Hamster Ova Research Articles

P–257 An unknown cause lead to polyspermy in IVF cycles and 0PN zygotes in ICSI cycles in male patient

Abstract Study question The patient sperm has normal morphology and motility, which paternal factors cause the abnormal fertilization in IVF/ICSI and what is the underlying mechanism? Summary answer A genetic mutation of BEX1 and decreased PLC-zeta has been found in patient, which may provide novel insights of polyspermy and pronucleus formation during fertilization. What is known already In mammals, pronucleus formation, a landmark event for fertilization, is critical for embryonic development. Abnormal fertilization refers to the abnormal number of pronucleus and polar bodies in zygotes during in vitro fertilization, with an incidence of 5–15%, among which the incidence of polyspermy and 0PN is about 2–10% and 30%. However, the mechanisms underlying pronucleus formation still unclear. More research has focused on oocyte activation, while paternal relevant abnormal fertilizations have been rarely established. The mechanism of how sperm and/or substances carried by sperm influence the physiological process of fertilization is also unclear. Study design, size, duration In our study, we first work on the preliminary observation and analysis of sperm morphology, structure and sperm chromosome number, and then made further analysis at the genetic level to find out the cause of this particular phenotype in this patient. We use of zone-free golden hamster ova test the fertilizing capacity and rescue the pronucleus formation with SrCl2. Participants/materials, setting, methods The patient, golden hamster, Papanicolaou stain, scanning electron microscope (SEM), Transmission Electron Microscope (TEM), Fluorescence in situ hybridization (FISH), Whole Exome Sequencing (WES), IVF, ICSI, Assisted Oocyte Activation (AOA). Main results and the role of chance During 2016–2018, they did 4 cycle assistant reproduction technology. Cycle1, conventional IVF(C-IVF), 9 MII oocytes, 9 3PN zygotes; Cycle2, ICSI, 10 MII oocytes, 10 0PN zygotes; Cycle3, donor-oocytes C-IVF, 6 MII oocytes, 6 3PN zygotes, and the donor did C-IVF get normal zygotes and embryos; Cycle4, donor-sperm C-IVF, 7 MII oocytes, 4 2PN zygotes, 3 useable embryos. Remarkably, clinical examination about male shows normal sperm semen parameters. Papanicolaou stain and SEM shows that the sperm of the patient has normal morphology. The TEM data shows that the spermatozoa with normal head morphology and intact 9 + 2 sperm flagella structure. In the sperm FISH analysis, Chromosome ploidy is haploid. We performed WES on the male, after exclusion of frequent variants and application of technical and biological filters, two homozygous missense mutations were identified in BEX1 (c.191G>A [p. W64X]), which has been few reports of male infertility. The western blot result show that the PLC-zeta was decreased in patient. After 10mM SrCl2 assisted oocyte activation, the zygote has the pronucleus formation in ICSI. Limitations, reasons for caution At present, we only observe sperm related factors (morphology, structure, chromosome number, genetic mutation). Next step is to detect the substances sperm carried (e.g. RNA-seq, proteomics). In this case, what is of great concern to us is the inconsistencies of the abnormal fertilization during the conventional IVF and ICSI cycles. Wider implications of the findings: Many studies of fertilization mechanism, the main focus is on the maternal cytoplasmic factors, such as the Ca 2+ release initiate the fast block of oocytes. There are few reports about abnormal fertilization due to sperm factors. Our case may offer new insights for the study of fertilization. Trial registration number Not applicable

The Integrated HIV-1 Provirus in Patient Sperm Chromosome and Its Transfer into the Early Embryo by Fertilization

Complete understanding of the route of HIV-1 transmission is an important prerequisite for curbing the HIV/AIDS pandemic. So far, the known routes of HIV-1 transmission include sexual contact, needle sharing, puncture, transfusion and mother-to-child transmission. Whether HIV can be vertically transmitted from human sperm to embryo by fertilization is largely undetermined. Direct research on embryo derived from infected human sperm and healthy human ova have been difficult because of ethical issues and problems in the collection of ova. However, the use of inter-specific in vitro fertilization (IVF) between human sperm and hamster ova can avoid both of these problems. Combined with molecular, cytogenetical and immunological techniques such as the preparation of human sperm chromosomes, fluorescent in situ hybridization (FISH), and immunofluorescence assay (IFA), this study mainly explored whether any integrated HIV provirus were present in the chromosomes of infected patients' sperm, and whether that provirus could be transferred into early embryos by fertilization and maintain its function of replication and expression. Evidence showed that HIV-1 nucleic acid was present in the spermatozoa of HIV/AIDS patients, that HIV-1 provirus is present on the patient sperm chromosome, that the integrated provirus could be transferred into early embryo chromosomally integrated by fertilization, and that it could replicate alongside the embryonic genome and subsequently express its protein in the embryo. These findings indicate the possibility of vertical transmission of HIV-1 from the sperm genome to the embryonic genome by fertilization. This study also offers a platform for the research into this new mode of transmission for other viruses, especially sexually transmitted viruses.

In vitro study on vertical transmission of the HIV‐1 gag gene by human sperm

HIV/AIDS is a major public health problem worldwide. To explore the feasibility of HIV vertical transmission by human sperm, plasmid construction and transfection, interspecific in vitro fertilization of zona-free hamster ova by human sperm, fluorescence in situ hybridization (FISH), RT-PCR, and immunofluorescence assay (IFA) were carried out. The FISH signals for HIV-1 gag DNA were observed in the nuclei and chromosomes of transfected human sperm, male pronuclei of zygotes, and nuclei of blastomeres of two-cell embryos, indicating that the HIV-1 gag gene could be transmitted via the sperm membrane and integrated into the sperm genome. In contrast, human sperm carrying the target gene achieved normal fertilization, and replication of the sperm-mediated target gene was synchronized with the host genome. Using RT-PCR, the positive bands for the target gene were observed in the transfected human sperm and two-cell embryos. These results further confirm that the target gene can be transcribed into mRNA in human sperm and embryonic cells. Positive signals for the HIV-1 p24 gag protein were shown by IFA in two-cell embryos containing the sperm-mediated target gene and not in the transfected human sperm, which indicated that the sperm-mediated target gene could be translated to make HIV-1 p24 gag protein in embryonic cells, but not in sperm cells. The results provide evidence for possible vertical transmission of the HIV-1 gag gene to the embryo by fertilizing sperm in vitro.

Comparison of the effects on penetration capacity of human spermatozoa between using Ham's F-10 medium and a medium based on the composition of human tubal fluid*: Vergleich des Einflusses der Penetrationskapazität menschlicher Spermatozoen bei Verwendung

The penetration ability of human spermatozoa on zona-free hamster ovum when using Ham's F-10 (HF-10) medium was compared with that when using a medium based on the composition of human tubal fluid. Forty semen specimens were each divided into two aliquots; one aliquot was washed with and incubated in HF-10 medium and the other in HTF medium. The penetration rate of sperm with motility between 30 to 60 percent was significantly increased when HTF medium was used (67.9 +/- 6.9%) compared with the rate when HF-10 medium was used (49.2 +/- 7.3%) (P less than 0.05). This study shows that the ZFHO penetration ability of human spermatozoa with poor motility can be improved by using HTF medium as compared with the use of HF-10 medium.

An improved experimental model for studying vertical transmission of hepatitis B virus via human spermatozoa

Study of the mechanism of transmission of hepatitis B virus is important for public health. An improved experimental model is described for studying vertical transmission of hepatitis B virus (HBV) via human spermatozoa. Recombinant plasmid pIRES2-EGFP-HBx which would express enhanced green fluorescent protein (EGFP) used as a marker for the expression of hepatitis B virus X (HBx) gene was constructed successfully and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize zona-free hamster ova in vitro. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBx DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive controls (PCR) indicating presence and expression of HBx gene. In contrast, HBx gene expression was not detected in the embryos without green fluorescence and negative controls (PCR and RT-PCR). This improved experimental model is more efficient, accurate and reliable for studying further perinatal transmission of HBV or other viruses causing chronic human disease possibly via the male germ line.

Construction of Wif1-IgG1/Fc Recombinant Vector and Characterization of Its Expression in CHO Cells.

Wnt signalling dysregulation has been implicated in cancers, including lymphoma. The Initiation of Wnt signalling modulated by soluble Wnt ant agonists, including soluble frizzled related proteins, dickkopf proteins, and Wnt inhibitory factor-1 (Wif1). These natural inhibitors can be used directly as theraputic reagents for cancers. But the recombinant antagonist only give very low blood levels, are cleared rapidly from the body (only several hours) and must be administered daily via subcutaneous injection. Therefore, development of new forms of Wnts inhibitors with prolonged circulating half-life is of significant interest. Human IgG immunoglobulins have longer circulating half-life (21 days in humans). When fused with a Wnt inhibitor, its dimeric structure further increases the effective size and circulating half-life. Thus administration of vector plasmid may be every 2~3 week. Moreover, IgG Fc can interact with FcR of CTL, NK, MΦ, and complements and the target cells can be killed through ADCC and CDCC. We constructed a Wif1-IgG1/Fc expressing vector and expressed it in Chinese hamster ovum cells in order to seek effective methods to inhibit lymphoma growth. Wif1 and IgG/Fc cDNAs were amplified by RT-PCR from human lymphocyte and cloned into the eukaryotic expression vector pcDNA3.0 by direct cloning and the resultant recombinant plasmid pcDNA Wif1-IgG1/Fc was transfected into 293 cells with lipofectmin. The constitutively expressing cells were obtained by G418 screening. Expression of the fusion protein was confirmed by Western blot, the fusion protein was purified with affinity chromatography, and its' biological activity was examined by flow cytometry, MTT colorimetry and ELISA. Our results show that the fusion protein significantly upregulated Wif1 density and promoted molt3 cell apoptosis, which was time dependent. The molt3 cell apoptosis induced by the fusion protein increased in the presence of NK cells or complements. Thus, we have successfully constructed and functionally expressed Wif1-IgG1/Fc fusion protein and shown that the purified fusion protein could kill molt3 cells through apoptosis and by ADCC and CDCC.

Effect of antibiotics on development in vitro of hamster pronucleate ova

Antibiotics are commonly added to embryo culture media, but effects on embryo development have not been examined thoroughly. Hamster ova were used to investigate whether penicillin, streptomycin or gentamicin affect embryo development in vitro. Ova were collected 10 h post activation by spermatozoa in vivo and cultured in five treatments: 1) Control: chemically-defined medium HECM-9 with no antibiotics; 2) HECM-9 with 100 IU/mL penicillin; 3) HECM-9 with 50 μg/mL streptomycin; 4) HECM-9 with 10 μg/mL gentamicin and 5) HECM-9 with both 100 IU/mL penicillin and 50 μg/mL streptomycin. Individually, penicillin, streptomycin and gentamicin did not affect embryo development to the 8-cell stage at 58 h post oocyte activation, or morula/blastocyst stages, or blastocysts alone at 82 h post activation. However, when penicillin and streptomycin were both present in the culture medium the percentages of 8-cell embryos at 58 h and blastocysts at 82 h were significantly lower than the control. No antibiotic treatment improved hamster embryo development in vitro. We caution against the use of penicillin and streptomycin together for hamster embryo culture, and show that it is not necessary to include any antibiotics in embryo culture media for up to 72 h if proper sterile technique is used with an oil overlay.

Adherence of human spermatozoa to trypsin-modified hamster ova.

Human spermatozoa do not attach to the zona pellucida of other species because of their high specificity compared to that of other animals in recognizing the zona pellucida. This study describes the recognition mechanism of human spermatozoa through trypsin treatment of the ovum of human and other species. This research has revealed two findings: (1) The "lattice-like" structure, the surface of the zona pellucida of the hamster ovum, was lost and became a uniformly coarse and flat structure. More than 200 human spermatozoa attached to it. The number of attaching spermatozoa increased significantly along with the prolongation of the incubation period and in direct dependence on the spermatozoa concentration. (2) Human spermatozoa did not attach to the zona pellucida of mouse, rat, and human ovum. This result confirmed that human spermatozoa attach to hamster ovum if modified with trypsin.

Expression of the BE-20 epididymal protein gene: in situ hybridization.

A protein designated as BE-20 was purified from cauda epididymal fluid of male rabbits and the amino acid sequence of the N-terminus was determined. A 23-mer oligonucleotide coding the N-terminal eight amino acids of the BE-20 protein was synthesized. The oligonucleotide was used as sense primer with rabbit epididymal mRNA as template in the RT-PCR system. The BE-20 cDNA consisted of 499 bp with an open reading frame of 285 bp encoding a deduced polypeptide composed of 95 amino acids. Digoxigenin-labeled BE-20 cDNA was prepared and used as a hybridization probe to detect the specific mRNA. The probe interacted with a 1.2-kb mRNA prepared from rabbit epididymis; mRNAs prepared from rabbit testis gave negative reaction. Using tissue sections, the BE-20 mRNA was located in the epithelial cells of the cauda epididymis and proximal segment of the ductus deferens by in situ hybridization method. Sections of the corpus and caput epididymis, testis, and liver gave negative reaction. Polyclonal anti-BE-20 antibodies were raised and found to inhibit in vitro the capacity of human sperm to penetrate zona-free hamster ova. The results suggest that BE-20 protein may influence maturation of spermatozoa during its movement through the epididymis and/or the capacity of sperm to fertilize ova.

The biologic significance of white blood cells in semen

To analyze the data available on the biologic significance of white blood cells (WBC) in semen of infertility patients. The relevant literature was reviewed. It is not possible to identify reliably WBC by conventional sperm staining techniques. The peroxidase method is sufficient for quantification of granulocytes, but immunocytology is the gold standard for the detection of all WBC populations in semen. Granulocytes are the most prevalent WBC type in semen (50% to 60%), followed by macrophages (20% to 30%) and T-lymphocytes (2% to 5%). The prevalence of leukocytospermia (> 10(6) WBC/mL semen) among male infertility patients is approximately 10% to 20%. There is controversy on the significance of WBC in semen. Whereas some authors did not observe sperm damage in the presence of leukocytospermia, others have found evidence that WBC are significant cofactors of male infertility: [1] seminal WBC numbers were higher in infertility patients than among fertile men; [2] leukocytospermia was associated with decreased sperm numbers and impaired sperm motility; [3] WBC damaged sperm function and hamster ovum penetration in vitro and were important prognostic factors for IVF-ET failure. Because of absence of clinical symptoms, the origin of WBC is difficult to determine. Normally, most WBC appear to originate from the epididymis because vasectomized men show very few WBC in semen. On the other hand, leukocytospermic samples show low citric acid levels, pointing to asymptomatic prostatitis as a source of WBC in semen. Surprisingly, approximately 80% of leukocytospermic samples are microbiologically negative. In some cases Chlamydia trachomatis might have triggered a persistent inflammatory reaction leading to leukocytospermia. Sperm damage by WBC can be mediated by reactive oxygen species, proteases and cytokines. Furthermore, genital tract inflammation facilitates the formation of sperm antibodies. As seminal plasma has strong anti-inflammatory properties and because there is only short contact between sperm and WBC in prostatitis and seminal vesiculitis, inflammations of the epididymis and testis are likely to have the largest impact on sperm. There is ample evidence that WBC can affect sperm function. Further studies are needed to define cofactors that increase or decrease the risk of sperm damage by WBC.

Identification of porcine sperm plasma membrane proteins that may play a role in sperm–egg fusion

Sperm plasma membrane (PM) proteins that demonstrate affinity for egg PM preparations have the potential to be biologically important during sperm-egg binding and/or fusion. In this study four such proteins have been identified. To provide quantitative evidence for possible biological function, the large natural variation among different porcine sperm populations with regard to their ability to interact with the egg was compared with the relative binding of egg PM material to individual proteins. An aliquot from each of 24 porcine ejaculates was evaluated by the zona-free hamster ova bioassay and the remainder processed to yield sperm PM vesicles. Aliquots of sperm PM were solubilised, separated by SDS-PAGE, western blotted and probed with partially purified, biotinylated egg PM protein. Bound egg PM proteins were visualised on western blots by an avidin/biotin/horseradish peroxidase system and analysed by scanning laser densitometry. Four sperm PM proteins (62, 39, 27 and 7 kDa estimated molecular mass) were the predominant binders of egg PM. The amount of egg PM bound to the 62 kDa protein was significantly correlated with the ability of sperm from the 24 ejaculates to penetrate zona-free hamster ova (percentage of ova penetrated, p = 0.01, R = 0.65; number of penetrated sperm per ovum, p = 0.02, R = 0.63).

Characterization of a Monoclonal Antibody to a Human Intra-Acrosomal Antigen that Inhibits Fertilization1

Among monoclonal antibodies (mAb) selected after the immunization of mice with human ejaculated spermatozoa, mAb I9G9 (IgG1 kappa) was found by immunoperoxidase staining to label most of the acrosome of human spermatozoa permeabilized with methanol-acetone. The antigen was poorly expressed on the surface of fresh ejaculated sperm, but was detectable on most viable sperm after 5-h incubation in medium containing human serum albumin (HSA) followed by 30-min incubation with the calcium ionophore A23187. This treatment resulted in acrosomal loss. Immunoelectron microscopy labeling with I9G9 mAb localized the antigen within the acrosome. Immunocytochemistry on testis sections showed that antigen was located in the round spermatids within the adluminal compartment of the seminiferous epithelium. Western blotting of sperm extract proteins showed that sperm intra-acrosomal (SIAA) recognized by I9G9 mAb had a polymorphism of immunogenic peptides from 16 to 35 kDa. Most of the antigenic peptides possessed an isoelectric point of approximately 5. When spermatozoa were treated with a series of protease inhibitors, the polymorphism of immunogenic peptides was reduced, suggesting that the multiple form of the antigen was due, at least in part, to proteolytic processing. In the testis, only a single peptide band of 35 kDa was detected with mAb I9G9. Studies of human tissue specificity by Western blotting showed that the epitope recognized by I9G9 mAb was present solely in ejaculated spermatozoa and the testis. I9G9 mAb did not agglutinate or immobilize sperm but inhibited the penetration of zona-free hamster ova by human sperm.

Interleukin-6 enhances the fertilizing capacity of human sperm by increasing capacitation and acrosome reaction.

The effects of human recombinant interleukin-6 (IL-6) on human sperm penetration of zona-free hamster ova (SPA) and human sperm cell capacitation and acrosome reaction were investigated. IL-6 at higher concentrations (60-600 pg/100 microliters) significantly (P < 0.001) increased rather than decreased the human sperm penetration rates in SPA. The effects were specific because immunoadsorption with the specific neutralizing antibody completely abolished the enhancing effects of IL-6. At the same concentrations, IL-6 also significantly enhanced spontaneous as well as calcium ionophore-induced acrosome reaction and release of acrosin from the human sperm cells. There was no effect of IL-6 on percent sperm motility, although it significantly affected various motility characteristics such as velocity, linearity, amplitude of lateral head displacement (ALH), and beat frequency of sperm cells--the motility parameters involved in hyperactivation phenomenon of sperm cells. These results demonstrate for the first time that IL-6 provides a positive signal in enhancing fertilizing capacity of human sperm by increasing capacitation leading to acrosome reaction, and thus it may have clinical applications in the treatment of male infertility in humans.

Tamoxifen treatment of oligozoospermia: a re-evaluation of its effects including additional sperm function tests.

Because of previous contradictory results, we reevaluated the effects of tamoxifen on 29 men presenting with idiopathic oligozoospermia. To determine whether a possible increase in sperm concentration might be correlated with an improvement of sperm quality, the hamster ovum penetration (HOP) test and the hypo-osmotic swelling (HOS) test were included as additional tests of sperm function. Patients were treated with tamoxifen (20 mg/day) for 3 months. From 4 weeks until the end of the study, tamoxifen had a significant effect (P < 0.001) on blood levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), and estradiol (E2). Prolactin (PRL) concentrations were not altered significantly (P > 0.05). There was no significant improvement (P > 0.05) of conventional semen parameters (volume, concentration, motility, morphology), and of HOP and HOS test results. The lack of correlation between a rise in hormone levels and improvement of sperm quality suggests that tamoxifen is of questionable value in men with idiopathic oligozoospermia.

Importance of total motile oval count in interpreting the hamster ovum sperm penetration assay.

A prospective study relating computer-assisted semen analysis (CASA) and technician-determined morphology to sperm penetration assay (SPA) outcome in patients with unexplained infertility or male factor was undertaken on 190 males aged 28-45 to determine the value of total motile oval count (TMO), compared to concentration, motility, and morphology considered independently, in predicting the outcome of the SPA. Prewash sperm count ranged 20-1,328 x 10(6), motility 0-93%, morphology 25-78% oval (%OVAL), and SPA scores 0-100%. Multiple regression analysis yielded two statistical models that identified significant predictors for % penetration (%P). Only TMO in one model and %OVAL in an independent effects model showed statistically significant correlation (P < 0.0001) to %P for all subjects. Discriminant function analysis showed the TMO model 85.4% accurate in classifying %P both in the abnormal range (< 20%P) and in the normal range (> or = 20%P). The independent effects model correctly classified 93% in the abnormal group, but projected 72 false negatives in the 101 subjects with %P > or = 20%, correctly classifying only 28.7%. Relative risk analysis showed TMO as a stronger risk factor affecting SPA outcome than %OVAL. It is concluded from this study that below 20%P, both TMO and %OVAL appeared to be comparable as predictive factors. Additionally, the TMO model was equally predictive for SPA > or = 20%P, where the independent effects model showed only 28.7% accuracy. SPA outcome appeared to be more profoundly affected by a decrease in TMO than by a decrease in %OVAL alone.

The microsperm penetration assay: development of a sperm penetration assay suitable for oligospermic males

To develop a specialized sperm penetration assay (SPA) for the evaluation of sperm from oligospermic patients. The development of the assay is in four parts: determine optimal sperm number; demonstrate quality control; establish statistical limits for fertile population; compare results to in vitro fertilization (IVF) outcome. A group of 63 patients with oligospermia and/or poor motility and a group of 17 fertile donors were compared using the optimized SPA and the micro-SPA. Sperm from a third group of 35 patients were simultaneously incubated with human ova (IVF) and hamster ova (micro-SPA). Both types of SPA scores are expressed as a sperm capacitation index (penetrations per ovum). Outcome of IVF is expressed as a percent of ova fertilized. Using 25,000 sperm was found to be optimal. The normal fertile range was statistically determined to have a lower limit (-2 SD) of 2.0 penetrations per ovum. When scores from 63 male factor patients were compared using the optimized SPA, only 43% had sufficient swim-up sperm. However, the micro-SPA could accurately test 100% of the samples because it requires only one tenth the number of sperm. The micro-SPA provides a valuable diagnostic test for the evaluation of the male factor patient.

Teratospermia in domestic cats compromises penetration of zona-free hamster ova and cat zonae pellucidae.

The ability of spermatozoa to bind and penetrate zona-free hamster ova and the zonae pellucidae of domestic cat oocytes in vitro was compared between normospermic (greater than 60% structurally normal spermatozoa per ejaculate) and teratospermic (less than 40% normal spermatozoa per ejaculate) domestic cats. The effects of culture media (Biggers, Whitten, Whittingham [BWW] versus modified Krebs Ringer bicarbonate [mKRB]) and simple dilution (DR), ejaculate centrifugation, and either resuspension (NS) or swim-up processing (SU) on penetration also were examined. High percentages of structurally normal spermatozoa were bound to zona-pellucida-free hamster ova regardless of the morphological forms in the inseminant. Mean percent normal spermatozoa bound to ova in DR, NS, and SU sperm aliquots from teratospermic male cats were not different (P greater than 0.05) from similarly treated normospermic aliquots. However, the percent penetration of hamster ova by normospermic ejaculates (10.5%) was superior (P less than 0.05) to that of teratospermic ejaculates (2.8%). Although swim-up processing improved percent sperm motility, progressive motility, and normal morphology in teratospermic ejaculates (P less than 0.05), no difference was observed in ovum penetration among the DR-treated, NS-treated, and SU-treated spermatozoa (P greater than 0.05). Culture medium had no effect on sperm binding in the hamster assay, but ovum penetration rate by spermatozoa in the normospermic ejaculates was enhanced (P less than 0.05) using mKRB (13.5%) when compared with BWW (7.6%) medium. Spermatozoa from teratospermic cats were capable of binding and penetrating cat zonae; however, sperm-zona interaction (defined as percent of oocytes with spermatozoa binding to or penetrating into the zona) was different (P less than 0.05) between normospermic (65.3%) and teratospermic (24.2%) cats.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-poisson distribution of sperm from grandfathers in zona-free hamster ova.

Earlier reports indicated that sperm from 25% of patients from infertile couples, but not from normal or fertile donors, show deviations from the theoretical Poisson distribution of the number of sperm penetrating zona-free hamster ova. Using semen samples from 15 grandfathers (aged 60 to 84 years) and 24 young fathers (aged 25 to 36 years), this study analyzed whether age also has an effect on the distribution. It was found that the overall fit to the Poisson distribution of the samples from grandfathers was very poor; in contrast, the samples from young fathers fit well. The observed deviations from the Poisson distribution among grandfathers may be a consequence of their long periods of sexual abstinence. Decrease in sexual activity produces age-different populations of sperm that probably differ in penetrating ability. Samples from older fathers also show a worse fit to the Poisson distribution than do those from younger fathers. These results suggest that the duration of sperm storage in the genital tract after maturation has an effect on sperm function.

Relationship Between Hypoosmotic Swelling Test, Semen Analysis, and Zona-Free Hamster Ovum Test

The hypoosmotic swelling test is a simple test for measuring the functional competence of the human sperm membrane. Fifty-four patients with idiopathic infertility were assessed by hypoosmotic swelling test and the results were compared with those of routine semen analysis and zona-free hamster ovum human sperm penetration test (ZSPT). Semen samples with abnormal semen parameters had lower percentage of swollen sperm in comparison with those with normal semen parameters. A positive correlation was observed between sperm concentration and sperm swelling (r = .50, p less than .05). A strong positive correlation was observed between the percentage of sperm motility and the percentage of swollen sperm (r = 0.60, p less than .01), and between motile sperm concentration and sperm swelling (r = .62, p less than .01). On the other hand, sperm swelling correlated only weakly with the percentage of sperm penetration. The results indicate that the hypoosmotic swelling test appears to evaluate different functional qualities of sperm than ZSPT.

Estimation of the quality of irradiated spermatozoa from cockerels using the classical method and zona‐free hamster ova

1. The quality of gamma irradiated spermatozoa from cockerels was investigated by measuring motility, oxygen consumption and ability to penetrate zona-free hamster ova. 2. Doses of 750 Gy slightly reduced motility and oxygen consumption; however, irradiated spermatozoa penetrated on average 56% (minimum 33%) of hamster ova. 3. The zona-free hamster ova test may be useful for estimating avian sperm quality.