Clinical and seminal parameters associated with testicular microlithiasis and its severity in males from infertile couples.

STUDY QUESTION: Is there an association of testicular microlithiasis (TM) and its severity with testicular dysfunction in men from infertile couples? SUMMARY ANSWER: The presence of ≥5 testis microcalcifications per sonogram at the scrotal ultrasonography (US) of infertile males was associated with a more severe testicular dysfunction as compared to males with limited, or without, TM. WHAT IS KNOWN ALREADY: TM, representing an incidental finding in the scrotal US, is associated with male infertility and a higher risk for testicular cancer as compared to that in infertile males without TM. Still, there are unresolved questions on the relation between TM severity and testicular dysfunction in infertile men, as well as on the identification of risk factors for TM. STUDY DESIGN, SIZE, DURATION: This study was an observational, retrospective, case-control investigation involving males who underwent clinical evaluation, measurement of reproductive hormones, seminal analysis and scrotal US as part of diagnostic work-up for couple infertility at an andrology clinic, between January 2004 and December 2018. One hundred patients, out of the 2112 scored men, were found to have TM during the US evaluation. One hundred male partners from 100 infertile couples without TM, comprising the control group, were selected through a matched analysis by age and date of evaluation to reduce the confounding effect of both age and technique variability all along the long period of observation. PARTICIPANTS/MATERIALS, SETTING, METHODS: TM was defined as limited TM (LTM) or classical TM (CTM), when the maximum number of hyperecogenic spots per sonogram was <5 or ≥5, respectively. CTM, LTM and control groups were compared for clinical variables, serum levels of FSH, LH, and total testosterone, as well for semen parameters and scrotal US features. MAIN RESULTS AND THE ROLE OF CHANCE: After the exclusion of cases with testicular nodules to eliminate the possible confounding effect of testis cancer on testicular dysfunction, cases with CTM showed a lower mean testis volume (P = 0.03) and a lower sperm concentration (P = 0.03) as compared to the other two groups. A higher FSH level was observed in the CTM group compared to the LTM group (P = 0.02) and in controls (P = 0.009). The multiple logistic regression analysis showed that only a smaller testicle volume exhibited an independent significant association with a higher odds of detecting CTM (odds ratio = 0.84, 95% CI: 0.75-0.94; P = 0.02). No significant differences were observed between groups in the prevalence of risk factors for testicular cancer, or in the prevalence of conditions associated with TM. LIMITATIONS, REASONS FOR CAUTION: The retrospective design of the study did not allow conclusions to be drawn about the possible underlying links in the associations of TM with defective spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS: Males from infertile couples who exhibit a reduced testicular volume should undergo scrotal US, independent of sperm parameters, to exclude CTM and, eventually, testis cancer, although the association of CTM and current or future testis cancer risk is not yet clear. Evidence is provided here demonstrating that the presence of LTM has no clinical relevance in males from infertile couples. STUDY FUNDING/COMPETING INTEREST(S): Investigation was funded by Ministero dell'Università e della Ricerca, PRIN 2018, Italy. The authors have not declared any competing interests. TRIAL REGISTRATION NUMBER: N/A.

Prevalence of anti-sperm antibodies and relationship of degree of sperm auto-immunization to semen parameters and post-coital test outcome: a retrospective analysis of over 10 000 men.

STUDY QUESTION: What is the prevalence and the relationship of anti-sperm antibodies (ASA), screened by means of IgG-mixed anti-globulin reaction (MAR) test, to semen quality and post-coital test (PCT) outcome? SUMMARY ANSWER: A 100% positive IgG-MAR test, detected in 2% of the study population, was associated with lower sperm output and progressive motility, and was the sole determinant of higher prevalence of a negative PCT outcome. WHAT IS KNOWN ALREADY: Although ASA may affect sperm fertilizing ability and the IgG-MAR test is recommended by the World Health Organization (WHO) as an integral part of semen analysis for screening the occurrence of ASA, the prevalence and clinical relevance of positive MAR test results remain controversial. STUDY DESIGN, SIZE, DURATION: A retrospective analysis of 12 296 consecutive men who attended a university/hospital andrology clinic for the evaluation of fertility potential was carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunological screening with the IgG-MAR test was performed on all ejaculates as an integral part of semen analysis. Positive samples (≥10%) were further tested for IgA-ASA. The prevalence of positive IgG-MAR tests results, along with the relationship of the degree of sperm auto-immunization to semen parameters and PCT outcome, were analyzed. MAIN RESULTS AND THE ROLE OF CHANCE: After excluding semen samples showing azoospermia or severe oligo-asthenozoospermia, the prevalence of a positive IgG-MAR test in the remaining 10 025 men was 4%, 3.4% and 2%, with 10%, 50% and 100% thresholds, respectively. The 100%-positive MAR tests exhibited significantly higher consistency over time, and were significantly associated with higher prevalence of a mixed pattern (i.e. when the majority of sperm exhibited beads attached on both the head and along the tail) of positivity as well as with the concomitant occurrence of IgA-ASA. Additionally, the 100%-positive MAR tests were significantly associated with a lower median value of the total number of spermatozoa and progressive motility, compared to samples with a lower degree of positivity or negative samples. In the PCT performed in 120 couples, where ASA were detected in the male partner, the 100%-positive MAR tests were significantly associated with a higher prevalence of negative PCT outcome, in comparison to the lower degree of positivity, independent of, and without any significant contribution from, other determinants (semen and cervical mucus quality). LIMITATIONS, REASONS FOR CAUTION: Only surrogate infertility-related end-points were analyzed in the present study. However, since the impairment of sperm penetration through the cervical mucus represents the primary mechanism of ASA-interference with fertility, PCT outcome may represent a suitable clinical end-point. WIDER IMPLICATIONS OF THE FINDINGS: The present study, being the largest reported to date, provides a reliable estimate of ASA prevalence. Moreover, it indicates that a 50%-positive MAR test, which is suggested by WHO as the clinically-relevant threshold, also includes patients with a degree of sperm auto-immunization that contributes to couple infertility only in the presence of other causal factors; conversely, the 100%-positive MAR test can represent the sole determinant of couple infertility, as it was the sole significant predictor of the highly prevalent negative PCT outcome. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the University of L'Aquila, Italy. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Mutations in dynein genes in patients affected by isolated non-syndromic asthenozoospermia.

BACKGROUND: Asthenozoospermia (AZS) is a common cause of male infertility characterized by reduced forward motility (WHO grade A+B sperm motility <50% or A < 25%) or absent sperm motility in fresh ejaculate. AZS may exist as an isolated disorder, in combination with other sperm anomalies or as part of a syndromic association. Up to date, only a few genes, constituting the cilia/flagella structure, have been associated with isolated AZS in humans, whereas several other genes are known to be involved in syndromic form of AZS, including primary ciliary dyskinesia (PCD) and Kartagener syndrome (KS). Axonemal ultrastructural defects, including absent or shortened arms of dyneins, can be found in >50% of PCD/KS patients. Approximately 90% of KS male patients are affected by AZS. The majority of KS patients can be ascribed to dynein genes mutations. METHODS: Mutation screening of DNAI1, DNAH5 and DNAH11 genes was performed in 90 patients with isolated non-syndromic AZS and 200 controls. RESULTS: We found three mutations (one in each gene) specifically associated with AZS in seven patients (7.8%). Mutations are inherited from the mothers and may be found in familial clusters. No ultrastructural axonemal anomaly was detected in sperm. CONCLUSIONS: We report for the first time a possible association between mutations in dynein genes and isolated AZS. Male carriers of the mutations always exhibit AZS, whereas female carriers manifest no alterations in either fertility or pulmonary clearance.

Semen leukocytes and oxidative-dependent DNA damage of spermatozoa in male partners of subfertile couples with no symptoms of genital tract infection.

The influence of seminal leukocytes on generation of oxidative damage to sperm DNA was here investigated on male partners of subfertile couples asymptomatic for a genital tract infection. The study included 111 ejaculates from men attending the Andrology Centre at University of L'Aquila. Semen leukocytes subset included round cells expressing pan-leukocyte CD45 antigen, monocyte/macrophage lineage antigen CD14, and activated macrophages HLA-DR antigen. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression identified spermatozoa with DNA oxidative adducts while terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end labeling (TUNEL) assay detected spermatozoa with DNA fragmentation. Flow cytometry and immunocytochemistry was used for determinations. Main outcome measure was the association of semen leukocyte subpopulations with spermatozoa showing oxidative-related DNA damage and with routine semen parameters. Leukocyte subpopulations were strictly correlated (p < 0.0001), but no association was found between the concentration of leukocytes, semen parameters, the percentage of TUNEL-positive and of 8-OHdG-positive spermatozoa. The percentage of 8-OHdG-positive spermatozoa was positively correlated with the percentage of TUNEL-positive spermatozoa (r = 0.48; p < 0.0001) and negatively correlated with sperm concentration (r = -0.44; p < 0.0001). Sperm concentration and the percentage of TUNEL-positive spermatozoa independently contributed (ß = -0.25, p = 0.008; ß = 0.23, p = 0.05, respectively) to the variation in percentage of 8-OHdG-positive spermatozoa after adjusting for age, abstinence time, and smoking. In conclusion, oxidative-dependent DNA damage in spermatozoa was associated to poor semen quality but not to different leukocyte subpopulations in ejaculates of men asymptomatic for a genital tract infection.

Fas and Fas ligand expression in fetal and adult human testis with normal or deranged spermatogenesis.

In mice, the Fas/Fas ligand (FasL) system has been shown to be involved in germ cell apoptosis. In the present study we evaluated the expression of Fas and Fas ligand (FasL) in fetal and adult human testis. Semiquantitative RT-PCR demonstrated the expression of Fas and FasL messenger ribonucleic acids in adult testis, but not in fetal testis (20-22 weeks gestation). In situ RT-PCR and immunohistochemistry experiments on adult human testis demonstrated the expression of FasL messenger ribonucleic acid and protein in Sertoli and Leydig cells, whereas the expression of Fas was confined to the Leydig cells and sporadic degenerating spermatocytes. The number of Fas-positive germ cells per 100 Sertoli cell nuclei was increased in 10 biopsies with postmeiotic germ cell arrest compared to 10 normal testis biopsies (mean, 3.82 +/- 0.45 vs. 2.02 +/- 0.29; P = 0.0001), but not in 10 biopsies with meiotic germ cell arrest (mean, 1.56 +/- 1.07). Fas and FasL proteins were not expressed in cases of idiopathic hypogonadotropic hypogonadism. Together, these findings may suggest that Fas/FasL expression in the human testis is developmentally regulated and under gonadotropin control. The increased germ cell expression of Fas in patients with postmeiotic germ cell arrest suggests that the Fas/FasL system may be involved in the quality control mechanism of the produced gametes.

Effect of thyroid hormone on the pre- and post-natal development of the rat testis.

The relationship between thyroid function and testicular development in the rat was investigated. Hypothyroidism was induced during fetal or post-natal life by adding methimazole (MMI) to the drinking water of pregnant or lactating mothers. A group of newborn rats was treated with MMI and i.p. injections of L-tri-iodothyronine (L-T3). Hypothyroidism was shown by the reduced serum levels of total T3 and of total thyroxine (T4) in pregnant mothers and in pubertal rats. Testes were studied using light microscopy at 18 and 21 days post coitum or during puberty (21, 35 and 50 days after birth); serum levels of gonadotrophins were also evaluated in pubertal rats. Hypothyroidism had no effect on testicular development during fetal life and when induced in newborn rats it was associated at puberty with reduced serum levels of FSH and LH and with delayed maturation of the testis compared with control rats. The delay in maturation consisted of a reduction in the diameter of seminiferous tubules, and a reduction in the number of germ cells per tubule; this was associated with increased degeneration and arrested maturation of germ cells. In addition, Sertoli cells demonstrated retarded development, as indicated by a delay in the appearance of cytoplasmic lipids and in the development of a tubule lumen. Hormonal and morphological abnormalities were absent in rats treated with MMI plus L-T3. In conclusion, hypothyroidism occurring soon after birth caused reduced levels of gonadotrophins in the serum and a delay in pubertal spermatogenesis, possibly due to retarded differentiation of the Sertoli cells.

Sperm acrosin activity and fluorescence microscopic assessment of proacrosin/acrosin in ejaculates of infertile and fertile men.

OBJECTIVE: To compare biochemically active with immunoreactive sperm acrosin in fertile and infertile men. SETTING: This study was conducted in a tertiary care center, the Andrology Clinic, Department of Internal Medicine, University of L'Aquila. PATIENTS: We evaluated the males in 40 infertile couples with no recognized cause of female infertility and 20 fertile men. INTERVENTIONS: Ejaculates were collected under standardized conditions of abstinence. MAIN OUTCOME MEASURES: Total sperm acrosin activity was measured on a spectrophotometer in washed sperm stored at -80 degrees C for 1 to 6 days. The percent of spermatozoa immunostained by an antiserum against proacrosin/acrosin by indirect immunofluorescence (IFL) was determined on methanol fixed sperm smears. RESULTS: Biochemically active acrosin was correlated to immunoreactive acrosin (P = 0.0028), and both were inversely correlated to the percent of spermatozoa with an abnormal head (P = 0.00024 for acrosin activity and P = 0.0013 for IFL). Biochemically active and immunoreactive acrosin were lower in infertile compared with fertile men (P = 0.0012 and P = 0.0009, respectively). Sixty-eight percent of ejaculates with an acrosin activity lower than the limit value observed in fertile men showed a normal sperm morphology and a normal immunoreactivity for acrosin. CONCLUSIONS: A low sperm acrosin activity in teratospermic ejaculates is because of a lack or a defect of the immunogenic and functional domains of the protein. A low sperm acrosin in infertile men with normal semen parameters results from a possible functional defect of the enzyme that is immunohistochemically detected in spermatozoa.

Ultrastructure of fetal human gonad before sexual differentiation and during early testicular and ovarian development.

The development of the human gonad was investigated by light and transmission electron microscopy in 20 embryos and fetuses between 4.5 and 11.5 weeks of gestation, i.e. during the stages of sex-indifferent gonad, initial testicular and ovarian development. The gonadal blastema in 4.5-week-old embryos appeared formed by poorly differentiated somatic mesothelial cells, and by specialized germ cells (PGCs) with signs of ameboidism, cellular structures suggesting active protein biosynthesis and mitotic activity. The sexual differentiation of the gonads was clearly observed in 7-week-old embryos and involved at the same time the testis and the ovary. The former contained seminiferous cords formed by palisades of poorly differentiated Sertoli cells, which were segregated from the mesothelium by a rudimentary albuginea. The interstitial tissue at this age contained mesenchymal cells. Between 8 and 11.5 weeks of gestation, there was a synchronous cytodifferentiation of both Sertoli and Leydig cells. The latter acquired features of steroidogenic elements. The ovaries of 7-week-old fetuses contained packed ovigerous cords formed by somatic and germ cells (oogonia). The former embraced the oogonia with thin overlapping cytoplasm projections, and acquired features similar to those of cells in primary follicles, already at this early fetal age. At the same time the sexual differentiation of the gonads involved somatic and germ cells. In the female, the oogonia continued to show the main features they had during migration and colonization, including a high mitotic rate, signs of ameboidism and a developed apparatus for protein synthesis. On the contrary Gonocytes enclosed in the seminiferous cords progressively entered a quiescent phase characterized by a reduced mitotic rate, a decrease of endoplasmic reticulum and nucleolar complexity. The chronological relationship between the cytodifferentiation of Sertoli and Leydig cells, and changes of germ cells, suggest that somatic components of the testis may contribute to a male type of differentiation of germ cells from the very beginning of sexual differentiation.

Within-subject variation of seminal parameters in men with infertile marriages.

As little information exists on the semen variability in infertile men, this study aimed at analysing the within-subject variability of semen from men with infertile marriages included in an intrauterine insemination (IUI) programme. Five ejaculates from each of 436 men (2180 specimens) were analysed. The within-subject coefficients of variation (CV(w)) were high for all parameters (semen volume, sperm concentration, forward motility and combined parameters), ranging from 0.73 for the total motile sperm count to 0.27 for the semen volume. Nevertheless, within-subject fluctuations were smaller than the between-subject variability, as indicated by high Intraclass Correlation Coefficient (ICC) values, which, however, significantly lowered when

Immunophenotypical characterization of contractile cells in caput epididymidis of men affected by congenital or post-inflammatory obstructive azoospermia.

Myoid cells of the human caput epididymidis are replaced by large cells with ultrastructural features of smooth muscle cells (SMC) in chronic obstruction of the male genital tract. To evaluate whether these cellular changes are associated with different functional phenotypes we analysed the immunohistochemical expression of myosin heavy chain isoforms and of extracellular matrix (EM) components in the human caput epididymidis contractile cells in normal and in obstructed epididymides. Normal caput epididymidis myoid cells expressed a scattered immunostaining for SM2, marker of differentiated contractile SMC, while no staining was detected for SMemb (the non-muscle-type myosin heavy chain isoform) and for its transcription factor BTEB2, markers of undifferentiated proliferating SMC. A faint immunoreaction (IR) for EM was observed in the peritubular wall of the normal caput. In the contractile wall of the obstructed caput epididymidis a strong IR was detected for all myosin heavy chain isoforms as well as for collagen type IV and for fibronectin, markers for a secretory function of SMC. These findings, unknown in other models of SMC pathophysiology, suggest that myoid cells resume the molecular machinery of both mature SMC and of differentiating/secretory cells in the chronic obstruction of the human caput. Contractile cells of the epididymal duct represent a unique model to study the plasticity of SMC.

Postnatal development and distribution of peptide-containing nerves in the genital system of the male rat. An immunohistochemical study.

The distribution and relative density of peptide-containing nerves was studied in the rat in order to assess the progression of neuronal changes during the postnatal development of the male genital system from the prepubertal age to adulthood. Testis, caput and cauda epididymis, ductus deferens, seminal vesicles, prostate and penis from 8-, 20-, 38-, and 70-day-old rats were sectioned and were immunostained with antisera to the neuropeptides calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), and to a general neuronal marker, protein gene product 9.5 (PGP 9.5). The testicular parenchyma and caput epididymis did not show any immunoreactivity. Very scattered CGRP-containing nerves were present in 8-day-old rats; numerous VIP-, CGRP-, and NPY-peptide-containing nerves were observed in the cauda epididymis, ductus deferens, accessory glands and penis of 20-day-old rats. The number of nerves increased in 35-day-old rats while no changes were observed in more adult rats. A parallel increase was seen for the immunostain for PGP 9.5. These data suggest that peptide-containing nerves appear in the genital system after birth and reach a full development before the completion of puberty. Peptide-containing nerves were visible first in the interstitial area and then spread in the muscular coat of the ducts, glands and of the blood vessels. While CGRP- and NPY-containing nerves were distributed in the vicinity of the muscle cells, VIP-containing nerves were also observed in the subepithelial regions, suggesting a possible role of this neuropeptide in the control of epithelial functions.

Quantitative parameters of seminiferous epithelium in secretory and excretory oligoazoospermia.

Testicular biopsy specimens from infertile men (sperm count, less than 10(6)/ml) were evaluated on 1-micron thick sections, and counts of stem cells and differentiated spermatogonia, primary spermatocytes, early and late spermatids, and Sertoli cells were compared to counts in six fertile men. Biopsy specimens were also compared for the appearance of seminiferous tubule wall, blood vessels, and interstitium. Infertile men were grouped according to the following diagnoses: hypospermatogenesis (n = 5), spermatocyte arrest of spermatogenesis (n = 5), and obstruction of the genital tract (n = 7). A low productivity of spermatogenesis in cases of hypospermatogenesis appeared to be due to an exaggerated degeneration of primary spermatocytes and to a yield of abnormal spermatids. A block of meiosis in spermatocyte arrest was associated with a degeneration of primary spermatocytes and with a reduced number of staminal spermatogonia. Abnormal spermiogenesis was observed in cases of obstruction of the genital tract and was associated with an increase in stem cell spermatogonia. A thickening of seminiferous tubule and blood vessel walls could be responsible for the limited functional capacity of Sertoli cells, causing altered spermiogenesis in cases of excretory azoospermia. A severe primitive failure of Sertoli cells in secretory oligoazoospermia could account for a deranged maturation and degeneration of premeiotic and postmeiotic germ cells.

Chromatin defects in normal and malformed human ejaculated and epididymal spermatozoa: a cytochemical ultrastructural study.

Cytochemical defects in chromatin were examined by transmission electron microscopy (TEM) after the staining by alcoholic phosphotungstic acid (PTA) of normal and malformed ejaculated spermatozoa from 35 male partners of infertile couples, and in six sperm samples retrieved from the caput epididymidis of men affected by obstructive azoospermia. PTA staining was also analysed in normal ejaculates of fertile men after incubation of the washed spermatozoa with dithiothreitol (DTT) to reduce disulfides to thiols, or with DTT followed by iodoacetamide, a blocking agent for thiol groups. PTA stained 63 (27-100)% of malformed heads and 25 (10-100)% of normal sperm heads (median (range) n = 35; P = 0.0001, Wilcoxon matched pairs test). The percentage of normal heads stained by PTA was negatively correlated with the percentage of heads of normal form, with condensed chromatin and a normal acrosome (Spearman r = 0.75; P = 0.0001), and positively correlated with the percentage of malformed heads after conventional TEM analysis (Spearman r 0.60; P = 0.0001). Staining with PTA in normal heads was not correlated with the presence of non-condensed chromatin in otherwise normal sperm heads evaluated by conventional TEM analysis. In spermatozoa recovered from the caput epididymidis, 15% of normal heads were stained with PTA, significantly fewer than in ejaculated sperm samples (P = 0.014). The reduction of disulfides to thiols was associated with PTA staining of all normal heads, and this was prevented by incubation with iodoacetamide. We conclude that PTA staining of the nuclei of human ejaculated spermatozoa may indicate a defect of chromatin condensation, owing to an excess of free thiol groups. The lower percentage of normal epididymal sperm heads that stained with PTA in cases of obstructive azoospermia compared with ejaculated sperm may be related to an overoxidation of thils owing to the ageing of spermatozoa.

Testicular changes after treatment with a GnRH analog (buserelin) in association with cyproterone acetate in men with prostatic cancer.

Twelve patients (age range: 53-78 years) with prostatic cancer were treated with Buserelin (1.2 mg/day) and cyproterone acetate (150 mg/day). Testicular biopsies performed after 13-96 weeks of treatment were compared to those obtained from 6 untreated men of similar age. Deranged spermatogenesis was observed in all but 1 treated patient. The appearance of immature Sertoli cells and atrophic Leydig cells suggests a condition of pharmacologic 'hypophysectomy'. The variable damage to the seminiferous epithelium and findings of an incomplete involution of Leydig cells suggested a decreased but still present testicular steroidogenesis.

Ultrastructural analysis of chromatin defects in testicular spermatids in azoospermic men submitted to TESE-ICSI.

BACKGROUND: Testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI) is offered to treat obstructive and non-obstructive azoospermia, but factors that influence the outcome of ICSI are not well defined. METHODS AND RESULTS: The percentage of elongated spermatids with normal chromatin condensation in azoospermic patients submitted for TESE-ICSI was determined. The quantitative analysis could be applied to nine of 19 biopsies classified as incomplete late maturation arrest (LMA) and compared with 10 biopsies with normal spermatogenesis. The percentage of elongated spermatids with normal chromatin was lower in LMA than in normal histology (mean 4.4%, range 0-20, and mean 52.9%, range 40-70 respectively; P = 0.0001). The percentage of elongated spermatids with normal chromatin was negatively correlated with the serum concentration of FSH (r = -0.86, P < 0.0001) and the number of degenerated germ cells per 100 Sertoli cells nuclei (r = -0.68; P < 0.0001), while it was positively correlated with the number of elongating spermatids per 100 Sertoli cell nuclei (r = 0.81; P < 0.0001). The percentage of elongated spermatids with normal chromatin was not correlated with the rate of oocyte fertilization, while the delivery rate/cycle was higher in cases with normal histology compared with cases of LMA. CONCLUSIONS: These preliminary data suggest that an altered chromatin condensation is a ubiquitous defect in spermatids of non-obstructed azoospermic men submitted for TESE-ICSI.

Carbohydrate binding activity in human spermatozoa: localization, specificity, and involvement in sperm-egg fusion.

Sperm carbohydrate binding activity is involved in gamete recognition. We identified a human sperm protein extracted under reducing conditions, and with a molecular mass of 65 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and which binds D-mannose coupled to albumin (DMA) in presence of cations and a neutral pH. Epifluorescence microscopy showed that fluorescein-DMA binds to dead or permeabilized sperm heads. The DMA-binding activity of human sperm heads was highly specific for a polysaccharide structure containing charged sugar residues. After capacitation, or induction of the acrosome reaction using solubilized zonae pellucidae, fluorescein-DMA was bound respectively to 10.3% (+/- 3.5%) and to 37.6% (+/- 2.1%) of viable sperm heads. The sequential analysis of viable spermatozoa for fluorescein-DMA binding and for rhodamine-Pisum sativum agglutinin binding, showed that DMA-binding sites are present in viable acrosome-reacted spermatozoa. Three dimensional analysis of fluorescence and ultrastructural studies showed that DMA-binding sites are mostly restricted to the sub-acrosomal space of the equatorial segment. Incubation of spermatozoa and zona-free hamster eggs in the presence of DMA was associated with a dose-dependent significant reduction in the number of spermatozoa bound to the oolemma, compared with a control, and to a dose-dependent inhibition of oocyte penetration. This effect was highly specific for DMA, suggesting that DMA-binding sites in human spermatozoa are involved in sperm-egg fusion.