bluuebunny/crossref_metadata_2025 huggingface dataset:
Sperm‐aggregating factor from Spisula oocytes
Authors: Sato Eimei , Segal Sheldon J. , Koide S. S.
Published: February 2005

A membrane protein possessing sperm‐aggregating activity was partially purified from Spisula oocyest. Spisula oocytes were incubated with three different media: A) 1 M urea, 5 mM EDTA, 10 mM Tris‐HCI, pH 7.4, B) 1 M urea, 10 mM Tris‐HCI, pH 7.4, and C) 5 mM EDTA in artificial sea water. Oocytes incubated in media A or B at 22°C were viable up to 15 min of treatment based on the trypan blue exclusion test. After this treatment period, oocyte viability gradually decreased as demonstrated by a progressive increase in the uptake of the dye. However, oocytes excluded the dye when incubated in medium C for 2 hr or longer. Oocytes incubated in medium A or B did not undergo germinal vesicle breakdown (GVBD) on exposure to sperm, while GVBD was induced on treatment with 70 mM KCI, suggesting removal or alteration of sperm receptors by the treatment.

When sperm were incubated with oocyte extract prepared by treatment with medium A or B, they aggregated and formed clusters. The clusters remained unchanged for at least 1 hr at 22–24°C and sperm within the aggreates were motile. Extracts of Spisula oocytes showed species specificity by not agglutinating sperm of Arbacia punctulata, Asterias forbesi, ovalipes ocellatus, or Chaetopterus peramentaceus. The factor was puridied by ammonium sulfate fractionation (30% saturation) and by gel filtration on a Sephadex G 100 column. Four major protein peaks were eluted. Fraction comprising the second and third peaks possessed sperm‐aggregating activity at an affective does od 2.5 μg of protein per ml. The factor is a heat‐stable protein with an estimated molecular weight (mol wt) of 15 to 25 kdaltons.
Comparative morphometrics of oligochaete spermatozoa and egg‐acrosome correlation
Authors: Jamieson B. G. M. , Richards K. S. , Fleming T. P. , Erséus C.
Published: February 2005

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.
Calmodulin intracelluar concentration and cAMP‐dependent protein kinase activity in human sperm samples in relation to sperm motphlogy and motlity
Authors: Pariset Claude C. , Roussel Claude , Weinman Serge J. , Demaille Jacques G.
Published: February 2005

Calmodulin concentration and cAMP‐dependent protein kinase activity have been determined in human sperm samples. No significant differences have been noticed in the motility index of two groups of sperm samples differing in their intracellular concentration of calmodulin. It was however found that a low intracellular clamodulin concentration is frequently associated with particular anomalies of the head and tail region. In contrast, a positive correlation has been demonstrated between the motility index and the whole extractable cAMP‐dependent protein activity of the different sperm samples. In suspension s, When the cAMP‐dependent protein kinase activity was measured on intact sperm suspensions, a positive correlation between the activity and the motility index was also found.
Inhibition of fertilization in the rabbit by anti‐acrosin antibodies
Authors: Dudkiewicz Alan B.
Published: February 2005

Rabbit acrosin was purified from detergent extract of epididymal spermatozoa by molecular‐sieve Chormatography (Sephadex G‐75) and Priflavin‐Sepharose affinity chromatography. Affinity Purified rabbit acrosin (Specific activity = 63.7 U Per mg Protein) exhibited one major band (Mr = 36,000) on SDS‐polyacrylamide‐slab gel electrophoresis. Sheep werweantibodies, Preared against acid‐glycerol‐stabilized acrosin, were Fractionated sequentitally with 50 and 33% ammonium sulfate. Anti‐acrosin immunoglobulins were specific for the acrosome as evidenced by indirect fluorescence lebeling, and they inhibited acrosin's proteolytic activity when essayed for their effect on fertilization invivo, antiacrosin‐treated sperm produced the lowest persent fertility (7.1%) followed by preimmune y‐globulins(69%), Krebs‐ringer hosphate‐glucose‐serum(96.4%) Analysis of antibody‐treated sperm revealed no significiant chang in their motility or agglutination patterns. Antiacrosin antibodies, therefore, can effectivily neutrlize acrosin and inhibit fertilization when placed at the site of sperm‐egg interaction in vivo.
The sperm head of the plains mouse, Pseudomys australis: Ultrastructure and effects of chemical treatments
Authors: Flaherty Sean P. , Breed William G.
Published: February 2005

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.
Ribosomal preparations may induce maturation in Xenopus laevis oocytes
Authors: Baltus E. , Hanocq‐Quertier J.
Published: February 2005

Xenopus laevis oocytes undergo maturation when they are injected with large quantities of crude ribosomes from various origins: X laevis full‐grown or matured oocytes, Xenopus ovaries and embryos, Xenopus liver or mouse liver. All have the same efficiency, whatever their origin: they include 50‐90% maturation in the injected oocytes at about the same speed as progesterone treatment. The ribosomal preparations are inactive wen injected into recipient oocytes pretreated with cholera toxin or cycloheximide. After dissociation with the high salt extract, but not with the subunits. Hypotheses concernning the mode action of this ribosomal extract are disussed.
Biochemical studies of mammalian oogenesis: Synthesis of 5S and 4S RNA during growth of the mouse oocyte
Authors: Boreen Stuart M. , Gizang Elena , Schultz Richard M.
Published: February 2005

The relative proportion of synthesis of 5S RNA to 5S plus 4S RNA is constant during growth of the mouse oocyte. In conjunction with results of a previous study [Brower et al, 1981], the synthesis of 28S, 18S, 5S, and 4S RNA is coordinate during mouse oocyte growth.
Relationship between the length of sperm preincubation and zona penetration in the golden hamster: A scanning electron microscopy study
Authors: Barros C. , Jedlicki A. , Bize I. , Aguirre E.
Published: February 2005

Hamster spermatozoa are able to fertilize a high percentage of zona‐intact hamster oocytes when they are preincubated for 2 hr in a chemically defined medium. From this time on, the longer the preincubation time the lower the percentage penetration. Spermatozoa preincubated for 6 or more hr are unable to cross the zona pellucida, retaining however their ability to fuse with zona‐free hamster oocytes.

Zona‐intact hamster oocytes, as described above, were observed with the scanning electron microscope. When the oocytes were inseminated with spermatozoa preincubated for 1 to 5 hr the outer surface of the zona showed the penetrating spermatozoa and the sperm tracks made by those that failed to cross it. With longer preincubation times no penetrating spermatozoa were observed, and very few sperm tracks were present on the outer surface of the zona.

Control experiments showed that neither eggs, spermatozoa, nor fertilization were affected by the medium recovered after long preincubations.

These results show that care should be taken regarding the preincubation time when using the in‐vitro fertilization technique.
Ultrastructural studies of the porcine zona pellucida during the solubilization process by Li‐3,5‐diiodosalicylate
Authors: Dietl J. , Czuppon A. B.
Published: February 2005

Isolated porcine zonae pellucidae were investigated using transmission electron microscopy. It was found that the fine structure of the zona is not homogenous. The region near the oocyte consists of a more tightly packed micellar structure than the structure near the external surface. However, no clear boundary between the two structural features could be detected. The assumption that the molecular structure of the external and internal surface of the zona is not the same is substantiated by the finding that antibodies against the whole zona structure react apparently only with the more loosely bound structure on the external surface. This effect is attributed to differences in the spacial arrangements of the micellar structures rather than to differences in chemical composition. Solubilization of the zona results in a reorientation of the otherwise randomly interconnected structure. At lower Li‐3,5‐diiodosalicylate concentration (0.05 M) the fibrils seems to expand so that the individual fibers lie almost parallel to each other. At a higher Li‐3,5‐diiodosalicylate concentration (0.2 M) the individual micelles begin to break up from the zona surface, while at a still higher concentration (0.3 M) the rigid structure of the zona is completely solubilized. In the latter case no residual material could be detected in the sediment following high speed centrifugation of the mixture. These electron microscopic results correlated with the protein concentrations in the supernatant indicating that the maximal protein content (35 ng/zona) is obtained at 0.3 M or higher Li‐3,5‐diiodosalicylate concentrations.
The onset and maintenance of hyperactivated motility of spermatozoa from the mouse
Authors: Cooper T. G.
Published: February 2005

Estimates were made of the proportion of freely motile mouse spermatozoa displaying hyperactivated motility by an objective photographic method employing stroboscopic illumination under dark‐field conditions and examining displacements of the sperm head and bend angles of the sperm tail. In media known to support in vitro fertilisation hyperactivation gradually appeared reaching about 40% by 6 hr incubation, and it was not promoted by 2 mM caffeine or 0.1 mM Bt2 cAMP or washing the cells free of epididymal fluid. Raising the osmolarity of the medium to 400 mOSM with electrolytes, but not nonelectrolytes, did promote hyperactivation (60% by 2 hr) suggesting that the ionic strength of the medium was important. Hyperactivation in high ionic strength media could be prevented by removing or chelating Ca2+, or replacing Ca2+ with Ba2+ or Mg2+, when nonhyperactivated motility was maintained, but Sr2+, like Ca2+, permitted hyperactivated motility. Hyperactivation in low ionic strength medium could be promoted by the ionophore A23187, suggesting that Ca2+ movement into the cells is important. Of a range of glycolytic substrates tested supporting nonhyperactivated motility in the presence of lactate, only glucose supported hyperactivation. Addition to glucose— or Ca2+ — free, high ionic strength media after 2 hr increased hyperactivation immediately (glucose) or after a lag of 2 hr (Ca2+) suggesting that glucose acts on a Ca2+ — primed system. Removal from high ionic strength medium, chelation of Ca2+ or inhibition of glucose metabolism did not prevent hyperactivation continuing once it had been initiated, indicating different requirements for initiation and maintenance of this form of motility.
Culture of mouse preimplantation embryos as a quality control assay for human in vitro fertilization
Authors: Ackerman Steven B. , Swanson R. James , Stokes Gordon K. , Veeck Lucinda L.
Published: February 2005

Techniques for culturing preimplantation mouse embryos from the two‐cell stage to blastocyst are described, and the importance of this system to quality control assay the media and supplements used in human in vitro fertilization (IVF) procedures is discussed. Embryos from B6CBAF1mice were cultured in a commonly used mouse culture medium, modified Krebs‐Ringer‐bicarbonate (Krebs'), or in a commonly used human culture medium, Ham's F10 nutrient mixture supplemented with human fetal cord serum (FCS), and results were not significantly different. Using the mouse embryo culture system, tests on 174 preparations of FCS resulted in 24.1% producing less than 75% morula or blastocyst stages after 72 h in culture, compared to 9.5% of the Krebs' control cultures. Results of the mouse embryo culture system using 98 FCS subsequently used in human IVF were compared to results from the VIP Human In Vitro Program of Eastern Virginia Medical School of Norfolk, Virginia, from June 1982 through June 1983. The data suggest that prescreening of media and supplements using this mouse embryo culture system may indicate sources of factors potentially detrimental to the success of human IVF procedures.
A thin‐layer isoelectric focusing method for the separation of proteins in follicular fluid and seminal plasma
Authors: Nayudu P. L. , Gook D. , Lopata A. , Cornell F. N. , McLachlan R.
Published: February 2005

A thin‐layer electrofocusing method has been developed for the separation of proteins in human follicular fluid, seminal plasma, and blood plasma/serum. The method has a number of unique features. It is simple and rapid, and it produces distinct, undistorted bands that can be readily quantitated. Any samples solubilized by standard methods can be analyzed, and it can be adapted to favor specific regions of the pH spectrum. The gels used are composed of agarose and sorbitol, and they are formed between a plastic template and a Mylar film. Sample volumes ranging from 0.5 to 5 μl can be used. Protein separation is usually completed in 30–40 min at constant power of 1 or 1.5 W. The use of low power ensures that little heat is generated during the run and hence only minimal temperature control is needed. Carbon rod electrodes are a special feature as they allow the inverted gel to rest in direct contact with the electrodes, eliminating the necessity for electrode solutions. The inverted gel position allows fluid to drain from the surface of the gel and so prevents distortion of protein patterns. This overcomes the problems inherent in conventional horizontal electrofocusing of unpurified biologic fluids such as plasma, follicular fluid, and seminal plasma. Rapid protein staining is carried out with fixed and dried gels. The excellent resolution and the lack of distortion make this method suitable for reliable quantitative analysis of differences between samples by means of laser beam densitometry. This has enabled us to assess differences in specific follicular fluid proteins between individual follicles in the human, and to correlate these differences with fertilizability and cleavage potential of oocytes derived from such follicles.
Architecture of the hamster oocyte‐cumulus complex
Authors: Talbot P. , DiCarlantonio G.
Published: February 2005

The structure of the hamster oocyte‐cumulus complex (OCC) has been analyzed to help understand how mammalian sperm penetrate the investing coats of the oocyte. At ovulation, oocytes of most mammalian species are surrounded by a zona pellucida, corona radiata, and cumulus layer. Cells of the cumulus and corona radiata are separated by an extracellular matrix (ECM) that contains hyaluronic acid. In hamsters, the diameter of mature follicular OCCs is 0.61 ± 0.12 mm, whereas in freshly ovulated OCCs in culture medium it is 0.78 ± 0.15 mm. This indicates the OCC expands at or after ovulation. The corona radiata is 1–4 cell layers deep, and corona radiata cells are closely packed (center‐to‐center distances between adjacent cells in follicular OCCs averaged 14 ± 3 μm). The cumulus layer is 5–8 cell layers deep, and intercellular spaces are much larger (center‐to‐center distances between adjacent cumulus cells in follicular OCCs averaged 50 ± 20 μm). An ovulated OCC has an approximate volume of 248 × 106 μm3 and was estimated to contain approximately 5,700 cells. Studies with stretched OCCs show that ink particles can readily penetrate the extracellular spaces of the cumulus and corona radiata layers and interact with the ECM, staining it black. At the periphery of the OCC, the ECM appeared discontinuous and formed “cords” containing cumulus cells, whereas closer to the corona radiata the matrix completely filled intercellular spaces. Ink penetrated the corona radiata, but the ECM was difficult to visualize because of the close packing of cells in this layer. Our observations on unfixed OCCs show that cell packing becomes more dense proceeding from the periphery of the OCC to the zona pellucida and that the ECM at the periphery differs from the matrix deeper in the OCC. These data suggest that an incoming sperm would find penetration of the investing coats to increase in difficulty (that is physical resistance would increase) as it got closer to the oocyte. We have also examined the effects of processing for microscopy on the structure of the OCC. Both standard fixation procedures and fixation in the presence of ruthenium red caused condensation of the ECM, especially in the cumulus layer, and thus produced a significant decrease in the diameter of the OCC.
Induction of the acrosome reaction in the spermatozoa of the goat by secretions of the male accessory glands and milk
Authors: Courtens J. L. , Nunes J. F. , Corteel J. M.
Published: February 2005

The balancing effects of bulbourethral gland secretion (BUS) and of seminal vesicle secretion (SVS) on goat semen quality were previously demonstrated. In the present study, electron microscope observations revealed a high frequency of spermatozoa with a reacted acrosome among spermatozoa from cauda epididymis exposed to BUS in the presence of milk. This frequency was significantly reduced when SVS had been added either before or after BUS. No reacted acrosome was observed in the absence of milk. All mount spermatozoa were incubated with milk or SVS or BUS or combinations of the three materials labeled with colloidal gold. SVS attached specifically on the plasma membrane covering the anterior part of the acrosome, whereas BUS spread all over the sperm head. Milk attached on the anterior half of the sperm head only when BUS was present in the sperm environment. It is concluded that BUS plays an active role in the induction of the acrosome reaction in the presence of milk and that SVS counteracts this role.
Evidence of phospholipase A2 in the sea urchin egg: Its possible involvement in the cortical granule reaction
Authors: Ferguson James E. , Shen Sheldon S.
Published: February 2005

Fertilization of the sea urchin egg involves an exocytotic event known as the cortical granule reaction (CGR). In many cell systems, phospholipase A2 is implicated in regulation of the secretory event. Indirect evidence suggests that phospholipase A2 mediates the CGR; however, there has been no direct demonstration of phospholipase A2 activity in the sea urchin egg. We report here evidence of phospholipase A2 activity in egg homogenate of the sea urchin Lytechinus pictus. The enzyme was calcium‐dependent and had a pH optimum near the intracellular pH of the unfertilized egg. Neither exogenous calmodulin nor trifluoperazine had any apparent effect on enzyme activity. Quinacrine, a phospholipase A2 inhibitor, blocked the enzyme activity in the egg homogenate. In intact eggs, quinacrine blocked the CGR in a dose‐dependent, egg‐concentration‐dependent manner. The inhibitory effect of quinacrine on the CGR could not be overcome by the phospholipase A2 activator melittin or by the calcium ionophore A23187. Quinacrine did not inhibit sperm‐egg binding or sperm incorporation. These results lend further support to the hypothesis that phospholipase A2 is involved in the CGR.
Fertilization in a Crab. III. Cytodifferentiation of vesicles enclosing ring‐shaped elements involved in the cortical reaction
Authors: Goudeau Marie
Published: February 2005

During the cortical reaction, Carcinus maenas eggs successively released a fine granular material and a massive amount of ring‐shaped elements that subsequently formed most of the fertilization envelope. The ring‐shaped elements came from egg cortical vesicles and, owing to their striking morphology, acted as naturally occurring markers in ultrathin sections, which permitted us to understand the pathway of their intracellular transport. In this respect it was established that the ring‐shaped elements and their enclosing vesicles originated in the endoplasmic reticulum both in ripe oocytes and early fertilized eggs maintained under in vitro conditions. The intracellular transport pathway of the endoplasmic reticulum‐derived vesicles seemed to bypass the Golgi apparatus. Accordingly, the ring‐shaped elements appeared to be released by direct exocytosis from the endoplasmic reticulum system.

Finally, a tentative scheme of oocytes functioning is suggested for crustacean decapods, based on the remarkable similarities between the structure and ER origin of the ring‐shaped elements involved in the cortical reaction and the disc‐shaped granules traditionally considered as endogenous yolk precursors. The scheme implies that the oocyte ER system might produce a precursor common to the cortical reaction exudate and to the endogenous yolk, in the form of the ring‐ or disc‐shaped elements.
The karyotype and ultrastructural characteristics of spontaneous preimplantation mouse parthenotes
Authors: Anderson Everett , Hoppe Peter C. , Lee Gloria S.
Published: February 2005

Karyotypic and light and electron microscopical analyses were made of spontaneous preimplantation mouse parthenotes from the LT/Sv inbred strain. It was found that the activated oocyte and developing embryos were diploid. We believe that diploidization is achieved by the oogonium undergoing a premeiotic mitosis without cytokinesis followed by two meiotic divisions, thus producing diploid parthenotes.

The developmental events with respect to membrane specialization, such as junctional complexes, were similar to those observed in fertilized embryos.

A unique feature of the developing parthenote was the failure of the mitochondria to change during the morula stage. The mitochondria retained a few irregularly oriented cristae rather than many transversely oriented ones observed in morulae developing from fertilized eggs. The significance of this observation is discussed.
The interactions between spermatozoa and uterine epithelium in the hibernating bat, pipistrellus kuhli natt
Authors: Andreuccetti Piero , Angelini Francesco , Taddei Carlo
Published: February 2005

In hibernating females of Pipistrellus kuhli, as well as in other bats previously described, the spermatozoa inseminated in autumn are stored in the uterus, both free and attached by their heads to the uterine epithelium, throughout winter.

With the electron microscope the apical region of the attached sperm heads appears to be contained in plicae of the endometrial cells. Furthermore, a great number of microvilli protruding from the uterine epithelium participate in the interaction with spermatozoa. In the region of contact between the two cell types, their plasma membranes run parallel with a constant 12‐nm gap. The sensitivity of these interactions to EDTA and hyaluronidase suggests an involvement of Ca++ and proteoglycans in these bindings, while their insensitivity to trypsin tends to exclude a role of proteins.

In addition, evidence of exocytosis of dense granules just at the bottom of the plicae containing the spermatozoon suggests a transfer of material from the uterine epithelium to the intercellular space, thus supporting a physiological role of these interactions.
The effect of anoxia on fowl spermatozoa: Recovery of fertilizing ability, motility, and cellular concentrations of potassium and ATP
Authors: Wishart G. J.
Published: February 2005

Fowl semen when diluted in a glutamate‐based medium without glucose, gradually lost its fertilizing ability during 4 hr of anaerobic incubation at 30°C. This incubation regime offered a system by which various in‐vitro tests of spermatozoal viability could be assessed for their usefulness as monitors of fertilizing ability. Widely used tests such as spermatozoal enzyme leakage, dye exclusion, and morphology as assessed by light microscopy showed no change in spermatozoal status as the fertilizing ability declined. However the ability of sperm, during a short aerobic incubation to restore their motility and ATP and K+ concentrations, declined as did their fertilizing ability. When glucose was added to this re‐aeration medium, spermatozoal motility, K+ and ATP concentrations, and fertilizing ability were restored to optimal levels. Thus the fertilizing ability of fowl sperm, following anaerobic storage at 30°C, appeared to be related to their ability to restore ATP and K+ concentrations and motility. An initial event in the loss of fertilizing ability was a loss in the ability of sperm to oxidise endogenous substrates. This could be restored by the addition of glucose.
Rat eggs normally exhibit a variety of surface phenomena during fertilization
Authors: Battaglia D. E. , Gaddum‐Rosse P.
Published: February 2005

The surface topography of the rat egg was examined during fertilization in vitro and in vivo. Using phase optics, 348 in vitro fertilized and 50 in vivo fertilized eggs were continuously monitored throughout the 7‐hour period of sperm incorporation. A myriad of different surface configurations were seen, with each egg exhibiting one or more of the following changes. A small number of eggs (4–6%) formed surface elevations over the sperm head after its detachment from the flagellum, 15–30 min after sperm‐egg fusion; 1 to 1.5 hr after fusion, 40–50% of the eggs produced the so‐called incorporation cone, a prominent surface elevation over the decondensing sperm nucleus. The vast majority of eggs (74–82%) formed surface elevations over the proximal tip of the flagellum 2–3 hr after sperm‐egg fusion. These had no association with the decondensing sperm nucleus. A few eggs (11–12%) exhibited multiple protrusions that were distributed randomly about the egg surface, whereas 14–20% did not manifest any surface elevations and remained spherical throughout the sperm incorporation period. Regardless of the type of surface change, all of the eggs resumed a spherical shape by the time sperm incorporation was complete. These observations are in contrast to the conclusions by previous authors that formation of the so‐called incorporation cone over the decondensing sperm nucleus is a ubiquitous event.
The transport of morphologically abnormal sperm in the female reproductive tract of mice
Authors: Nestor Anne , Handel Mary Ann
Published: February 2005

Mice of the PL/J strain exhibit a high percentage of morphologically abnormal sperm and provide a model for studying the function of abnormal sperm. The ability of such sperm to reach the site of fertilization within the female reproductive tract has been investigated. We have found a decrease in the percentage of structurally abnormal sperm within the population that reaches the oviduct. This observation suggests either that there is an active selection against abnormal sperm or that they are physiologically disadvantaged in reaching the site of fertilization.
Possible involvement of a sialylated component of the sperm plasma membrane in sperm‐zona interaction in the mouse
Authors: Lambert Hovey , Van Le Anh
Published: February 2005

We have studied the molecular mechanisms of gamete interaction in vitro in the laboratory mouse, Mus musculus. In particular, we were interested in whether this interaction is similar to a lectin‐hapten‐mediated process. Inhibition of sperm‐zona binding was examined using various concentrations (.25 mM to 50 mM) of different sugars (sialic acid α‐methylmannose, glucose, fucose, galactose, and N‐acetyl‐glucosamine). Sperm‐zona binding was significantly decreased when eggs were pretreated with 10 mM of sialic acid or α‐methylmannose but not by other sugars tested. Furthermore, treatment of capacitated sperm with neuraminidase destroyed their ability to bind and fertilize eggs. We have also used a specific lectin for sialic acid from the horseshoe crab (Limulus polyphemus) to agglutinate mouse sperm. The lectin (.120 ng/ml) mediated agglutination of mouse sperm (105 sperm/ml) was routinely observed to increase from a 10% agglutination immediately following their isolation from the epididymis to 100% agglutination 90 minutes later. Collectively, these results suggest the appearance of specific sugar moieties on the surface of the sperm plasma membrane which, in this particular species of mouse, are sialylated glycoproteins acting as ligands for specific receptors on the surface of the egg. These are the first data to indicate that sperm‐egg recognition and attachment is a lectin‐hapten‐mediated process in the mouse.
Improved survival of zygotes in superovulated rats following surgical or pharmacological block of ovarian steroid secretion
Authors: Sherman D. , Shinnar Nili , Lelyveld Y. , Kraicer P. F.
Published: February 2005

The purpose of this study was to test the hypothesis that the preimplantation losses of zygotes in pregnant superovulated juvenile rats was due to an imbalance of ovarian hormones. Twenty‐seven or twenty‐eight‐day‐old rats were injected with 20 iu of PMSG, 25 iu of hCG 50 hr later and mated overnight. From a mean ovulation rate of 52 ± 2 only 16 ± 4 zygotes survived after 4 days. After ligation of the cervical ends of the uteri on the day following fertilization the mean yield of zygotes was 22 ± 6. Ovariectomy on the day of fertilization increased the yield of zygotes to 39 ± 6, but the recovery of the zygotes was seriously complicated by postoperative adhesions and deformations of the adnexa. Inhibition of steroidogenesis with aminogluthehimide phosphate also increased the yield of zygotes. The optimal dose was 45 mg in six divided doses over 3 days, which gave a mean recovery of 57 ± 3 zygotes (of which 75% were blastocysts), that is 100% salvage. A lower dose (30 mg) reduced the recovery to the level of untreated animals, while increasing it to 60 mg resulted in maternal mortality and morbidity, as well as in developmental retardation of zygotes.
Entry of immotile spermatozoa into zona‐free hamster ova
Authors: Williamson Roger A. , Koehler James K. , Smith W. Dianne , Karp Laurence E.
Published: February 2005

We studied six men whose spermatozoa were immotile and possessed a variety of sperm tail structural abnormalities by electron microscopy. The semen of all six subjects had a normal percentage of oval forms and sperm undergoing capacitation and acrosome reaction. Despite the absence of motility, when incubated sperm from these subjects was added to a microdrop of medium containing zona pellucida‐free hamster ova, sperm penetration or entry into the cytoplasm of from 1–9% of the eggs was evident with phase contrast microscopy. This latter finding suggests that, at least in this system, oocytes actively facilitate sperm incorporation. Penetration was absent when sperm of fertile men were rendered immotile, though still viable, by heat treatment.
Fertilization in crabs: IV. The fertilization potential consists of a sustained egg membrane hyperpolarization
Authors: Goudeau Henri , Goudeau Marie
Published: February 2005

The electrophysiological properties of immature and mature oocytes of two crabs were analyzed. Growing immature oocytes of Carcinus maenas and fully grown immature oocytes of Maia squinado had essentially K+ dependent resting potentials, Em, of −61 ∓ 1 mV, n=19, and −67.3 ± 0.5 mV, n=68, respectively. Fully grown immature oocytes of Carcinus maenas showed an Em of −40 ± 1.5 mV, n=19, that was k+ and Cl− dependent. In mature oocytes of both species, the plasma membrane became exclusively permeable to cl− and the Em attained–41 ± 1 mV, n=49 and −34 ± 1.5 mV, n=27 for Carcinus maenas and Maia squinado, respectively. After in vitro insemination, a dramatic increase in egg membrane permeability to K+ was observed. This instantaneously caused a sustained hyperpolarization constituting, for these crabs, the fertilization potential. We observed that concurrently with this electrical response to fertilization, sperm reinitiated the oocyte meiotic maturation previously arrested at the first metaphase. The triggering mechanism of the fertilization potential as well as the possible occurrence of a physiological polyspermy are discussed.
Acrosomal reaction and early events at fertilization in Marthasterias glacialis (Echinodermata: Asteroidea)
Authors: Sousa Mário , Azevedo Carlos
Published: February 2005

When the spermatozoon of M glacialis contacts the mature oocyte jelly it adheres to it. Following this, there is a slight tumefaction of the acrosome, which is followed by the disruption of the apical acrosomal vesicle and cytoplasmic membranes. Acrosomal vesicle contents are liberated and spread along the outer surface of the oocyte jelly. Meanwhile, the acrosomal process begins to extend, penetrates all the jelly extension, then the vitelline layer, and finally contacts the cytoplasmic egg membrane. Nevertheless, the sperm cell continues lying at the outer border of the jelly. From the beginning of the acrosome reaction the dense and finely fibrillar subacrosomal material is connected, by some expansions, to the basal acrosomal vesicle membrane. Both nuclear and mitochondrial diameters have diminished.
Inhibitors of glycoprotein biosynthesis block fertilization in the hamster
Authors: Ahuja Kamal K.
Published: February 2005

The nature and control of changes in surface carbohydrates in capacitating hamster spermatozoa were analysed by using five inhibitors of glycoprotein biosynthesis in an in vitro fertilization system. Epididymal spermatozoa were treated with amphomycin, bacitracin, tunicamycin, 2‐deoxyglucose, and 2‐deoxy‐2‐fluoro‐D‐glucose either during the entire period of capacitation or briefly at the end of capacitation before exposing to Con A‐coated agarose beads or hamster eggs with or without their zonae pellucidae. Untreated 4½‐5‐hr spermatozoa exhibited nearly 100% fertilization and became bound to Con A‐agarose beads mainly along the length of their flagellae, resulting in the formation of clumps on the beads. In the presence of inhibitors of glycosylation, spermatozoa did not bind to Con A‐agarose beads or zona‐intact oocytes and they did not fuse with the zona‐free oocytes. Sperm‐zona binding was also inhibited by UDP‐galactose and UDP‐N‐acetylglucosamine, but not by UDP‐glucose. Sperm motility was not damaged by these inhibitors, and zona‐intact and zona‐free oocytes pretreated with these inhibitors underwent normal fertilization with untreated spermatozoa. These results further strengthen the view that glycoproteins on the sperm surface may be required during different stages of fertilization, including sperm‐egg fusion.
Studies on the stability of 125I‐purified porcine zona antigen (PPZA) obtained by chloramine‐T glucose oxidase with lactoperoxidase and N‐bromosuccinimide lodination procedures
Authors: Subramanian Marappa G. , Yurewicz Edward C. , Sacco Anthony G.
Published: February 2005

A purified pig zona antigen (PPZA) was labeled with 125I by chloramine‐T (CL‐T), glucose oxidase with lactoperoxidase (GO‐LP), and N‐bromosuccinimide (N‐BS) iodination procedures. The 125I‐PPZA fractions thus obtained were reconstituted to a concentration of 25,000 dpm/0.1 ml and tested at weekly intervals for binding capacity to a fixed dilution (1:400,000) of PPZA antibody. The 125I‐PPZA obtained by the N‐BS procedure showed greater stability compared to that obtained by CL‐T and GO‐LP methods. The nonspecific binding levels for the N‐BS and GO‐LP methods were comparable and significantly lower than for the CL‐T method.
Dispersion of the cumulus oophorus and dissolution of the zona pellucida in oocytes of DDK and other strains of mice
Authors: Wabik‐Sliz Barbara
Published: February 2005

The time needed in vitro for granulosa cell dispersal with hyaluronidase and for lysis of the zona pellucida with α‐chymotrypsin was estimated for unfertilized eggs from three inbred strains of mice, DDK, C57BL, and C3H, and one outbred strain, Q. Granulosa cells were dispersed most rapidly in the DDK and most slowly in the C3H strain.

The time needed for dissolution of the zona pellucida was also shortest for DDK eggs and longest for C3H eggs. The results suggest that the high sensitivity to enzymes of the granulosa layer and the zona pellucida of DDK eggs may allow more than one spermatozoon to penetrate through these barriers before the block against polyspermy is completed. The percentage of unfertilized eggs containing spermatozoa under the zona pellucida on the day of copulation was abnormally high in matings of females from the DDK inbred strain with males from DDK and C57BL strains. This may suggest some disturbances in establishing contact between the egg plasma membrane and fertilizing spermatozoa.
Separation of mouse testis cells on a Celsep™ apparatus and their usefulness as a source of high molecular weight DNA or RNA
Authors: Wolgemuth Debra J. , Gizang‐Ginsberg Elena , Engelmyer Eric , Gavin Brian J. , Ponzetto Carola
Published: February 2005

The use of a self‐contained unit‐gravity cell separation apparatus for separation of populations of mouse testicular cells is described. The apparatus, a Celsep™, maximizes the unit area over which sedimentation occurs, reduces the amount of separation medium employed, and is quite reproducible. Cells thus isolated have been good sources for isolation of DNA, and notably, high molecular weight RNA.
Changes in guinea pig sperm intracellular sodium and potassium content during capacitation and treatment with monovalent ionophores
Authors: Hyne R. V. , Edwards Kim P. , Lopata A. , Smith J. D.
Published: February 2005

Guinea pig spermatozoa were collected from the caudae epididymides in various isotonic solutions and the intracellular sodium and potassium content was determined by atomic absorption spectroscopy. The sperm intracellular Na and K content was found to be influenced by large variations in the extracellular concentrations of these ions. Treatment of spermatozoa suspended in a saline‐based solution with the monovalent ionophores monensin or nigericin caused an approximate 2‐fold increase in the intracellular Na content and a 3–6 fold decrease in the intracellular K content. Incubation of the spermatozoa in a K+‐free minimal culture medium (MCM‐PL) at a pH of 7.6 or 8.3 for 2 hr caused an approximate 2‐fold increase in the sperm intracellular Na content and a 5‐fold decrease in the intracellular K content. The motile spermatozoa incubated for 2 hr at pH 7.6 showed less than 5% acrosome reactions, compared with 30–40% acrosome reactions after incubation at pH 8.3, in response to the addition of 5 mM Ca2+. Changes in the sperm intracellular elemental composition during culture in vitro, which may lead to an acrosome reaction, are discussed.
Scanning electron microscope study of human cervical mucus
Authors: Barros C. , Argüello B. , Jedlicki A. , Vigil P. , Herrera E.
Published: February 2005

The ultrastructure of human cervical mucus was studied by scanning electron microscopy. In fertile women, naturally occurring midcycle cervical mucus showed an arrangement of parallel fibers oriented along the main axis of the mucus sample. Sperm migration through a column of cervical mucus in vitro was also studied. It was found that sperm present among the longitudinal fibers were oriented parallel to the them and to the main axis of the mucus sample.
–SH and S–S involvement in Chlamydomonas mating
Authors: Forest Charlene L.
Published: February 2005

Reagents that block or cross‐link sulfhydryl (–SH) groups and those that reduce disulfide (S–S) bonds have been tested for their effects on mating in Chlamydomonas reinhardii. Wild‐type (wt) gametes of mating type + (mt+) and mt−, and a fusion‐defective mt− mutant, gam‐11, were studied. Differential sensitivities of mt+ vs mt− and of wt mt− vs gam‐11 mt− were analyzed. Concentrations of reagents that did not disrupt flagellar agglutination, the first stage of the mating reaction, were generally used. Pretreatment of mt+ gametes with the membrane permeable –SH reducing agent dithiothreitol (DTT) inhibits flagellar sexual signaling at concentrations that do not inhibit any part of the mating reaction of mt− gametes. Wt mt− is more sensitive than wt mt+ to inhibition by low concentrations of p‐chloromercuribenzoate sulfonate (pCMBS), an organic mercurial. The membrane‐impermeable reducing agent, reduced glutathione (GSH), also preferentially inhibits wt mt−. Gam‐11 mt−, a fusion‐defective mutant, which has been used to study the sensitivity of the adhesion of the plasma membrane‐associated mating structures, is less sensitive to GSH and pCMBS inhibition that is wt mt−. DDT and pCMBS cause an increase in mating structure adhesion in pretreated gam‐11. The differential inhibition of pair and group formation during gam‐11 × wt mt+ matings has suggested a possible mechanism for mating structure adhesion.
Aneuploidy in male germ cells induced by alcohol ingestion
Authors: Alvarez Marvin R.
Published: February 2005

The effects of ethyl alcohol ingestion, at levels comparable to human drinking, have been evaluated by cytogenetic analysis of mouse spermatogonia and primary spermatocytes. The alcohol was administered intragastrically at different doses and for various periods of time.

An increase in mitotic and first division meiotic aneuploidy was observed in direct proportion to the alcohol dose. Approximately a three‐fold increase in mitotic and meiotic aneuploidy over control levels was observed in animals exposed to a daily 3 gm/kg bw dose for 6 to 28 days. The level of aneuploidy was found to return to control levels by 14 days after cessation of alcohol ingestion.

Analysis of the kinetics of decrease in aneuploidy following cessation of alcohol suggests that the primal event resulting in altered chromosome numbers occurs at a stage of the cell cycle other than anaphase and therefore is not due to nondisjunction nor to anaphase lagging.
Cholinergic modulation of progesterone‐induced maturation of Xenopus oocytes in vitro
Authors: Dascal Nathan , Yekuel Rivkah , Oron Yoram
Published: February 2005

Mixed and muscarinic cholinergic agonists (acetylcholine, carbamylcholine, methacholine, oxotremorine, and pilocarpine) accelerated in a dose‐dependent manner the progesterone‐induced maturation of Xenopus laevis oocytes. None of these agonists induced oocyte maturation in the absence of progesterone. The accelerating effect of cholinergic agonists was blocked in a dose‐dependent manner by specific muscarinic antagonists (atropine and scopolamine) but not by specific nicotinic antagonists (d‐tubocurarine and hexamethonium). The specific nicotinic agonist, dimethylphenylpiperazine, alone induced maturation in the absence of progesterone. The optimal promoting effect of acetylcholine was observed when oocytes were exposed to acetylcholine for 30 min, 5 min after the addition of progesterone, and was markedly better than when oocytes were exposed to acetylcholine throughout their incubation with progesterone. The effect of acetylcholine was observed in both follicle‐enclosed and in defolliculated oocytes, indicating that follicular cells were not the target of the cholinergic drugs.
Maturation of the human oocyte in vitro: Nuclear events during meiosis (an ultrastructural study)
Authors: Sathananthan A. Henry
Published: February 2005

States of meiotic maturation have been traced in human oocytes cultured in vitro from prophase I to telophase II, using electron microscopy.

Two hundred ten oocytes, aspirated at laparoscopy after appropriate hormonal stimulation, were cultured for 1–72 h and examined. Most oocytes were inseminated and the final phases of maturation were studied in fertilized ova.

The majority of oocytes had resumed meiosis and had matured to metaphase II. Some were arrested at metaphase I, and a few remained inactive at the germinal vesicle stage. Maturation of oocytes to metaphase II was achieved 3 h after laparoscopy; to telophase II, 3 h after insemination. Maturation of some oocytes to metaphase II may have occurred in the ovary.

Immature oocytes had eccentric nuclei containing dense fibrillar nucleoli. Corona cell processes formed complex junctions with the oolemma and a type of interaction analogous to neural transmission was observed. Maturing oocytes showed typical barrel‐shaped spindles, but no centrioles or asters. Chromosomes were attached to microtubules via kinetochores. Midbodies were elaborated in the ooplasm and determined the plane of cleavage of polar bodies. Some aspects of oocyte degeneration, aberrations of oocyte maturation, and consequences of aging are also reported.

The significance of cytoplasmic maturation that occurs concurrently with nuclear maturation is discussed in relation to assessment of oocytes for in vitro fertilization.
The action of gossypol on rat germinal cells
Authors: Baccetti Baccio , Bigliardi Elisa , Burrini Anna G. , Renieri Tommaso , Selmi Gloria
Published: February 2005

Adult male Sprague‐Dawley rats were fed daily with 25 mg/kg of gossypol acetic acid for 55 days. The examination of the testis revealed the constant integrity of the blood‐testis barrier. The earliest damage to the germinal cells appeared on the 14th day of treatment in spermatids in stage 18‐19, and consisted of mitochondrial swelling and cristae disorganization, which was manifested in an altered assembly of the mitochondrial helix and possible axonemal alterations. It appeared that the target cell was a 30–37‐day‐old spermatid that showed the first alterations 6–14 days later, when it reached stage 18. The mitochondrial and eventual axonemal damage was conserved after the migration into the epididymis, affecting 6% of the total sperm population on the 14th day of treatment and reaching the maximum (100%) on the 25th day of treatment. On the 5th day of treatment, epididymal spermatozoa revealed, moreover, the presence of other tail alterations: lack of half of the axonemal components (also in sperm with unaffected mitochondria), loss of midpiece plasma membrane, and breaking of the outer accessory fibers. The conclusion is that a direct action of gossypol on epididymal spermatozoa is superimposed on the action earlier exerted on the spermatids.