Human sperm-cervical mucus interaction and the ability of spermatozoa to fuse with zona-free hamster oocytes

Samples of semen and cervical mucus were provided by 18 couples. Cervical mucus was obtained for each day possible and stored at 4 degrees C until all the samples were collected. Flat capillary tubes were loaded with the mucous samples and spermatozoa from the husband's semen sample were allowed to migrate through the cervical mucus (3 cm column) into culture medium. The spermatozoa recovered after migration through cervical mucus were assayed in vitro with zona-free hamster oocytes. Control experiments were carried out using spermatozoa from the same semen sample but prepared by the swimming-up technique. Altogether, 557 eggs in the control group and 1236 eggs in the experimental group were analysed, and the results demonstrated that the % of sperm penetration, the mean number of sperm decondensations per penetrated egg and the mean number of spermatozoa adhering per egg all had higher values (P less than 0.05) for the control samples than for the experimental samples. We suggest that cervical mucus modifies human spermatozoa, as measured by their interaction with zona-free hamster oocytes.     

Dynamics of the global tyrosine phosphorylation during capacitation and acquisition of the ability to fuse with oocytes in human spermatozoa

Phosphorylation of tyrosine residues in cellular proteins represents a major event during sperm capacitaton, but its relationship with the acquisition of sperm-fertilizing ability is still unclear. In this study we explored the relationship between the kinetics of the global tyrosine phosphorylation, monitored with a flow cytometric assay, and the acquisition of the human sperm ability to fuse with oocytes, evaluated with the progesterone-enhanced hamster egg penetration test. Sperm tyrosine phosphorylation appeared to be an early event in the capacitation process, with a 3.6-fold mean increase within 1 h of capacitation, but at this time sperm-oocyte fusion was extremely poor compared with that observed at 5 h of capacitation. Capacitation in calcium-free medium produced a 2-fold mean increase in tyrosine phosphorylation compared with that seen in complete capacitation medium both at 1 h and 5 h of capacitation, whereas sperm-oocyte fusion significantly increased only at 1 h, remaining unchanged at 5 h of capacitation. The cAMP analog, N,2-O-dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP), prevented the inhibitory effect of seminal plasma on tyrosine phosphorylation but not on sperm-oocyte fusion. In conclusion, these results suggest that the acquisition of sperm-fertilizing ability is always associated with an increase of the global tyrosine phosphorylation, but tyrosine phosphorylation does not necessarily reflect the acquisition of the sperm-fertilizing ability. Flow cytometry assay, a reliable technique to quickly quantify the global levels of the human sperm tyrosine phosphorylation, could be useful for a further elucidation of the biological meaning of this process, with the perspective of its clinical use as a measure of the sperm-fertilizing potential.     

Effects of various enzymes on the ability of hamster egg plasma membranes to fuse with spermatozoa

To investigate which component of the plasma membrane of the hamster egg plays the central role in the sperm–egg fusion, the egg membrane was treated with a variety of proteolytic, carbohydrate-hydrolyzing, lipid-hydrolyzing, and other enzymes. The only enzyme that markedly effected the ability of the egg membrane to fuse with spermatozoa was phospholipase C. The lipid moieties of the egg plasma membrane (and possibly of the sperm membrane) must be of primary importance in sperm–egg fusion at fertilization.     

Affinity binding of hamster oviductin to spermatozoa and its influence on in vitro fertilization

The aim of this study was to determine the influence of hamster oviductal glycoprotein (Oviductin) on in vitro gamete interaction. Oviductin was purified from the oviducts using lithium 3,5-diiodosalicylate, followed by phenol extraction. Immunocytochemistry using indirect fluorescence staining revealed that oviductin binds to the sperm anterior acrosomal region. The specific binding of oviductin resulted in inhibition of in vitro fertilization in studies using cumulus-free oocytes. The inhibitory effect was dependent on the concentration of oviductin and occurred in both ovarian and oviductal oocytes but not zona-free oocytes, indicating that sperm-zona interaction was interferred by oviduction. However, the inhibitory effect of oviductin sperm-zona interaction was reduced when cumulus-enclosed oocytes from ovaries and oviducts were used, indicating that the egg investment including cumulus oophorus has some effect on oviductin-sperm complex and maintaining the fertilizing ability. © Wiley-Liss, Inc.     

The binding of sterol sulfates to hamster spermatozoa.

Spermatozoa obtained from the cauda epididymidis possess twice the ability to take up sterol sulfates in vitro when compared to sermatozoa obtained from the caput. This would suggest that a modification of the membrane composition of the spermatozoa occurs during passage through the epididymis. Free sterols are taken up in a similar pattern. Radioautographic studies reveal that, for sterol sulfates, this uptake occurs selectively in the regions of the head and mid-piece of the spermatozoa whereas the free sterols are distributed evenly throughout the length of the spermatozoa. The binding of sterol sulfates to spermatozoa appears to involve sites that are unsaturable. The possibility exists that sterol sulfates, previously implicated in membrane stabilization, may play a similar role in spermatozoa.     

Fertilizing ability in vitro of golden hamster spermatozoa after acute testicular X-irradiation]

The present study is concerned with the effect of radiation to the testis on fertilizing ability in vitro using golden hamster spermatozoa. Male hamsters at 6 and 8 weeks of age were given acute testicular X-irradiation (200 kVp, 20 mA, 0.47-0.48 Gy/min). Spermatozoa were collected from the cauda epididymides at different times after irradiation and then they were suspended in fertilization medium. After preincubation for 4-5 hr, the spermatozoa were cultured with the eggs collected from mature hamsters treated with PMSG-hCG. Fertilized eggs were examined for incidence of sperm penetration and formation of pronuclei at 4-5 hr after insemination. The fertilization rate (47.7%) at the 6th week after irradiation with a dose of 2 Gy was much lower in comparison with the control value (92.6%). However, the fertilization rates at the 3rd and 9th weeks after irradiation were 97.7 and 90.6%, respectively. In these period, no difference was found between the irradiated groups and the control groups. From the changes in sperm concentration after irradiation with a dose of 2 Gy, it was found that the fertilization rate was the lowest at the 6th week. The sensitive stage to radiation during spermatogenesis with reference to the reduction of fertilizing ability after irradiation coincides with that of decrease in the sperm concentration and sperm motility. The results of fertilization rate at the 6th week after different doses of X-irradiation (0.25-6 Gy) indicated that the reduction of fertilization rate is nearly expressed as a dose-response relationship.(ABSTRACT TRUNCATED AT 250 WORDS)     

The status of acrosomal caps of hamster spermatozoa immediately before fertilization in vivo

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.     

Relationship between heparin binding characteristics and ability of human spermatozoa to penetrate hamster ova

Heparin binding site affinity and density on human spermatozoa were compared between fertile and infertile men with normal or abnormal results in the zona-free hamster ova-sperm penetration assay (SPA). A portion of fresh semen from fertile donors and potentially infertile men was processed through the SPA while the remainder of the ejaculate was used to quantitate heparin binding on spermatozoa. Saturation binding assays with [3H]heparin (15-375 nM) were analysed for 3 groups of men: (1) infertile patients with abnormal SPA results, (2) infertile patients with normal SPA results and (3) fertile donors. The heparin binding site density was significantly higher in men who possessed normal SPA results (infertile men and fertile donors) than in infertile men with abnormal scores in the SPA. There was no difference in heparin binding affinity between the three groups. These findings suggest that the heparin binding-site density may be related to the ability of human spermatozoa to undergo successfully the acrosome reaction.     

The ability of cryopreservatives to prevent motility loss and freeze-thaw damage to the acrosome of chicken spermatozoa

Proteolytic membrane digestion and motility were used to determine the effects of cryopreservatives and freezing on acrosomal damage and survival of chicken spermatozoa. None of the cryopreservatives, glycerol, DMSO, or ethylene glycol caused a decrease in proteolytic membrane digestion by the chicken spermatozoon before freezing. After rapid freezing, high levels (16 and 24%) of glycerol prevented significant freeze-thaw reductions in proteolytic digestion. High levels of ethylene glycol (16 and 24%) and DMSO (12 and 14%) significantly reduced the ability of frozen spermatozoa to completely digest the protein membrane. Motility after freezing was highest (63%) in 16% glycerol.Glycerol at high levels appeared to be the best cryopreservative under these conditions.     

Double fertilization in maize: the two male gametes from a pollen grain have the ability to fuse with egg cells

In flowering plants, two male gametes from a single pollen grain fuse with two female gametes, the egg and central cells, to form the embryo and endosperm, respectively. The question then arises whether the two male gametes fuse randomly with the egg and central cells. We investigated this question using two nearly isogenic maize lines with supernumerary B chromosomes (TB10L18) or without (r-tester). B chromosomes regularly undergo non-disjunction at the second pollen mitosis, producing one sperm cell with zero B chromosomes and one with two. We first confirmed earlier studies showing an excess of transmission of the B chromosomes to the embryo rather than to the endosperm. We then tested the possibility of a directed fertilization. For TB10L18 pollen, we could demonstrate the existence of a size dimorphism between the two sperm cells, correlated to the content in B chromosomes, as detected by fluorescence in situ hybridization (FISH). However, no directed fusion of B chromosome containing sperm to egg cells could be detected when using in vitro fertilization. The absence of directed fusion in vitro could also be demonstrated for control lines. We conclude that both male gametes have the capacity to fuse with the egg cell in maize, although sexual reproduction results in a preferential transmission of supernumerary B chromosomes.     

CD9 protein appears on growing mouse oocytes at the time when they develop the ability to fuse with spermatozoa

CD9 is a member of the tetraspanin superfamily proteins and is the only protein on the mouse oocyte which is known to be indispensable in sperm-egg fusion. Here, using indirect immunofluorescence we show that CD9 appears on the oolemma during the early stages of the growth of the oocyte, when it measures 13-22 microm in diameter. When the oocyte reaches a diameter of 17-22 microm, the density of CD9 in its oolemma is similar to the density of this protein in the cell membrane of the fully grown secondary oocyte. The appearance of CD9 in growing oocytes correlates with the previously reported time of the acquisition of fusibility between the spermatozoon and the egg. Accordingly we propose that during oogenesis the development of the ability of the oolemma to fuse with sperm may be regulated by synthesis of CD9 by the oocyte.     

In-vitro development of the fertilizing ability of hamster epididymal spermatozoa after co-culture with epithelium from the proximal cauda epididymidis

The epididymides of adult male hamsters were surgically ligated at the junction of the distal corpus and proximal cauda regions. After 3 days, spermatozoa recovered from the distal corpus displayed greater progressive motility and head to head agglutination in capacitating medium than did those from intact controls, but had low fertilizing ability (3% fertilization rate) in vitro or in vivo. When these spermatozoa were incubated for 6 h with epithelial cells from the proximal cauda epididymidis, previously cultured for 3 days, they maintained motility and exhibited a significant increase in fertilizing ability (30% and 29% in vitro and in vivo respectively). The fertilizing ability of distal corpus spermatozoa incubated with 3-day-old cultures without androgens, or 8-12-day-old epithelial cells with fibroblast overgrowth, or without epithelial cells, remained low (5%). Increase in sperm fertilizing ability was associated with increased sperm binding to the zona pellucida in vitro. These results demonstrate that, under suitable culture conditions, the final stages in the development of hamster sperm fertilizing ability can be achieved in vitro. Factors secreted by cultured epithelium from the proximal cauda epididymidis are implicated in this maturation process.     

The nucleolus and its modifications during oogenesis of Torpedo marmorata

The structural organisation of the nucleolar apparatus during oogenesis of the spotted ray Torpedo marmorata was investigated. The observations showed that unlike other cartilaginous fishes, in T. marmorata the nucleolar apparatus was always represented by one or two conspicuous nucleoli, whose organization significantly changed during oocyte development. In the smallest follicles (follicles <300 μm in diameter) the nucleolus was made up of granular and fibrillar components, and actively incorporated ³H uridine; later it becomes more and more electron‐dense so in follicles of 400 μm in diameter its components and ³H uridine incorporation were no longer evident. These results indicate that in T. marmorata the nucleolar apparatus significantly changes and undergoes a possible impairment in rRNA synthesis. After nucleolus inactivation, the synthesis of rRNA may be substained by granulosa.     

Time requirement for capacitation of boar spermatozoa assessed by their ability to penetrate the zona-free hamster egg.

Boar spermatozoa were preincubated for various times in the isolated uterus and oviduct from a maturing gilt and used to inseminate zona-free hamster eggs. The proportions of eggs penetrated and activated were increased, and the interval between insemination and sperm penetration was shortened when the spermatozoa were preincubated for 4--5.5 h instead of 2--.5 h. Overall penetration rates were higher and sperm penetration occurred about 1 h earlier when the eggs were inseminated with spermatozoa preincubated in the uterus than in the oviduct. It is concluded that the change in ability of boar spermatozoa to penetrate zona-free hamster eggs is due to capcitation which requires 4--4.5 h and 5--5.5 h of preincubation in the isolated uterus and oviduct, respectively.