Studies on biological actions of sperm immobilizing antibody on human fertilization in vitro

Effects of sperm immobilizing antibodies on sperm penetration through human zonae pellucidae have been studied. Exposure of human spermatozoa obtained from fertile donors to seven serum samples with sperm immobilizing antibody impaired sperm penetration completely in six cases and incompletely in one case. During the course of treatment of a patient with circulating sperm immobilizing antibody by means of an in vitro fertilization and embryo transfer program, it was found that fertilization was completely blocked in the presence of the patient's serum, but three matured ova fertilized successfully when umbilical cord serum was used instead of autoserum from the patient. Furthermore, when spermatozoa were exposed to an IgG fraction of sera containing sperm immobilizing antibody, sperm binding and penetration were markedly inhibited. The spermatozoa, preincubated with sperm immobilizing antibody, showed penetrability across the zona pellucida. However, exposure of possibly capacitated sperm to the antibody completely blocked sperm binding to and penetration through the zona pellucida. These results suggest that sperm immobilizing antibodies cause infertility by preventina sperm binding to and penetration through the zona pellucida, possibly by interfering with the step of fertilization beyond sperm capacitation.     

Subcellular immunochemical localization of acrosin in human spermatozoa during the acrosome reaction and zona pellucida penetration

The changes in acrosin immunoreactivity in human spermatozoa undergoing spontaneous or chemically induced acrosome reactions were studied by electron microscopic immunocytochemistry with an acrosin-specific monoclonal antibody. Migration of limited amounts of acrosin to the sperm surface was the earliest event characterizing the beginning of the acrosome reaction. The acrosome of such spermatozoa remained morphologically intact, swelled, or showed intraacrosomal vesiculation without any disruption of the plasma and acrosomal membrane integrity. Massive release of acrosin coincided with the fusion of the plasma and outer acrosomal membranes. However, even fully acrosome-reacted spermatozoa always retained some acrosin on the exposed inner acrosomal membrane and in the equatorial segment of the acrosome. This residual acrosin was also detected on spermatozoa within the zona pellucida of human oocytes inseminated in vitro, while the previously released bulk of acrosin remained attached to the surface of the zona pellucida at the site of sperm entry. These findings are compatible with multiple functions of acrosin in human sperm-egg interaction, including sperm-zona pellucida binding, dispersal of acrosomal contents, and facilitation of zona pellucida penetration.     

The potential diagnostic use of human zona-free eggs prepared from oocytes that failed to fertilize in vitro

Human oocytes that failed to fertilize in a clinical in vitro fertilization (IVF) program were freed from the zona pellucida and reinseminated with spermatozoa from subfertile patients or from fertile donors. Penetration results were, in general, significantly worse when patients spermatozoa were used as compared with donor spermatozoa. When inseminated with donor spermatozoa, oocytes from cases of male infertility gave better penetration results as compared with those from cases of idiopathic infertility with no apparent sperm defect. No differences in penetration results were found between oocytes aged 1 day and 2 days in culture before zona removal and reinsemination, even though less pronuclei reached full functional maturity in the more aged group of eggs. These results indicate that human zona-free eggs prepared from oocytes after unsuccessful IVF attempts may be used to assess the fertilizing ability of human spermatozoa. Moreover, when used with donor spermatozoa, this system may help to determine the contribution of the oocyte to fertilization failure.     

In vitro fertilization in the rat: observations on living eggs

The purpose of this study was to observe and record some of the key events during in vitro fertilization in the rat. Freshly ovulated eggs were incubated with epididymal spermatozoa at 37 degrees C and removed 4.5 to 7.5 hours later for microscopic examination. The head of the fertilizing spermatozoon penetrated the zona pellucida with its long axis perpendicular to the zona: this orientation was maintained during subsequent incorporation into the vitellus. Sperm motility was drastically reduced soon after sperm-egg fusion. Simultaneously, the flagellum, most of which was still outside the zona, assumed a characteristic curved posture. Time-lapse cinematography demonstrated that the vitellus frequently underwent surface movements during the tail incorporation process, suggesting that its cortex was undergoing significant changes. This study presents the first long-term observations on the fertilization of living rat eggs in vitro.     

Effects of human seminal plasma on fertilizing capacity of human spermatozoa.

Using zona-free hamster eggs and salt-stored human eggs for assessing the fertilizing capacity of human spermatozoa, the effects of human seminal plasma on fertilizing capacity of human spermatozoa were investigated. The persistent presence of seminal plasma prevented sperm attachment to and penetration into the zona pellucida and vitellus. A series of experiments with zona-free hamster eggs revealed that, once the spermatozoa were preincubated in a seminal plasma-free environment known to induce the acrosome reaction, the seminal plasma no longer interfered with sperm-egg fusion. The native seminal plasma appears to interfere with both the acrosome reaction and vigorous motility of the spermatozoa, and this could be the reason fertilization fails when the plasma is consistently present around the spermatozoa. The fertilization-disturbing factor or factors in the seminal plasma appear to be macromolecular substances or substances associated with macromolecules.     

Blocking effect of sperm immobilizing antibodies on sperm penetration of human zonae pellucidae

To investigate the mechanism of the blocking effect of sperm immobilizing antibodies on human fertilization, an in vitro zona penetration test was carried out using media containing the IgG fraction extracted from sperm immobilizing antibody-negative or-positive serum. The sperm penetration rate of the test was 100% (6/6) when spermatozoa were treated with the IgG fraction derived from sperm immobilizing antibody-negative serum, whereas it was only 17% (1/6) when spermatozoa were treated with the IgG fraction derived from sperm immobilizing antibody-positive serum. Electron microscopic observation of the sperm immobilizing antibody-negative and-positive serum-treated spermatozoa showed that the number of acrosome-reacted spermatozoa was significantly greater in the sperm immobilizing antibody-negative serum than in the antibody-positive serum. Therefore, it appears that one of the blocking mechanisms of the spermatozoal penetration of the zona pellucida by sperm immobilizing antibodies may be due to inhibition of the acrosome reaction in the spermatozoa.     

Oocyte-sperm interaction in the course of IVF: a scanning electron microscopy analysis

Interaction between the spermatozoon and the zona pellucida during the first steps of fertilization was analysed on approximately 500 polyploid and unfertilized IVF oocytes by scanning electron microscopy (SEM). Oocytes demonstrate high inter- and intra-individual variations in size and morphology which do not correlate either with the maturity of the oocytes or with the age of the women. During gamete interaction, corona radiata cells are widely dispersed around the zona but are still in contact with it through cytoplasmic filaments. Channels between granulosa cells guide spermatozoa towards the zona. In the course of fertilization, different types of attachment of the spermatozoon to the oocyte occur. Most commonly, a flat, tangential attachment of the sperm head to the surface of the zona appears, which is then followed by intrusion into the zona in precisely this horizontal position. However, vertical binding with penetration by the tip of the head first also occurs. In oocytes where large, cluster-like numbers of bound spermatozoa are visible, vertical binding and penetration is the most usual position. In the process of gamete interaction, both spermatozoa and zona pellucida are actively involved. Spermatozoa, including their tails, which are attached to the zona, are overgrown by filaments of zona material. These filaments of the zona are made of granules, which are the basic components of zona material. After the removal of the zona pellucida by laser, the oolemma becomes visible. It is covered by microvilli of highly variable numbers. Between these microvilli, cortical granules are evident, and appear even before sperm penetration.     

Significance of D-mannose as a sperm receptor site on the zona pellucida in human fertilization

The role of monosaccharides in human fertilization was studied by testing their effects on penetration of spermatozoa into mature human oocytes (zona penetration test). When oocytes were pretreated with concanavalin A, wheat germ agglutinin, or Ricinus communis agglutinin-I at a concentration of 100 micrograms/ml, no spermatozoa were found to bind to or penetrate through the zona pellucida. Penetration of spermatozoa was restored when the zona pellucida pretreated with wheat germ agglutinin and Ricinus communis agglutinin-I were rinsed with N-acetyl-D-glucosamine (wheat germ agglutinin inhibitor) and D-galactose (Ricinus communis agglutinin inhibitor), respectively. Conversely, the blocking effect of concanavalin A on sperm penetration was not reversed by treatment with D-mannose (concanavalin A inhibitor). Furthermore, pretreatment of spermatozoa with D-mannose (50 mmol/L) completely inhibited sperm penetration through the zona pellucida. However, sperm penetration was clearly demonstrated when the zona pellucida was pretreated with D-mannose. These data suggest that D-mannose residues are essential in, or sterically closely related to, the sperm receptor site on the human zona pellucida.     

Is the sperm-binding capability of the zona pellucida linked to its surface structure? A scanning electron microscopic study of human in vitro fertilization

The structure of the zona pellucida and the early interactions between human oocytes and spermatozoa were investigated in an in vitro fertilization program. Thirty-five mature (preovulatory) oocytes, 10 immature oocytes lacking a germinal vesicle, and 11 atretic oocytes which had not undergone fertilization at 10–20 hr after insemination were studied by light and scanning electron microscopy. Observed through employment of these techniques, the zona pellucida showed two basically different patterns: a mesh-like, spongy structure having wide and/or close meshes; and a compact, smooth surface. The smooth-surfaced zona was most commonly seen in the cultured oocytes belonging to the immature and atretic groups. These observations seem to show that the spongy appearance of the zona pellucida is related mainly to oocyte development and maturity. In this study, greater numbers of penetrating spermatozoa were noted on oocytes showing the mesh-like zona, in contrast to the presence of a few sperm flattened against its surface or the frank absence of sperm associated with oocytes having the more compact, smooth zona. It is likely that the condensation of the outer aspect of the zona pellucida causes a disorientation of sperm-binding sites, which would probably result in markedly reduced binding and penetration capacity with spermatozoa. These changes might ultimately lead to impairment of in vitro oocyte fertilizability.Dedicated to Professor Johannes Lang on his 65th birthday.     

Generation of mouse oocyte monoclonal isoantibodies: their effects and those of antisperm monoclonal antibodies on in vitro fertilization

Monoclonal isoantibodies to mouse oocyte antigens were generated by modified hybridoma techniques similar to those described for mouse sperm monoclonals. Following isoimmunization with mouse oocytes and cell fusion, hybrid cells were cultured initially in a semi-solid medium containing methylcellulose. Seven to ten days after cell fusion about 350 hybrid clones were recovered for subculture. By an indirect immunofluorescence assay using frozen or fresh mouse oocytes, twenty hybridomas were shown to produce antibodies that bind to various oocyte components including antigens of the zona pellucida. However, they did not cross-react with mouse spermatozoa or lymphocytes.A system was established to evaluate whether monoclonal antibodies to gamete-specific antigens have any inhibitory effects on the fertilization of mouse oocytes in vitro. A monoclonal antibody against zona antigen(s), ME 56, was shown to block fertilization of mouse oocytes via the inhibition of sperm binding to the zona pellucida. On the other hand, three out of four antibodies reacting with mouse sperm acrosomes were also inhibitory to mouse in vitro fertilization, perhaps mainly due to the inhibition of sperm acrosomal reactions. Using a sodium dodecylsulfate gel/protein blot radioimmunobinding method, the molecular weight of zona antigen(s) that react with ME 56 was determined to be in the range of 95,000, whereas that of the acrosomal antigen(s) reacting with the fertilization-inhibiting antibody, MS 207, was about 30,000. The results of this preliminary study suggest that monoclonal antibodies to certain gamete antigens can be a valuable tool for the analysis of sperm-egg interactions during the fertilization processes.     

Inhibition of sperm penetration through human zona pellucida by antisperm antibodies

An in vitro penetration test using human spermatozoa, sera, and eggs stored in a highly concentrated salt solution was designed for examination of the effect of antisperm antibodies on the process of fertilization. Spermatozoa from a healthy fertile donor incubated in modified Biggers, Whiiten and Whittingham (BWW) medium containing 7.5% antisperm-antibody-negative serum, could penetrate through the zonae pellucidae of the stored eggs, but not when the spermatozoa from the same donor had been incubated in modified BWW medium containing 7.5% antisperm-antibody-positive serum. After the antisperm-antibody-positive serum was absorbed with washed spermatozoa, the sperm penetration was not blocked. Therefore, antisperm antibodies appear to block human sperm penetration through the human zona pellucida.     

Molecular events that regulate mammalian fertilization

Mammalian fertilization is the net result of a highly programmed sequence of molecular events that collectively result in the union of two radically different looking haploid cells, sperm and egg, to form a diploid zygote. For successful fertilization, sperm cells undergo continuous modifications during their formation in the testis, maturation in the epididymis, and capacitation in the female genital tract. Only capacitated acrosome-intact spermatozoa are capable of binding to the egg's extracellular coat, the zona pellucida (ZP) in a receptor-ligand manner. The species-specific irreversible binding of the opposite gametes elevates intrasperm Ca2+ and triggers a signal transduction cascade that results in the fusion of the sperm plasma membrane and outer acrosomal membrane at multiple sites (i.e., induction of the acrosomal reaction) and the secretion of acrosomal contents. The hydrolytic action of the acrosomal enzymes (i.e., glycohydrolases, proteinases etc.) released at the site of sperm-egg binding along with the hyperactivated beat pattern of the bound spermatozoon, are important factors that regulate its penetration of the ZP and fertilization of the egg. In this article, we intend to discuss data from this and other laboratories that provide useful insights into biology underlying sperm development in the testis, maturation in the epididymis, capacitation in the female genital tract, sperm-egg interaction, and induction of the acrosome reaction (AR) before the acrosome reacted sperm can fertilize an egg. Our intention is also to discuss how Ca2+ signaling cascades regulate sperm functions and male fertility. Finally, we will discuss sperm molecules that are under intensive research to regulate male fertility.     

人精子甘露糖受体的表达与精卵融合的关系

目的:研究人精子甘露糖受体(MR)的表达与精卵融合的关系。方法:正常人活精子获能培养后与去透明带金黄地鼠卵行精子穿透试验(SPA)监测精卵相互作用,同时用异硫氰酸荧光素偶联的甘露糖化牛血清白蛋白(FITC-DMA)检测精子甘露糖受体的表达并分析SPA各项指标与精子甘露糖受体表达的关系。结果:SPA指标中穿透指数、结合指数均与精子MR表达率呈正相关。结论:人精子MR与精-卵膜融合密切相关,可能起某种介导作用。     

Zona pellucida penetrability of metaphase I and II human oocytes after aging and salt treatment

Human oocytes that failed to fertilize in vitro were examined for the degree of meiotic maturity and reinseminated either in the living state or after treatment with a concentrated salt solution as used for storage of human zonae pellucidae for diagnostic tests of sperm function. No dependence of zona pellucida penetrability on the oocyte maturity status was observed in either treatment group. Zonae pellucidae of metaphase I and metaphase II oocytes were equally penetrable after the salt treatment, whereas penetration occurred only exceptionally when the oocytes were living. These results show that, after in vitro aging and subsequent salt treatment, eventual differences in oocyte maturity status will not influence results of zona pellucida penetration tests. On the other hand, successful penetration of salt-stored zonae pellucidae by spermatozoa does not necessarily imply that these spermatozoa are able to penetrate the zona pellucida of a fresh living oocyte.     

Spermiologic aspects of in vitro fertilization

Some publications of the last three years, which deal with the influence of the spermatozoa on the success rates of the in vitro fertilization (ivf) were analysed: 1. Semen samples with 10(7) spermatozoa per ml and more than 30% motile and normally configurated sperm cells are regarded as the lower limit of the usability. 2. Microbiological investigations, determination of the concentration of white blood cells, assessment of the motility during an in vitro incubation, evaluation of the acrosomal enzymes, the resistance of the spermatozoa to osmotic stress and the ability of the penetration of pre-ovulatory mucus represent additional criteria for the selection of semen samples. 3. The correlation between the results of the penetration test of the zona pellucida-free hamster oocytes and of the ivf is discussed. A lower limit of the conventional semen parameters concerning a success of the ivf is not detectable by this test. 4. The applied method of the separation of the spermatozoa should depend on the sperm quality. "Swim up"-method led to the best rates of fertilization. 5. Improvement of the male conditions for the ivf is possible. 6. The infertility caused by antisperm-antibodies is an accepted indication of ivf in contrast to oligozoospermia. 7. The fertilization of the oocyte by the spermatozoon prerequisites several spermatozoal alterations (capacitation, acrosome reaction), which are presented in this article in parts.     

Effect of aging and cold temperature storage of hamster ova as assessed in the sperm penetration assay

The penetration of zona pellucida-free hamster ova by human spermatozoa has been used to quantitate sperm penetration potential. However, since mammalian eggs in vitro have limited viability, the effect of in vitro aging on the ability of hamster ova to be penetrated by human spermatozoa was examined. Zona-free ova maintained at room temperature (25 degrees C) lost their ability to be subsequently penetrated with a half-life of 50.1 +/- 8.8 minutes. This was partly the result of removing the zona pellucida by trypsin digestion, since zona-free oocytes in the presence of trypsin inhibitor or zona pellucida-intact oocytes had half-lives of 99.1 +/- 15.2 and 120.5 +/- 17.4 minutes, respectively. Reduction in penetration rates associated with ovum aging did not appear to be due to loss of viability and could be completely prevented by maintaining the ova on ice (4 degrees C). In the presence of TEST-yolk buffer at 4 degrees C, ova retained (100%) their ability to be penetrated for up to 24 hours and were morphologically indistinguishable from fresh ova. These observations show that ovum aging in vitro at 25 degrees C is much greater than previously anticipated. This may result in artifactually low and variable scores in the penetration bioassay.     

Inhibition of mouse in vitro fertilization by an antibody against a unique 18-amino acid domain in the polysulfate-binding domain of proacrosin/acrosin

OBJECTIVE: To determine the contribution of the polysulfate-binding domain (PSBD) of acrosin during sperm penetration. DESIGN: To inhibit the in vitro fertilization of mouse zona-intact oocytes by using a polyclonal antibody raised against an 18-amino acid peptide of proacrosin (anti-PSBD). SETTING: Unit of Reproduction and Development, Faculty of Biological Sciences, Pontifical Catholic University of Chile. PATIENT(S): None. INTERVENTION(S): A polyclonal antibody against the 43IFMYHNNRRYHTCGGILL(60) peptide was raised in New Zealand female rabbits. The specificity of the antibody was evaluated by an ELISA. Zona-intact mouse oocytes were coincubated with capacitated spermatozoa for 3 hours in the presence of 0.63 mg/mL of the antibody or preimmune serum. As a control, we used zona-free mouse oocytes under the same experimental conditions. MAIN OUTCOME MEASURE(S): We evaluated the fertilization rate of zona-intact and zona-free mouse oocytes by phase-contrast microscopy. An oocyte was considered fertilized when at least one decondensed sperm head was found within the egg cytoplasm. We evaluated 50-60 mouse oocytes in each group in three independent experiments. RESULT(S): The anti-PSBD antibody inhibited the fertilization of zona-intact, but not zona-free, mouse oocytes, by capacitated spermatozoa. In addition, the binding of the anti-PSBD to proacrosin/acrosin in a solid-phase assay was inhibited in the presence of polysulfates (fucoidan). CONCLUSION(S): The anti-PSBD directed against the PSBD of proacrosin/acrosin inhibited the penetration of capacitated mouse spermatozoa through the zona pellucida. This antibody may be a useful tool to define the roles of the different domains of proacrosin/acrosin during gamete interaction.     

Treatment of sperm with high-ionic strength medium increases microsurgical fertilization rates of rabbit oocytes fertilized by subzonal placement of sperm

This study was conducted to investigate the requirement for sperm processing in microsurgical subzonal placement of sperm in rabbit oocytes. Fertilization rates with standard in vitro fertilization and microsurgical subzonal sperm placement were found to be similar (56 and 55%) when sperm treated with high-ionic strength Brackett's defined inedium to initiate capacitation were used. Statistically significant reductions in fertilization rates for both standard in vitro fertilization and subzonal placement were noted when twice-washed spermatozoa were used. Initiation of capacitation of spermatozoa results in higher fertilization results even when the zona pellucida is bypassed during fertilization.     

Sperm-zona pellucida interaction and immunological infertility

Immune reactions against gametes appear to be physiologically important for the maintenance of homeostasis in reproduction. In contrast, aberration of the immune homeostasis might give rise to ‘immunological infertility’. Antisperm antibodies cause infertility by blocking fertilization. The mechanism can be explained as inhibiting the acrosome reaction of sperm by their blocking effect on capacitation through inhibiting an increase of fluidity of the sperm membrane. Autoantibodies against zona pellucida also cause infertility by blocking sperm-zona pellucida interaction, though the definitive mechanism has not been elucidated. Pretreatment of spermatozoa with D-mannnose completely inhibited sperm penetration through, but not binding to, the zona pellucida. Furthermore, very rapid kinetics between sperm extracts and D-mannnose by a BIAcore apparatus suggest that a D-mannose ligand of the sperm surface is easy to bind to and dissociate from a D-mannose residue in the sperm receptor site on the zona pellucida. Thus, D-mannnose on the human zona pellucida might be an essential molecule acting as a second sperm receptor, through which sperm penetrate into the zona pellucida. Because these antibodies appear to not cause any deleterious clinical symptoms, sperm and zona pellucida antigens are promising candidates in the development of an immunocontraceptive.     

整合素配体玻连蛋白在人精子的表达及其与受精的关系

目的 :进一步研究整合素配体玻连蛋白 (Vn)在人精子的表达及其与受精的关系。方法 :选用 1 4例生育力正常的成年男性及 8例精液常规分析正常的不明原因不育男性患者的精液标本 ,液化后提取上游精子。精子体外获能后用兔抗人 Vn多克隆抗体及羊抗兔 Ig G-FITC行免疫染色。然后用流式细胞仪计数 Vn表达阳性精子百分数。部分获能精子同时与去透明带金黄地鼠卵行异种体外受精 (SPA)以检测其受精力 ,比较两组获能精子表面Vn表达阳性精子百分率及受精率差异并分析受精率与 Vn表达阳性的获能精子百分数之间的相关性。结果 :生育组与不育组获能精子 Vn表达水平分别为 2 1 .2 4± 1 1 .70 %与3.6 4± 3.2 7% ,不育组明显低于生育组 (P<0 .0 5)。生育组受精率大于 1 0 % ,不育组受精率小于 1 0 % ,符合划分生育力正常与异常的标准。所有标本 Vn表达阳性的获能精子百分率与精子受精率间具有相关性 (r=0 .476 )。结论 :人获能精子表面存在一定水平的整合素配体玻连蛋白表达 ;Vn参与受精过程 ;Vn表达异常可能与一些不明原因的不育有关