.1 to 100 μg/ml. Sperm were stained and examined for the degree of acrosome reaction and viability after 9 h of incubation. There were significant dose and treatment effects of the induction of the acrosome reaction. Viability did not differ significantly betweendoses or treatment. Heparin enhanced the acrosome reaction between concentrations of 0.1 to 1.0 μg/ml, whereas higher levels depressed the percentage of sperm undergoing the acrosome reaction. Seminal plasma added to sperm cultures depressed the stimulatory effect of GAGs. Treatment of chondroitin-4-sulfate with chondro-4-sulfatase prohibited the stimulatory effect. It is concluded that GAGs, components of the female reproductive tract, may promote the acrosome reaction so that successful fertilization can occur.

Parameters influencing ovum pickup by oviductal fimbria in the golden hamster

Abstract: 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.

Gametes and fertilization in the Chinese hamster

Abstract: 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.

Effects of egg aging on in vitro fertilization and first cleavage division in the hamster

Abstract: The postovulatory fertile life of mammalian eggs is remarkably short (approximately 6-36h). Anomalies of embryogenesis may result from fertilization of aged, defective eggs. Attempts to study this problem using whole-animal models are complicated by chances in the natural milieu of the gametes. In the present study, postovulatory hamster eggs were allowed to agein vivo then fertilized in vitro. Cumulus-intact eggs recovered from superovulated hamsters either 2 or 9 h after the estiated time of ovulation (12 h postHCG) were incubated for 4 h with preincubated sperm suspentions in a modified Tyrode's solution devised for in vitro fertilization. Eggs were either fixed or cultured for another 20h in fresh medium to allow cleavage to occur, then examined by light microscopy (phase and interference-contrast). No significant difference was found in the ablities of fresh and aged eggs to be penetrated by spermatozoa (94% vs 90%, respectively; 8 replicated experiments), but only 59% of penetrated aged eggs were found to undergo morphologically normally fertilization (2 polar bodies, 2 prounclei) compared with 75% of fresh eggs (difference significant, P< 0.01). About 13% of eggs were polyspermic in both categories. The most common anomaly in aged fertilied egges was failure to extrude the second polor body (23% off eggs vs 8% of fresh eggs, P < 0.01). Only 21% of aged eggs underwent first cleavaage, and only 74% of these appeared morphologically normal, compared with value of 68% and 98%, respectively, for fresh eggs. These data show that in the hamster, abnormal fertilization and cleavage failure can, in part, be directly attributed to postovulatory deterioration of eggs. We also infer that the apparently very short penetrable life of hamster eggs in vivo shown by previous investigators is an indirect effect of postovulatory changes in the female reproductive tract that are unfavorable for sperm-egg interactions.

Effects of selenium deficiency on the shape and arrangement of rodent sperm mitochondria

Abstract: Selenium-deficient male mice were obtained by feeding a selenium-deficient diet for three successive generations to Swiss-Webster mice. Examination of epididymal sperm by transmission electron microscopy revealed progressively increasing alterations in the shape and arrangement of mitochondria within the midpiece. Other midpiece anomalies included acute bends, disorientation of the axoneme and dense fibers, and cytoplasmic masses at atypical locations. Some cross sections showed both the principal piece and midpiece within the same plasma membrane. Negatively stained whole mounts of mitochondrial ghosts prepared from epididymal sperm of normal and first-generation selenium-deficient mice and rats indicated that the selenium-deficient ghosts were smaller, less curved, and more fragile than those of normal sperm mitochondria. Thus, selenium appears to be required for the normal development or stabilization of mitochondria1 shape during spermiogenesis in these rodents.

Active immunization of cynomolgus monkeys (Macaca fascicularis) with porcine zonae pellucidae

Abstract: Cynomolgus monkeys (Macaca fascicularis) were immunized with porcine zonae pellucidae to assess the possible antifertility effects of the zona antibodies. Serum antibody titers were evaluated utilizing a rapid solid-phase radioimmunoassay. Six of twelve monkeys conceived 6 to 10 wk after vaccination. All monkeys reached maximal antiserum titers by the time of conception, although the six animals that did not conceive had considerably lower antibody titers. Further pregnancies did not occur until antibody level had declined markedly, 8 mo after last immunization. The menses of all but one of the remaining six monkeys were interrupted intermittently. Also, the usual midcycle elevated estradiol levels were absent for several cycles. Both menses and midcycle estradiol peaks were reestablished in all but one monkey 3 to 5 mo after the last booster was given. Two monkeys conceived when serum antibody levels dropped to one fourth of maximal, but both had a still birth. Histological observations showed accumulation of luteal tissue and massive atresia of small follicles at the end of the study (18 mo). We conclude that through heteroimmunization with porcine zona pellucida monkeys can become infertile and that this condition is reversible. Because the zona preparation used in this study appeared to contain traces of nonzona material, it was not possible to determine whether the menstrual irregularities and oocyte atresia that we observed were owing to immunological effects on the zona itself or to the production of antibodies against other ovarian components.

cAMP‐dependent phosphorylation associated with activation of motility of Ciona sperm flagella

Abstract: Demembranated spermatozoa of Ciona do not become motile when provided with MgATP, unless their motility is activated in vivo before demembranation or unless the demembranated spermatozoa are activated in vitro with cAMP or with the catalytic subunit of a cAMP-dependent protein kinase. CAMP causes a greater than fivefold enhancement of 32P incorporation by demembranated spermatozoa. Analysis by one-dimensional PAGE and autoradiography shows several axonemal protein bands that become 32P-labeled during in vitro activation with cAMP and identifies protein bands whose labeling is specifically reduced if motility of the spermatozoa is activated before demembranation, suggesting that these proteins also become phosphorylated during activation of motolity in vivo. These phosphorylated proteins appear to include dynein heavy-chain components, but axonemal tubulin is not phosphorylated. Partially phosphorylated spermatozoa can be activated by an increase in KCI concentration, which appears to dissociate one phosphorylated component from the axoneme.

Progressive defects observed in mouse sperm during the course of three generations of selenium deficiency

Abstract: Three successive generations of mice were fed a Torula yeast based Se-deficient diet with or without 01 ppm Se in the drinking water The Se-deficient mice, in the course of three generations, showed a decrease in body weight, testis weight, epididymal weight, and sperm production The percentage of morphologically abnormal sperm increased in successive generations The majority of sperm defects were found in the midpiece region of the tail Many of these aberrant sperm were motile A progressive decrease in fertility was noted during the first two generations of Se deficiency This system confirms the essential role of Se in spermatogenesis and provides a model for the evaluation of the primary effect of Se deprivation on the structural development of sperm

Spermatogenic cell differentiation in vitro

Abstract: Culture conditions that support the in vitro development of many spermatogenic stages from the frog Xenopus laevis are described. Spermatogenic cells were dissociated with collagenase and preelongation stages aseptically isolated by density gradient centrifugation in Metrizamide. The cells were then cultured in modified forms of defined nutrient oocyte medium (DNOM). The development of spermatogenic cells was affected significantly by changes in fetal calf serum concentration, cell density, energy sources, and NaCl concentration. Optimum in vitro spermatid development was obtained when spermatogenic cells were cultured at relatively high densities (3–7 × l07 cells/25 cm2) in DNOM modified to contain 10% heat-inactivated, dialyzed fetal calf serum, 2 mM 1-glutamine, 0.1 % glucose, 15 mM HEPES buffer (pH 7.4), and 38.3–48.3 mM NaCl. These culture conditions also supported the differentiation of preelongation spermatids and spermatocytes isolated by density-gradient centrifugation in Metrizamide and subsequent unit gravity sedimentation in gradients of bovine serum albumin. Approximately 95 % of such isolated spermatids and spermatocytes continued differentiating in vitro for 14 days at in vivo rates. Phase-contrast and electron microscopy of the cultured cells demonstrated that in vitro differentiation was morphologically normal between the leptotene and elongate spermatid stages. Autoradiographic studies of preleptotene development demonstrated that spermatogonia proliferated and preleptotene spermatocytes developed to zygotene in 12-day cultures. The results suggest that many spermatogenic stages in Xenopus can develop independent of Sertoli cells, and demonstrate that spermatogenic cell cultures can now be used for in vitro studies of spermatogenesis.

Metabolism of lipid and carbohydrate in sea urchin spermatozoa

Abstract: When the dry sperm of the sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial sea water at 0°C and at 20°C, they became motile and the levels of ATP and creatine phosphate decreased rapidly. The level of ADP hardly changed, and the AMP level increased after the dilution. After the dilution, the respiratory rate at 2°C was almost one fifth that of 20°C. Both phospholipid and glycogen were used for the energy sources in sea urchin sperm. The level of phospholipid was 10-fold higher than that of glycogen in the dry sperm. The phospholipid level decreased after dilution at 20°C, though the level hardly changed at 0°C, suggesting that phospholipid was hardly metabolized the lower temperature. The level of α -glycerophosphate increased at 20°C after the dilution but did not change at 0°C. The level of glycogen decreased after the dilution, regardless of the temperature. The glycolysis was also activated after the dilution. Of the intermediates of the tricarboxylic acid cycle, the citrate concentration increased at 0°C and the malate concentration also increased at 0°C and especially strongly at 20°C.

Reproduction in mice: the fate of spermatozoa not involved in fertilization.

Abstract: Mechanisms were sought through which the control of preimplantation mouse embryo development by spermatozoa might be effected. A potential route for the transmission of sperm-dependent stimuli to C3HeB/FeJ females was uncovered. It was found that within 24–48 hr after artificial insemination with spermatozoa, in which the DNA had been labeled with tritiated thymidine, a minimum of 9% of the radioactivity was transported across the uterine walls. It was deposited among the maternal tissues in a pattern that differed from the patterns of isotope distribution obtained when either free tritiated thymidine or Escherichia coli cells containing DNA labeled with tritiated thymidine were used instead of labeled sper-matozoa. In sperm-treated animals the ovaries, the adrenals, and a mesenteric lymph node exhibited strikingly large accumulations of radioactivity. The heart, spleen, and uterus manifested lesser accumulations of label, but were higher than liver, kidney, lung, brain, muscle, and intestine. The specific activity of the lymph node was found to decrease during the 12–72-hr period following insemination. This result led to the hypothesis that the lymphatic system could serve as a route for the dissemination, to maternal tissues, of radioactivity originally associated with spermatozoa deposited in the uterus. Heat-inactivated spermatozoa, which have the potential for facilitating the first cleavage of fertilized embryos, exhibited a distribution pattern indistinguishable from untreated spermatozoa. Sperm protein kinase was found to survive the heat inactivation of spermatozoa. This stability was interpreted as being compatible with the kinase functioning as an intermediary in the transmission of sperm-dependent stimuli that control preimplantation embryo development in mice.

The evolution of the nematode spermatozoon

Abstract: The main features of the Nematode sperm cell are the absence of a flagellum and of an acrosome. Transition forms have never been described, as in other animal phyla also reaching the aflagellate condition, like Platyhelminths and Arthropods. The absence of the flagellum must be considered as a definitive acquisition in the group. In addition, centrioles have been demonstrated to be lacking in most cases. The absence of the acrosome is the second general feature of the Nematode sperm cell. Among other features, more or less common to the Nematodes, the most important and general is the presence in the cell periphery of spheroidal membranous vesicles, originated from the Golgi complex but not involved in fertilization or in the production of ascaridin granules. These are absent only in the Ascarid Aspiculuris and the Dorylaimiid Xiphinema, both kinds of sperm having a peculiar shape. These granules are possibly involved in cell motility. Some Nematode sperm have proteinaceous crystalline inclusions originated from the rough endoplasmic reticulum, called ascaridin granules, the role of which remains obscure. A third important feature is the absence of a nuclear envelope, characterizing all described Nematode spermatozoa, the only exception being the Enoplid Mesacanthion, which seems to be for this reason the most primitive model in the group. Other features are the reduced number, or total absence of, the chondriome, an amoeboid movement not owing to an actomyosin system and a dense halo of 10-nm filaments surrounding the perinuclear cytoplasm. In this apparently homogeneous picture, three main evolutionary steps can be recognized. The first one, represented by the primitive Enoploid Mesacanthion, is that of a sperm conserving the nuclear envelope, surrounded by a few mitochondria and many membranous vesicles. The second, the most typical of the group, present in high Enoplida, and in Rhabditida, Strongylida, Ascarida, Spimrida, Trichinellida, is that of roundish, amoeboid spermatozoa devoid of a nuclear envelope but containing mitochondria, membranous vesicles, filaments, microtubules, sometimes centrioles, and sometimes ascaridin granules. The third step is apparently a simplification of the second; in fact, in Tylenchida and Dorylaimiida, the sperm is devoid of membranous vesicles, while in Mononchida and Dioctophymatida it is devoid of mitochondria. Aspiculuris, also devoid of membranous vesicles and having a big mitochondrial derivative, can be assigned to the same level. Nematode sperm evolution does not seem therefore to be a progressive acquisition of new characters, but rather a radiation from an already perfect model of some further simplifications occurring in parallel in most of the orders.

Studies on the polymorphic spermatozoa of a marine snail. 2. Genesis of the eupyrene sperm

Abstract: Spermiogenesis of the eupyrene sperm in the snail, Fusitriton oregonensis, was studied with light and electron microscopes. Endoplasmic reticulum, which encircles the nucleus in each spermatid, appears to connect with the Golgi body and to interconnect between adjacent spermatids via cytoplasmic bridges. It is suggested that as the Golgi body migrates around the nucleus the endoplasmic reticulum may circulate with it. The alignment of the proacrosome with the nucleus is effected by a 180° rotation of the Golgi body, after which it separates and migrates posteriorly with the residual cytoplasm. Each sperm possesses a well-developed intracellular digestive system as indicated by multivesicular bodies, residual bodies, and myeloid figures. Autophagy begins in the residual cytoplasm before it is released from the middle piece. Microtubules are found outside the nucleus and mitochondria during the final stages of spermiogenesis, when elongation is almost complete. These microtubules appear to be involved in the final shaping and twisting process, in which torsion is locked in the nucleus and the mitochondria spiral around the axoneme. The annulus attaches the distal centriole to the plasma membrane in the early spermatid and as flagellar production begins they move towards the implantation fossa at the base of the nucleus. There are two centrioles in the early spermatid, the distal centriole and procentriole. The small procentriole fuses with the distal centriole in the intranuclear canal to form the centriolar cap of the basal body. This cap is pushed through the end of the nuclear tube and is separated from the subacrosomal space by only the nuclear membranes.

Nuclear and chromatin structure in rat spermatozoa

Abstract: The nuclei of mature mammalian spermatozoa contain a highly ordered, lamellar substructure, presumably constituting the nucleoprotein of the haploid chromosomal complement. With a view toward constructing a plausible model of chromatin packing in sperm, we have determined some of the quantitative parameters associated with these “nuclear lamellae” in rat spermatozoa. Epididymal sperm from white, Sprague-Dawley rats were examined by conventional sectioning methods, freeze fracture of fixed and unfixed specimens, and by whole mount replica techniques. Fixation and glycerolation did not significantly alter nuclear structure as seen by freeze fracture. Numerical data obtained from cross fractures of sperm heads indicate that the number of lamellae are quite constant at 10.4 ± 1.8 and that the linear measure of the lamellae is 7.2 ± 2.3 μm per cross fracture. The total area of cross fracture, assuming an elliptical profile is 2.3 k 0.7 μm2 and the thickness of the lamellae is 18.2 ± 3.5 nm with a range of 13.5 to 25.5 nm. An estimate of the total surface area of the nuclear lamellae could be made from measurements of projected nuclear area (from replicas and sections) as 173 ± 15 μm2. From these data and the known amount of DNA in the rat sperm nucleus, a model can be proposed for the organization of the nucleoprotein in these lamellar sheets. It is suggested that the chromatin is arranged in a coiled-coil configuration closely associated together in a side-by-side fashion and continuous in extent. Approximate calculations based on this simple model are within a factor of 2 or 3 of predicting the correct amount of DNA in the sperm nucleus.

Nucleolar transformations in the human oocyte after completion of growth

Abstract: Nucleolar ultrastrure was studied in fully grown human oocytes obtained from multilaminar preantral follicles and from follicles at different stages of antrum formation. Selective staining for ribonucleoproteins and 3H-uridine labeling were used in attempt to better understand the nature and functional significance of homogeneous dense nucleoli found in oocytes from large antral follicles. There was an apparent increase in the radio of nucleonema to nucleolar interstices, accompanied by a gradual degranulation of the nucleolonema during early stages of antrum fromation. The process of nucleolar homogenization continued in oocytes from medium-size antralfollicles, island of more tightly packed fibrils being hybothesized to represent persisting active transcription units. Entirely filamentous and homogeneous nucleoli were typical for oocytes from large antral follicles. They were demonstrated to ribonucleoprotein filaments embedded in pale- staining matrix. They were demonstrated to contain newly synthesized RNA after a 30-min pulse with 3H-uridine. The described nucleolar transformations are interpreted as acorrelate of nucleolar transition from a site of RNA synthesis into a site of RNA Storage during in human oocyte preovulatory development.

Selective binding of specific rat epididymal secretory proteins to spermatozoa and erythrocytes

Abstract: 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.

Dithiothreitol, a disulfide‐reducing agent, inhibits capacitation, acrosome reaction, and interaction with eggs by guinea pig spermatozoa

Abstract: Capacitation of guinea pig spermatozoa in vitro was inhibited by the disulfide-reducing agent dithiothreitol (DTT). Even a brief treatment with DTT inhibited capacitation unless an oxidizing agent (glutathione disulfide) was present in the posttreatment medium. Precapacitated spermatozoa were unable to undergo the acrosome reaction in the presence of DTT, indicating that this reagent also blocks the acrosome reaction. Acrosome-reacted spermatozoa were incapable of attaching to and penetrating the zona pellucida in the presence of DTT. Even when acrosome-reacted spermatozoa were directly brought to the surface of zona-free eggs, they were unable to bind to and fuse with the egg plasma membrane so long as DTT was present in the medium. These observations suggest that the tertiary and quaternary structures of sperm surface proteins regulated by their thioldisulfide status are of critical importance in the physiology and function of spermatozoa preliminary to and in the process of fertilization.

Binding to spermatozoa of a major soluble protein secreted by the epididymis of the lizard Lacerta vivipara

Abstract: A major soluble protein occurring in two forms, a monomer (protein L) and a polymer, has been identified in the voluminous secretory granules produced by the epithelium of Lacerta vivipara epididymis. An antiserum was raised against protein L and used as an immunohistochemical probe. Indirect immunofluorescence microscopy has indicated that protein L is able to bind to the heads of the spermatozoa. By incubating spermatozoa with buffers of increasing ionic strength and by using nonionic detergents it was not possible to remove completely the protein L. Therefore it was concluded that the binding of protein L to the spermatozoa was not labile and might play an important role in the physiology of the spermatozoa, which is presently under investigation with this convenient model.

The sperm head of the plains mouse, Pseudomys australis: Ultrastructure and effects of chemical treatments

Abstract: The ultrastructure of the sperm head of the plains mouse, Pseudomys australis, and the effects of chemical treatments on the sperm head components has been investigated to determine the nature of the material in the hooks on the apical margine of the sperm head. Ultrastructural studies indicated that the dorsal hook contained nuclear, subacrosomal, and acrosomal material, whereas the two ventral hooks were largely composed of an extention of the subacrosomal material with two thin acrosomal projections at their base. Acrosomal material was dispersed by mild detergent treatment, where as the bulk of the material in the ventral hooks were generally found to be similar to the subacrosomal material in the dorsal hook in their resistance to the various chemical treatments. Treatment of sperm with NaOH or guanidine-hydrochloride and DTT revealed two layers of material in the ventral hooks.

Surface and internal antigens of rat spermatozoa distinguished using monoclonal antibodies

Abstract: 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.

Postovulatory aging of human ova: II. Spontaneous division of the first polar body

Abstract: Two oocytes recovered from the Fallopian tube at 24 and 50 h following the LH peak in plasma were examined by light microscopy and transmission electron microscopy. The possibility of fertilization was excluded by clinical and morphologic evidence. In the earliest specimen, the first polar body showed condensed nuclear chromatin, without nuclear envelope, and is interpreted as entering interphase. In the other specimen, the first polar body was undergoing division, which is interpreted as spontaneous second meiotic division.

Fine structure of the mature spermatozoon of rhynchocinetes typus, crustacea decapoda

Abstract: Rhynchocinetes typus spermatozoa obtained from the vas deferens have the shape of a round-headed nail. The head measures 30 μm in diameter and 14 μm of height. At the center of the flat face of the head emerges a single rigid spike of 53 μm in length. Cross sections of this spike show that it has a wall of 0.4 μm in thickness and a core of 0.6 to 0.8 μm. The outer surface of the spike has a longitudinal striation. When the spermatozoa are placed in sea water it is possible to observe the unfolding of rays. The number of rays in different spermatozoa of the same individual varies from 9 to 13. Each ray is formed by a channel-like sheath that contains a rigid rod that occupies about 1/3 the length of the ray. This rod has a transverse striation with a periodicity of 185A. The rays are bound among them by a thin membranous sheet that is highly folded in vas deferens spermatozoa. At the distal end of each ray there is a rigid spine of 50 μm in length. The nucleus is coplanar to the radial plane and it extends through the rays. The structure and ultrastructure of R typus spermatozoa depart from that reported for spermatozoa of other Caridea species.

Comparative morphometrics of oligochaete spermatozoa and egg‐acrosome correlation

Abstract: Ultrastructural variation between spermatozoa of nine lumbricid species has examined for Prearson r correlations, by principal components and coordinates analysis, and graphically, with particular reference to the acrosmoe. Results are compared with a survey of acrosome in seven other oligochaete families. Acrosome lengths correlates highly with length of the primary acrosome vesicle (PAV) though absolute lengths of the two in an acrosome may differ greatly. High correlation ofacrosome lenghts with thickness of the zone pellucida of the oocyte in lumbricida and other families is consistent with the hypothesis that intertaxon variation in acrosome length in an adaptation to the thickness of the zona. This variation is brought about by variation in the length of the acrosome tube, which is a unique structure of the Clitellasta. Correlation with zona thickness is considered premarily to be that of the acrosome (and tube) length and not of the PVA. Other sperm components do not show high correlation with zona depth, except an enigmatic correlation re of axial rod width. Lumbricid arcosome lenght shows a moderate positive allometry relative to zona depth. The short acrosome of Tubificidae and Enchytraeidae (microdriles) correlate with short oocyte microvilli and narrow zonae. Acrosome lenght‐zona correlation is not a secondary expression of body size (which varies indiscriminately in lumbricids) or egg size; microdriles have larger eggs than megadriles. Mwgadrile features of the acrosome of the tubificidan Phreodrilus, including greater length, are attributed to postulated internal fertilization. It is suggested that the oliochatee acrosome penetrates the zone between, and possibly manipulated by, its microvilli before the acrosome reaction (which is briefly charaterized electron microscopically) occurs or progresses far. Copyright

Adenylate cyclase activity in porcine sperm in response to female reproductive tract secretions

Abstract: Adenylate cyclase activities were studied in porcine sperm in the presence and absence of Mn++ before and after incubation in vivo and in vitro. Incubation of sperm in vivo for 30 min increased the Mg++-stimulated adenylate cyclase activity from 35.1 pmoles cyclic AMP formed per mg protein per 10 min to 50.4 pmoles. The activity stimulated by Mg++ and Mn++ increased from 392 to 729 pmoles after 30 min of in vivo incubation. Activity after incubation in vivo for 120 min was not different from activity after 30 min. In vitro incubation of porcine sperm in Ca++-free Ringer-fructose resulted in no change, but incubation in oviductal and uterine flushings obtained from gilts soon after ovulation increased Mg++-stimulated activity by 24% and Mg++−+ Mn++-stimulated activity by 49%. In vitro incubations in preovulatory flushings plus follicular fluid or in bovine serum albumin also increased adenylate cyclase activity.

Comparison of strains and culture media used for mouse in vitro fertilization

Abstract: Success rates of superovulation in response to gonadotropic hormone treatment and in vitro fertilization (ie, mitotic cleavage following insemination) of mouse eggs from outbred CD-1, hybrid CB6Fl, or hybrid B6CBAF1, mice were compared using either a mouse inseminationmedium, modified Krebs-Ringer-bicarbonate (m-KRB), or a human insemination medium, Ham's F10 nutrient mixture. Inseminations were performed in either organ culture dishes or screw-top, flat-side tissue culture tubes. Mean superovulation rates (± SD) were 24.2 (5.1) for CD-1, 33.0 (5.8) for CB6F1, and 16.3 (6.6) for B6CBAF1 mice. For in vitro cleavage the best combination of mouse strain, insemination medium, and culture container was achieved using CB6F1, mice, m-KRB medium, and culture tubes. However, Ham's medium used with either hybrid mouse strain was shown to be employable for fertilization of mouse eggs in vitro as a quality control assay and/or experimental model system for testing the human in vitro fertilization procedure.