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PubMed1993

[The characteristics of monoclonal antibodies and their antigens associated with human sperm acrosome reaction. II. Effects of the monoclonal antibodies on sperm function, sperm antigen localization and immunoblotting].

By using microassay, the strong human sperm agglutination were caused by 9 of 23 monoclonal antibodies (McAbs) obtained by immunization with fresh human acrosome-reacted (AR) sperm, which was induced by ionophore A 23187, but no one of them demonstrated the ability to immobilize human sperm. In contrast to most studies reported by other laboratories, the antigens recognized by most of McAbs in the present study were located in equatorial segment and midpiece of human sperm, and no one in acrosome region of human sperm. The binding pattern of some McAbs on non-treated and AR sperm was found to be different. The human sperm proteins ranged in the size of 16-146 kDa were demonstrated to be reacted with 9 of 23 McAbs by immunoblotting. Moreover, the cross reaction of 23 McAbs with mouse and tree shrew sperm was detected by indirect immunofluorescent assay. These results suggest that AR sperm possesses peculiar immunogenic properties.

Acrosome↗

PubMed1995

Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation.

In the present study we investigated the relevance of sperm immobilization prior to intracytoplasmic sperm injection (ICSI) in the fertilization process. Using supravital staining of the spermatozoa with eosin and studying sperm decondensation with 2 mM dithiothreitol (DTT) in conditions imitating sperm handling during ICSI, we demonstrated that immobilization of the spermatozoon by squeezing its tail between the glass pipette and the bottom of the dish damages the sperm plasma membrane. Polyvinylpyrrolidone (PVP), which is usually present in the drop with the spermatozoon to facilitate its handling, was found to impede the access of both eosin and DTT to the sperm nucleus. We conclude that (i) sperm immobilization prior to ICSI damages the sperm plasma membrane, that (ii) this damage is sufficient for thiol-reducing agents to gain access to the sperm nucleus, and finally that (iii) PVP possibly interferes with sperm nucleus decondensation.

Cell Membrane↗

PubMedDate not supplied

Sperm immunity in infertile couples: antibody titers are higher against the husbands' sperm than to sperm from controls.

Titers of hemagglutination and cytotoxic antibodies to sperm were determined in the sera, seminal plasma, cervical mucus, and vaginal secretions from 69 infertile couples, using sperm from the husbands and from normal control subjects. Titers of hemagglutination and cytotoxic antibodies were significantly higher (p = 0.002 and p less than 0.001, respectively) against autologous sperm as contrasted with sperm from control subjects in sera from 55 autoimmune males. Hemagglutination and cytotoxic antibody titers were also significantly higher (p = 0.044 and p less than 0.001, respectively) against their husbands' sperm as contrasted with control sperm in sera from 46 females with isoimmunity to sperm. Ninety percent of males and females with sperm immunity were positive for histocompatibility antigens HLA-B7, HLA-B8, and/or HLA-BW35. In males, the presence of increased serum cytotoxic antibody titers against autologous versus control sperm was significantly associated with the presence of HLA-B7 allele (p = 0.0017) and with HLA-B7, HLA-B8, and/or HLA-BW35 in general (p less than 0.05). In the females, increased serum antibody titers to their own husbands' versus control sperm were not preferentially associated with HLA-B7, HLA-B8, or HLA-BW35 antigens.

Antibodies↗

PubMed1999

Effects of bull, sperm type and sperm pretreatment on male pronuclear formation after intracytoplasmic sperm injection in cattle.

This study investigated the effects of the bull, sperm type (dead, immotile or motile) and sperm pretreatment (i.e. mechanical (tail-cutting or tail-scoring) or chemical (heparin, heparin + caffeine, calcium ionophore A23187 or dithiothreitol)) on male pronuclear formation after intracytoplasmic sperm injection (ICSI) in cattle. Three experiments were conducted. In Experiment 1, spermatozoa from three bulls (A, B and C) were used for both ICSI and in vitro fertilization (IVF). The results were that sperm from bull B yielded a higher penetration/male pronuclear formation rate than that of bull C when used for IVF (89.6% v 25.6%, P<0.01). However, when injected into oocytes by ICSI, sperm from bull C had a higher male pronuclear formation rate than that of bull B (34.6% v. 16.1%, P<0.05). The effects of sperm type and mechanical pretreatment were examined in Experiment 2. No significant difference was found in the male pronuclear formation rate when the three types of sperm were injected into oocytes. Tail-scored sperm achieved a higher male pronuclear rate than that of non-mechanically treated ones (38.2% v. 13.2%, P<0.005). In Experiment 3, chemical pretreatments were tested and compared. Higher male pronuclear rates, compared with the control, were obtained when sperm were pretreated with heparin + caffeine, calcium ionophore A23187 and dithiothreitol (48.2%, 62.5% and 64.5% v. 25.0%, P<0.05, 0.005 and 0.005, respectively). These results indicate that (1) there is a bull variation in male pronuclear formation with ICSI, and the male pronuclear rate by ICSI is not coincident with the results by IVF, (2) immobilization of a spermatozoon by tail-scoring before ICSI can improve the formation of the male pronucleus, and (3) an appropriate chemical pretreatment of spermatozoa is necessary to achieve a higher rate of male pronuclear formation.

Animals↗

PubMed1992

Importance of mammalian sperm metalloendoprotease activity during the acrosome reaction to subsequent sperm-egg fusion: inhibitor studies with human sperm and zona-free hamster eggs.

We have previously shown that each of the metalloendoprotease (MEP) inhibitors phosphoramidon, diethylenetriaminepentaacetic acid, and carbobenzoxy-L-phenylalanine, when present only during the human sperm acrosome reaction (AR), will not inhibit the AR or sperm motility but will decrease the number of sperm that penetrate zona-free hamster eggs. The present study was designed to investigate whether this inhibition of penetration is due to an effect on sperm binding to the egg plasma membrane and/or to an effect on the actual membrane fusion event. In these studies we used ionomycin to initiate the AR and assayed binding in a Ca(2+)-free medium and fusion in Ca(2+)-containing medium in the same experiment. Eggs were loaded with the fluorescent dye Hoechst 33342, and the appearance of fluorescence in a sperm head indicated that fusion had occurred. The three MEP inhibitors reduced binding only slightly but inhibited the actual fusion step by 50-60% (determined with an equation that corrected for any inhibition of fusion due to inhibition of binding). MEP inhibitors present only during gamete interactions had little or no effect on fusion. We also found that phosphoramidon-inhibitable MEP activity was released during the ionomycin-initiated AR. Incubation of AR supernatant containing MEP activity with previously acrosome-reacted, phosphoramidon-treated sperm resulted in a large reversal of the phosphoramidon-inhibitory effect on sperm-egg fusion. These results support the hypothesis that the acrosomal phosphoramidon-inhibitable MEP released during the AR acts directly or indirectly during that event to increase the fusibility of the sperm plasma membrane region required for subsequent sperm-egg fusion.

Acrosome↗

PubMedDate not supplied

Detection of sperm surface related antibodies by indirect immunofluorescence test on sperm suspensions. Indirect IFT on sperm suspensions.

An indirect immunofluorescence test (IIFT) using sperm suspensions was carried out on 20 sera with sperm agglutinins (SA) and on 25 negative controls. IIFT gave results highly correlated with the occurrence of sperm agglutinating activity. Moreover, a relation was found among class of Ig involved in IF reactivity, fluorescent stain pattern and type of sperm agglutinations. In all sera with "mixed" or "tail-tail" sperm agglutinating activity, IgG were involved in IF reactivity; the fluorescent stain constantly appeared in a granular pattern along the sperm tail and most often on the head surface too. In relation to high titres of "head-head" sperm agglutinating activity, IgM were involved in IF reactivity; here the fluorescent stain appeared to be localized on acrosomal surface. The results indicate that indirect IFT on sperm suspensions specifically detects sperm surface related antibodies.

Antigens, Surface↗

PubMed1996

Measurement of the sperm motility index via the sperm quality analyzer and its relationship to other qualitative sperm parameters.

Sperm parameters such as the concentration and percentage of motile spermatozoa are commonly used to assess semen quality. The sperm quality analyzer (SQA) is a device that detects variations in the optical density of motile spermatozoa, providing a sperm motility index (SMI) that is based on various sperm parameters including the concentration, morphology and acrosomal status of motile spermatozoa. The relationship between SMI values of frozen-thawed bovine spermatozoa undergoing swelling in a hypoosmotic medium (100 mOsm/L) and other sperm parameters were evaluated. Frozen semen specimens from 3 bulls were thawed and washed with Ham's F-10 supplemented with 3% BSA and split into 3 (0.2 mL) aliquots. The aliquots were diluted with 1.0 mL of Ham's F-10 (Aliquot 1), isotonic sodium citrate (Aliquot 2), and hypotonic sodium citrate (Aliquot 3). The osmotic pressure of the media used for dilution of Aliquots 1 and 2 was 300 mOsm/L, while that for Aliquot 3 was 100 mOsm/L. Following dilution, the aliquots were incubated for 30 min and manually assessed at 5-min intervals for the percentage and grade of motility (Grades 0 to 4) as well as for the percentage of swollen spermatozoa. Sperm samples were simultaneously evaluated by SQA to obtain the SMI values at the same 5-min intervals during the 30-min incubation. Significant correlations were observed between SMI values and other sperm parameters in Aliquot 3 (P < 0.05). The results indicated that the SMI values obtained from frozen-thawed bovine spermatozoa exposed to a 100 mOsm/L diluent, which causes optimal swelling of spermatozoa, are highly correlated to other sperm parameters. The SQA unit, as applied in this study, can be used for rapid and reliable screening of sperm samples.

Journal Article↗

PubMed2003

Efficient utilization of very dilute aquatic sperm: sperm competition may be more likely than sperm limitation when eggs are retained.

Fertilization success may be severely limited in marine invertebrates that spawn both male and female gametes. In a diverse group of aquatic organisms only sperm are released, with sperm-egg fusion occurring at the mother. Here, we report fertilization kinetics data for two such 'brooding' or 'spermcast' species--representing each major clade of the animal kingdom. High levels of fertilization were achieved at sperm concentrations of two or three orders of magnitude lower than is common with broadcast spawning species. At a concentration of 100 sperm ml(-1), fertilization rates of a bryozoan and colonial ascidian were near maximum, whereas most broadcast spawners would have displayed near complete reproductive failure. A further experiment looked at the rate of uptake of sperm under natural conditions. Results suggested that sperm released at ca. 0.9 m from an acting female could be collected at a rate of 3-12 times greater than the minimum required simply to avoid sperm limitation. Thus, evolutionary pressures on gametic and other reproductive characteristics of many species that release sperm but retain eggs may be quite different from those of broadcast spawners and may confer on the former an enhanced scope for sperm competition and female choice.

Animals↗

PubMed1999

Destruction of protamine in human sperm inhibits sperm binding and penetration in the zona-free hamster penetration test but increases sperm head decondensation and male pronuclear formation in the hamster-ICSI assay.

PURPOSE: Our purpose was to investigate the fertilizing ability of human protamine-damaged sperm in a heterologous system using hamster oocytes. METHODS: The protamine of the sperm were damaged by exposure to dithiothreitol, a disulfide-reducing agent. Their ability to penetrate and form male pronuclei were investigated using the zona-free hamster penetration test and the hamster-intracytoplasmic sperm injection assay, respectively. RESULTS: The zona-free hamster penetration test revealed that protamine-damaged sperm are unable to bind and penetrate the hamster oocyte. On the other hand, hamster-intracytoplasmic sperm injection assay results showed that 56.9% and 39.2% of the injected oocytes developed male pronuclei in protamine-damaged and live-intact sperm groups, respectively, with a significant difference in these rates (P < 0.01). CONCLUSIONS: This study shows that protamine-damaged sperm are able to undergo sperm head decondensation and male pronuclear formation only when injected into the ooplasm, although they cannot bind and penetrate through the zona and enter the ooplasm.

Acridine Orange↗

PubMed2005

Serine protease activity, bovine sperm protease, 66 kDa (BSp66), is present in hamster sperm and is involved in sperm-zona interaction.

Bovine sperm protease, 66 kDa (BSp66) is a serine protease previously characterized in bovine spermatozoa. Like other proteases, it may be present in sperm from other mammalian species different from bovine, playing a role in the fertilization process. In this study, we looked for BSp66 in hamster spermatozoa using heterologous antibodies against bovine BSp66. An immunoreactive protein was detected by Western blotting in mature and immature sperm. The detected protein had two isoforms similar to the ones reported in bovine sperm. Furthermore, indirect immune detection by fluorescence and electron microscopy assays, showed BSp66 signal at the acrosomal region similar to bovine sperm. As it was determined in bovine sperm, the acrosomal reaction displays the antigen within the acrosomal content. When live hamster sperm was incubated with polyclonal antibody against bovine BSp66 a decrease in the number of sperm bound to zona pellucida in homologous IVF and an impairment of head-head agglutination, were observed. These results suggest that a protease homologous to bovine BSp66 is present in golden hamster spermatozoa, with a conserved molecular mass and cellular location. Moreover, hamster BSp66 is probably involved in zona pellucida recognition.

Animals↗

PubMed2003

Efficiency of percutaneous testicular sperm aspiration as a mode of sperm collection for intracytoplasmic sperm injection in nonobstructive azoospermia.

PURPOSE: We determined the feasibility of obtaining mature spermatozoa for intracytoplasmic sperm injection (ICSI) by percutaneous testicular sperm aspiration in men with nonobstructive azoospermia. We also compared the results of ICSI using spermatozoa recovered by open excisional biopsy versus percutaneous testicular sperm aspiration. MATERIALS AND METHODS: A total of 84 men with nonobstructive azoospermia underwent percutaneous testicular sperm aspiration to recover testicular spermatozoa for ICSI on the day of ova retrieval from the wife. Percutaneous testicular sperm aspiration was performed with the patient under general anesthesia in the upper and lower poles of each testis. It was followed by immediate microscopic search of the aspirate to confirm the presence of spermatozoa. In the absence of spermatozoa open excisional biopsy was performed in the same setting. RESULTS: Percutaneous testicular sperm aspiration resulted in the recovery of mature spermatozoa in 45 men (53.6%). Of the remaining 39 men (46.4%) requiring open biopsy adequate spermatozoa were recovered in 28 (71.8%). Although the fertilization rate was significantly higher in the sperm aspiration group, the cleavage and pregnancy rates were similar in the 2 groups. CONCLUSIONS: Percutaneous testicular sperm aspiration was a successful initial approach to collect mature spermatozoa in a high proportion of men with nonobstructive azoospermia. It is safe, minimally invasive and well tolerated by all patients.

Adolescent↗

PubMed2002

Live birth after posthumous testicular sperm aspiration and intracytoplasmic sperm injection with cryopreserved sperm: case report.

PURPOSE: To determine if a viable pregnancy is possible after aspiration of sperm from the testes of a man several hours after his death. METHOD: Following cryopreservation of the aspirated sperm, in vitro fertilization (IVF)-embryo transfer (ET) with intracytoplasmic sperm injection (ICSI) was used. The sperm selected were either those with motility or plumper non-motile sperm. RESULTS: Fertilization of oocytes and ET occurred following all five IVF-ET cycles. A clinical pregnancy was achieved in cycle 1 with non-motile sperm and a viable pregnancy resulted from cycle 5 with ICSI performed with viable sperm. CONCLUSION: Viable pregnancies following IVF-ET and ICSI are possible even when using testicular sperm obtained posthumously.

Adult↗

PubMed1976

[Sperm agglutination, sperm immobilization and sperm immunofluorescence test in married couples with unexplained infertility (author's transl)].

Thirty-four married couples with clinically unexplained infertility and normal spermiograms in all male partners were investigated for sperm antibodies by means of the sperm agglutination, sperm immobilization and sperm immunofluorescence test. In 50% of the couples, sperm antibodies were found in at least one of the partners and with at least one of the tests, e.g. ten times in the female partner alone, six times in the male partner alone, and once in both partners. The correspondance of the three tests was astonishingly low; it is assumed, therefore, that they indicate different types of antibodies. On the basis of the present investigations it is not possible to assess which type of antibody is responsible for the infertility of the female or male partner respectively. This seems only possible when the decrease of certain antibody types can be determined after a period of no antigen contact (avoidance of sperm contact) and when conception has taken place. The significance of autoantibodies against sperm in clinically healthy men with normal spermiograms remains open.

Antibodies↗

PubMedDate not supplied

Sperm motility should be assessed in fresh sperm and after a sperm washing procedure.

A prospective study was carried out on semen samples from 118 consecutive unselected men attending our infertility clinic to determine whether sperm motility may be affected by seminal plasma. The incidence of asthenozoospermia as defined by fewer than 50% of spermatozoa with forward progressive motility in the untreated semen was 37.4%. This value was significantly reduced to 23% after washing and removing seminal plasma. Men with asthenozoospermia in untreated semen but normal in the washed sample had a percentage of normal sperm morphology and a percentage of swollen tails in the HOS test similar to those of controls, and higher than those of asthenozoospermics in both the untreated and washed sample. Sperm velocity was also significantly improved after the washing procedure. Spermatozoa selected by swim-down procedure and applied to a seminal plasma medium reduced sperm motility affecting negatively the HOS test. Sperm motility should be assessed after a sperm washing procedure, since seminal plasma contains constituents that decrease sperm motility without affecting membrane integrity.

Adult↗

PubMed2005

The mouse T complex gene Tsga2, encoding polypeptides located in the sperm tail and anterior acrosome, maps to a locus associated with sperm motility and sperm-egg interaction abnormalities.

Previous studies of sperm from mice heterozygous for a t haplotype (t) and heterospecific combinations of the t complex identified two tightly linked genetic factors responsible for t/t male sterility related to expression of the flagellar waveform aberration, curlicue. Dnahc8, an axonemal dynein heavy chain gene, is a strong candidate for the proximal factor, Ccua, but the identity of the distal factor, Ccub, is unknown. In the present study, we employ motility assays of sperm from males heterozygous for t and novel heterospecific combinations of the t complex to demonstrate that Ccub is a composite of at least two synergic elements, Ccub1, positioned within a genomic interval spanning approximately 0.6 Mb immediately distal to Dnahc8, and Ccub2, situated in a region approximately 4-7 Mb distal to Ccub1. We also show that Tsga2, a testis-restricted gene, fulfills many of the prerequisites required to make it a strong candidate for Ccub1. These include: 1) its location within the aforementioned genomic interval; 2) a highly reduced level of testis expression by its heterospecific allele relative to the level of expression of its t allele; 3) determination that TSGA2(t) carries numerous nonsynonymous mutations in residues otherwise highly conserved in all known orthologous proteins; 4) the detection of major TSGA2 polypeptides in sperm protein extracts; and 5) the apparent distribution of these polypeptides in major sperm tail structures. Surprisingly, these TSGA2 isoforms appear to localize in the vicinity of the anterior acrosome, as well, suggesting that Tsga2 may also play a role in sperm-egg interaction. Finally, our results indicate that a TSGA2 polypeptide with apparent similarities to the smaller of the two sperm isoforms is expressed by epididymal cells.

Acrosome↗

PubMed2007

Effects of cytoplasmic genes on sperm viability and sperm morphology in a seed beetle: implications for sperm competition theory?

Sperm competition theory predicts that sperm traits influencing male fertilizing ability will evolve adaptively. However, it has been suggested that some sperm traits may be at least partly encoded by mitochondrial genes. If true, this may constrain the adaptive evolution of such traits because mitochondrial DNA (mtDNA) is maternally inherited and there is thus no selection on mtDNA in males. Phenotypic variation in such traits may nevertheless be high because mutations in mtDNA that have deleterious effects on male traits, but neutral or beneficial effects in females, may be maintained by random processes or selection in females. We used backcrossing to create introgression lines of seed beetles (Callosobruchus maculatus), carrying orthogonal combinations of distinct lineages of cytoplasmic and nuclear genes, and then assayed sperm viability and sperm length in all lines. We found sizeable cytoplasmic effects on both sperm traits and our analyses also suggested that the cytoplasmic effects varied across nuclear genetic backgrounds. We discuss some potential implications of these findings for sperm competition theory.

Adaptation, Physiological↗

PubMed1993

Novel parameters of human sperm hypoosmotic swelling test and their correlation to standard spermatogram, total motile sperm fraction, and sperm penetration assay.

OBJECTIVE: To improve the accuracy of human sperm hypoosmotic swelling test by introducing new morphological evaluation parameters for this test. DESIGN: Individual semen samples were processed, and the standard spermatogram, total motile sperm fraction, sperm penetration assay (SPA), and hypoosmotic swelling test were performed. SETTING: Male infertility clinic and andrologic laboratory in a university medical center. PATIENTS: One hundred eighteen subfertile men undergoing an infertility work-up. MAIN OUTCOME MEASURES: The results of hypoosmotic swelling test were subdivided into four groups (hypoosmotic swelling test 1, 2, 3, 4) according to the different shapes of hypoosmotically affected spermatozoa tails. RESULTS: The hypoosmotic swelling test 3 had the best correlation with the different sperm parameters: motility, total motile sperm fraction, concentration, and SPA. The hypoosmotic swelling test 1 showed a satisfactory interrelation for concentration and for total motile sperm fraction but no correlation for motility and SPA. The hypoosmotic swelling test 2 and the hypoosmotic swelling test 4 demonstrated no correlation at all with motility, concentration, morphology and SPA. CONCLUSIONS: The precision of hypoosmotic swelling test may be improved by using the hypoosmotic swelling test 3 type of spermatozoa tail shape as the parameter for a normally functioning spermatozoa.

Female↗

PubMedDate not supplied

Pregnancy rates in a donor sperm program using cryopreservation: effects of total number of motile sperm inseminated and of a procedure to concentrate sperm.

OBJECTIVE: To determine the relationship between the total number of motile sperm per insemination (TMSI) and pregnancy rates in a donor insemination program. To determine the effect of sperm concentration and resultant increase of TMSI on pregnancy rates. DESIGN: Retrospective analysis of pregnancy rates. SETTING: University hospital with tertiary service. PATIENTS: 179 women undergoing donor insemination. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Calculation of pregnancy rates according to TMSI. RESULTS: The pregnancy rate with insemination of thawed donor sperm increases when the TMSI of unconcentrated sperm is > or = 80 x 10(6). Pregnancy rates with previously concentrated sperm are as good as or better than rates of unconcentrated specimens. CONCLUSION: TSMI is important in determining pregnancy rates in donor insemination programs, and concentration of sperm prior to freezing may enhance pregnancy rates by increasing the TMSI.

Cryopreservation↗