Human fertilization: epididymal hCRISP1 mediates sperm-zona pellucida binding through its interaction with ZP3.

Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm-ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm-egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein-protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved not only in sperm-egg fusion but also in the previous stage of sperm-ZP binding through its specific interaction with human ZP3.

Avances recientes en la preservación de la fertilidad en el paciente oncológico / Recent advances in fertility preservation of the oncologic patients

En ésta revisión se discuten las posibles intervenciones para proteger la fertilidad futura de aquellos pacientes oncológicos prepúberes o en la pubertad temprana. En el varón, además de la conocida técnica de crioconservación del semen, se proponen alternativas útiles para niños prepúberes y aquellos adolescentes y jóvenes en los que la enfermedad ha deteriorado significativamente la calidad del semen. Se discute la criopreservación de biopsias testiculares que podrán ser utilizadas para: transplante subcutáneo de tejido, auto y xenotransplante de células germinales y para espermatogénesis in vitro. En la mujer se examina la crioconservación de embriones y ovocitos y la conservación de biopsias de ovario destinadas al auto y xeno transplante y la maduración folicular in vitro. Estadísticas recientes indican que 1 de cada 650 niños sufrirá de alguna forma de cáncer y que aproximadamente el 70 por ciento resultará curado. Sin embargo, éste éxito terapéutico trae aparejada una fuerte incidencia de alteraciones graves de la futura función reproductiva, tanto en hombres como mujeres...

Evidence for the participation of beta-hexosaminidase in human sperm-zona pellucida interaction in vitro.

Mammalian sperm-zona pellucida (ZP) interaction is mediated by sperm lectin-like proteins and ZP glycoproteins. We have previously reported the participation of binding sites for N-acetylglucosamine (GlcNAc) residues in human sperm function, including sperm interaction with the ZP. Additionally, previous results from our laboratory suggested that some of these events may be mediated by the glycosidase N-acetylglucosaminidase (beta-hexosaminidase, Hex, in mammals). In this study, we report the possible participation of Hex in human sperm-ZP interaction. Human recombinant Hex (hrHex) was obtained by expression in a stable transfected CHO cell line. When the recombinant enzyme was present during hemizona (HZ) assays, the number of sperm bound per HZ was significantly reduced. The same result was obtained when HZ were preincubated with hrHex. Additionally, the presence of a Hex-specific substrate during the HZ assay produced the same inhibitory effect. These results suggest the participation of a sperm Hex in the interaction with human ZP in vitro.

Strontium supports human sperm capacitation but not follicular fluid-induced acrosome reaction.

The ability of strontium (Sr(2+)) to replace calcium (Ca(2+)) in maintaining human sperm function has still not been completely characterized. In the present study, acrosome reaction (AR) inducibility in response to human follicular fluid (hFF) was compared in spermatozoa incubated in either Ca(2+)- or Sr(2+)-containing media. Other events related to sperm capacitation, such as protein tyrosine phosphorylation and hyperactivation as well as zona pellucida (ZP) recognition under both conditions, were also analyzed. Spermatozoa incubated overnight in the presence of Sr(2+) were unable to undergo the AR when exposed to hFF. Nevertheless, when spermatozoa were incubated under this condition and then transferred to medium with Ca(2+), sperm response to hFF was similar to that of cells incubated throughout in the presence of Ca(2+). The sperm protein tyrosine phosphorylation patterns and the percentages of sperm motility and hyperactivation were similar after incubation in Ca(2+)- or Sr(2+)-containing media. Under both conditions, the same binding capacity to homologous ZP was observed. Similar results were obtained when EGTA was added in order to chelate traces of Ca(2+) present in Sr(2+) medium. From these results, it can be concluded that Sr(2+) can replace Ca(2+) in supporting capacitation-related events and ZP binding, but not hFF-induced AR of human spermatozoa.

Transport in vitro fertilization and intracytoplasmic sperm injection: results of a collaborative trial.

OBJECTIVE: To determine whether the use of a central assisted reproduction laboratory, with gamete transport to the facility (transport assisted reproduction), would decrease oocyte quality or performance in IVF-ET and intracytoplasmic sperm injection (ICSI). DESIGN: Retrospective clinical study. SETTING: Public and private fertility clinics. PATIENT(S): A total of 467 couples underwent transport IVF, whereas 108 underwent transport ICSI. A group of 60 couples underwent conventional IVF during the same period. All methods and protocols used were similar among centers. Oocyte pick-up was performed by ultrasound-guided vaginal puncture. INTERVENTION(S): Oocytes were transported under controlled conditions, from the site of follicular aspiration to a central laboratory. MAIN OUTCOME MEASURE(S): The fertilization and cleavage rates and clinical pregnancies were compared among the study populations. RESULT(S): The differences between the fertilization and cleavage rates of ova and the rates of clinical pregnancies produced by transport and conventional methods were not statistically significant. CONCLUSION(S): Gamete transport to a central laboratory was not harmful for oocytes or for the outcome of assisted reproduction. Transport makes the use of IVF and ICSI available to physicians who are not affiliated with an assisted reproduction program, reduces costs, and increases acceptability of the procedures to patients.

Glycosidic residues involved in human sperm-zona pellucida binding in vitro.

Glycosidic residues of the mammalian zona pellucida (ZP) are known to be involved in sperm binding, suggesting the presence of complementary carbohydrate binding sites on spermatozoa. However, in previous studies, in which sperm suspensions were incubated with monosaccharides, no inhibitory effect was observed. Results of studies in which sperm were treated shortly after swim-up suggest that the use of non-capacitated cells may explain the apparently conflicting results. In the present report, we studied the effect of preincubation of capacitated spermatozoa with different monosaccharides on their ability to bind to ZP. After 5 h under capacitating conditions, spermatozoa were incubated in medium with or without a monosaccharide, resuspended in fresh medium and used for hemizona (HZ) binding assay. When ZH were incubated with spermatozoa treated with N-acetyl-D-glucosamine, D-mannose, D-fucose, L-fucose or D-galactose, a significant decrease in the number of spermatozoa bound was observed (level of inhibition: 62, 58, 82, 68 and 48% respectively) while treatment of spermatozoa with D-glucose produced no inhibition. Sugar treatment neither altered sperm motility nor the rate of acrosome reaction. These results suggest that N-acetylglucosamine, mannose, fucose and galactose residues are involved in human sperm-zona pellucida binding in vitro.

A new predictive test for in-vitro fertilization based on the induction of sperm acrosome reaction by N-acetylglucosamine-neoglycoprotein.

Neoglycoproteins with N-acetylglucosamine residues (BSA-GlcNAc) induced specifically the acrosome reaction (AR) in human spermatozoa. Our objective was to investigate the relationship between this phenomenon and the invitro fertilization (IVF) rate. Sperm suspensions from IVF protocols were incubated with BSA-GlcNAc (t), using calcium ionophore (i) or medium alone (c) as positive or negative controls. When the normalized AR percentage ratio (STIM) (% ARt-%ARc):(%ARi-%ARc) was compared with fertilization rate in 31 couples from our IVF programme, a positive correlation was found (r = 0.46, P < 0.01). The fertilization rate in patients with STIM > or = 0.2 was higher than in non-responders (STIM < 0.2); 72 +/- 7% compared with 5 +/- 3%. The overall predictive value of this test for adequate fertilization rate (> 30%) was 87%, sensitivity 91% and specificity 78%. False positives were 9% and false negatives 22%. For successful fertilization rates (> 60%), the results were: overall predictive value, 84%; sensitivity 100%; specificity 64%. False positives were 23% and no false negatives were found. The results indicated that the induction of AR in human spermatozoa by GlcNAc-neoglycoproteins could be used to predict their fertilizing ability in vitro.

Participation of human epididymal sperm coating antigens in fertilization.

A polyclonal antiserum directed against human sperm coating proteins of epididymal origin (anti-KCl) was tested for its ability to alter sperm function. Spermatozoa from normal ejaculates were selected by swim-up separation and capacitated by overnight incubation at room temperature. Exposure of these cells to anti-KCl (0.39 mg protein/ml), prior to their use in the hamster ova penetration test, reduced the penetration of denuded oocytes by 65% (P less than 0.005). Significant inhibitions of lesser magnitude were observed at lower serum concentrations (to 0.098 mg/ml). In an effort to understand the mechanism of this inhibition, other sperm function parameters thought to be related to oocyte penetration were studied. The inhibitory effect was exerted without noticeable changes in sperm motility (determined by the percentage of motile cells and their linear velocity), and in the absence of major sperm agglutination. Anti-KCl did not inhibit the occurrence of spontaneous or induced (by human follicular fluid) acrosome reaction in capacitated spermatozoa. In contrast, exposure to anti-KCl reduced the ability of capacitated spermatozoa to bind tightly to the hamster oolemma. None of these effects were elicited by a control preparation obtained from pre-immune rabbit sera. Exposure of zona-free oocytes to the antiserum did not alter their penetrability by normal sperm. These results suggest that the antigens recognized by anti-KCl participate in some specific step of the sperm-ovum interaction.

Identification of a major secretory glycoprotein from rat epididymis: interaction with spermatozoa.

A polypeptide with molecular mass of 17 kDa has been partially purified and identified as a major secretory glycoprotein in the rat epididymis. It is phosphorylated and contains high mannose-type oligosaccharides with 5 and 6 mannose units predominantly. These sugar residues are sufficiently exposed in the molecule to be released by endo-beta-N-acetylglucosaminidase H without prior denaturation or protease digestion. Specific binding of the glycoprotein to testicular spermatozoa was demonstrated with Ka 0.2 x 10(9) M-1 and 17,200 sites per cell, while no binding to epididymal spermatozoa was detectable. Direct labeling of surface proteins on cauda epididymis spermatozoa revealed the presence of a major band of 16.2 kDa, which may be equivalent to GP17. The interaction of the epididymal secretory protein with sperm suggests a possible role in the maturation process.

The effect of in vitro androgen stimulation upon androgen metabolism and trophic parameters in cultured human epididymis.

Previous results obtained with a model system of human epididymal tubules maintained in organ culture suggested that androgenic stimulation of this tissue resulted in responses similar to those obtained in epididymides of experimental animals under physiological conditions, as well as in other human androgen-dependent tissues. In this instance we have explored the possible influence of androgens on the activity of the androgen-converting enzymes 5 alpha-reductase and 17 beta-dehydrogenase. Activity of the former enzyme increases significantly during the culture period but no differences were found among the cultured groups, regardless of the addition of androgen. On the other hand, the activity of 17 beta-dehydrogenase was unchanged in all the experimental conditions tested. The co-culture of the tissue with explants of human testis was without effect. More than 85% of the activity of both enzymes was found localized in a fraction enriched in epithelial cells. Histological observation of the cultured tissues showed a marked disorganization of the pseudostratified epithelium in the absence of androgen while the inclusion of DHT in the media partially prevented these changes. We conclude that, under the conditions employed in these experiments, the activity of the enzymes studied is not influenced by androgens.

Studies on the mechanism of the antiandrogenic effect of a putative 5 alpha-reductase inhibitor.

The mechanism of the antiandrogenic effect of 5,10-seco-19-norpregnane-4,5-diene-3,10,20-trione (secosteroid), reputedly an irreversible inhibitor of 5 alpha-reductase, was investigated. Its addition (10 microM) to culture media effectively suppressed the synthesis of rat epididymal proteins specifically induced by 0.1 microM testosterone (T) or dihydrotestosterone (DHT). Under the same conditions, secosteroid did not change the rate at which labeled T was metabolized to 5 alpha-reduced compounds. In a comparative study, secosteroid inhibited 5 alpha-reductase in an isolated microsomal fraction while not affecting the enzyme activity in minced tissue. Secosteroid was shown to be a competitor of the binding of [3H]T and [3H]DHT (both at 4 nM) to the epididymal cytosol androgen receptor, with ID50 of 1 microM for the former and 4 microM for the latter, thus explaining the mechanism involved in its antiandrogenic properties.

Abnormal distribution of epididymal antigens on spermatozoa from infertile men.

An antiserum raised against human epididymal proteins associated with ejaculated sperm was used to test the hypothesis that the amount and/or localization of these antigens may be altered in men with infertility. With the use of immunofluorescence we found that in sperm from fertile donors 88.4% of the cells had the antigens localized over the acrosomal cap only and 1.3% had most of the antigens at extraacrosomal sites. Fifteen of the 26 infertile men (P1) studied had a similar relative distribution of antigens, but the remaining 11 patients (P2) had a 38-fold increase in cells with extraacrosomal localization of the antigens (40%, P less than 0.005). Using flow cytometry to quantitate immunofluorescence, content of antigen on sperm from patients from population P1 (680 +/- 60 V X 10(-4)) was not different from that of control (835 +/- 53 V X 10(-4], whereas it was significantly lower in sperm from patients from population P2 (554 +/- 64 V X 10(-4), P less than 0.005). Differences could not be correlated with parameters measured by routine semen analysis. Our results suggest a possible relationship between the decreased amount of epididymal antigens or their altered localization on sperm and the infertility of patients from population P2.

Further characterization of a model system for the study of human epididymal physiology and its relation to sperm maturation.

Some preliminary speculations about the possible participation of epididymal antigens in sperm function may be supported by the above data. On the one hand, the reduction in the amount of antigens and their abnormal localization on spermatozoa from infertile patients may be coincident with our view about participation of epididymal antigens in the development of zona pellucida binding ability and fertilizing capacity by spermatozoa during maturation. This hypothesis is derived from experiments showing that immature hamster spermatozoa gain their ability to recognize and bind to zona pellucida and to penetrate homologous oocytes when exposed to preparations enriched in androgen-dependent epididymal secretory proteins or preincubated in conditions that favor their interaction with these proteins. Supporting our viewpoint for such a role in humans is evidence showing the progressive development of the ability to interact with hamster denuded oocytes as human spermatozoa pass along the epididymis. On the other hand, the apparent correlation between the loss of epididymal antigens during capacitation and the increased fertilization of human oocytes in vitro may be reminiscent of the removal of a decapacitation or acrosome stabilizing factor known to occur in many species and that must be removed prior to fertilization. Pending further understanding of their physiological role, the androgen-dependent epididymal proteins may become a useful marker of epididymal function and/or of sperm capacitation in humans. Within this context, we wish to stress the potential value of the model system that we have developed for the study of human epididymal physiology.

Effect of androgen on androgen receptors in cultured human epididymis.

Optimal assay conditions were developed to determine androgen receptor concentrations in samples of human epididymis. Pretreatment of cytosol with mersalyl, as well as the inclusion of molybdate in the homogenization buffers, resulted in a substantial increase in the number of soluble sites detected. A high yield of nuclear and microsomal binding sites was obtained by prolonged incubation (12 h) in 0.6 mol NaCl/l. Organ culture for 6 days resulted in a major loss of androgen-binding sites. In the absence of androgen in the culture media, only 20% of the original sites were found in cultured tissue. Inclusion of dihydrotestosterone (0.1 mumol/l) in the media resulted in samples containing twice as many sites as controls. It is concluded that androgens influence the number of androgen-binding sites in cultured human epididymis in a manner analogous to that described for rat epididymis in vivo.

Androgen metabolism in the human epididymis. Effect of in vivo estrogen administration.

Androgen metabolism in human epididymis was studied by incubating tissue fragments with isotopically labeled testosterone (T) and androstenedione (A) under batch and superfusion conditions. Epididymides were obtained from 16 patients with prostatic cancer, 5 of them treated with diethylstilbestrol (2.5 mg/d) for several months prior to castration. Results from batch incubations with [3H]T (100 nM) for 2 h at 25 degrees C indicated a markedly lower 5 alpha-reductase activity in tissues from estrogen-treated patients, as evaluated by measuring the amounts of radioactive 5 alpha-dihydrotestosterone, 5 alpha-androstanediols and 5 alpha-androstanedione present in tissue and medium at the end of the incubation period. Superfusion experiments confirmed this estrogen effect and also showed a shift of the interconversion between A and T towards the reductive direction and a diminished tissue retention of DHT after estrogen treatment. These effects may contribute to the marked regression of the epididymal epithelium that was noted in the estrogen-treated patients, which is thought to be mainly the result of the inhibition of androgen biosynthesis caused by chemical hypophysectomy.

Identification of androgen-induced proteins in human epididymis.

Androgenic stimulation (0.1 microM testosterone or 5 alpha-dihydrotestosterone in the medium) of cultured human epididymal tubules increased the synthesis of five proteins, identified by their mobility relative to albumin (Ra) in polyacrylamide gels as 0.31, 0.43, 0.67, 0.81 and 1.01. This effect was inhibited by the simultaneous presence of 10 microM cyproterone acetate in the medium. The caput epididymidis was the most active region in the production of these proteins and a gradient of decreasing activity was found in successive segments. The appearance of induced proteins in the culture medium suggests their secretory nature, while some data indicate that androgens may also affect the secretory process. Bands corresponding to Ra 0.31, 0.43, 0.68 and 1.01 were found in caput and cauda epididymidis fluids, while bands coincident with Ra 0.31 and 0.43 were consistently found in extracts (0.5 M NaCl) of caudal spermatozoa. Preliminary determinations of molecular weight and isoelectric point for the different bands yielded the following results: Ra 0.31, 38,000 and 5.8; Ra 0.43, 21,000 and 6.2; Ra 0.68, 69,000 and 5.1; Ra 0.81, 13,900 and 6.8; and Ra 1.01, 29,000 and 6.8.

Immunochemical localization of secretory antigens in the human epididymis and their association with spermatozoa.

Ejaculated spermatozoa were washed and extracted with 0.6 M NaCl (2 h at 0 degree C) and the extract used to immunize rabbits. The crude antibody reacted with epididymal fluid and cytosol and with prostatic cytosol but did not recognize blood serum and testicular cytosol. After adsorption with prostatic proteins, the serum was specific for epididymis. Using immunoelectrophoresis and affinity chromatography, it was found that the antibody reacted with antigens which co-electrophoresed with androgen-dependent proteins (mobility relative to albumin, Ra) 0.3, 0.43 and 1.0, previously identified in human epididymis. Weak immunofluorescence in the epithelium of proximal caput tubules was detected on tissue sections. In contrast, distal caput and corpus tubules displayed a strong fluorescence in the cytoplasm of basal and principal cells as well as in spermatozoa present in lumen. Intense fluorescence was limited to the luminal content and the apical border and sterociliae of principal cells in caudal tubules. When applied to isolated spermatozoa, the reaction was negative for testicular sperm, while 49%, 82% and 100% of spermatozoa from caput, corpus and cauda, respectively, had a fluorescent acrosomal cap. An apparent gradient of increasing fluorescent intensities was also observed in this sequence. The reaction was strongest over the acrosomal cap, apparently absent in the postacrosomal region and weaker over the midpiece and principal piece. These results are interpreted as suggestive of the progressive coating of human spermatozoa with androgen-dependent epididymal proteins during epididymal transit.

Antibodies against epididymal glycoproteins block fertilizing ability in rat.

Antiserum against rat androgen-dependent secretory epididymal protein DE (raised in rabbit) was added to suspensions of rat spermatozoa from the cauda epididymidis which were used for artificial insemination. While control spermatozoa fertilized 41.6% of oocytes, those exposed to antiserum to protein DE fertilized only 6.6% (P less than 0.01). An equal amount of normal rabbit serum (NRS) did not cause inhibition (33.1%). To study the entry of antibodies into the epididymis, caudal tubules were cultured for 24 h and the fertility of the contained spermatozoa was assessed by artificial insemination. Culture in Medium 199 alone or with NRS resulted in spermatozoa which fertilized 52% of oocytes while the presence of antiserum to protein DE in the culture medium yielded spermatozoa which fertilized only 16.6% of oocytes (P less than 0.01). These results suggest (1) that the epididymal protein DE might be part of a sperm structure involved in the fertilization process, and (2) that, at least under the present culture conditions, immunoglobulins penetrate the epididymal epithelium in sufficient numbers to reduce fertility significantly.