Lynne Vigue

Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status

OBJECTIVE To test, both in semen and washed-sperm fractions, whether hyaluronic acid (HA) binding is restricted to sperm that have completed cellular maturation. DESIGN Comparisons of sperm in semen and in HA-bound sperm fractions. SETTINGS University-based diagnostic and research andrology laboratory. PATIENT(S) Semen samples originated in men being tested for infertility. INTERVENTION(S) The attributes of sperm maturity were tested by immunocytochemistry with creatine kinase and HspA2 antisera (highlights cytoplasmic retention in diminished-maturity sperm), aniline blue chromatin staining (detects persistent histones), pisum sativum lectin staining (reveals acrosomal integrity), and the FertiLight viability kit (highlights viable and nonviable sperm). RESULT(S) All markers of sperm maturity and immaturity supported the hypothesis that HA-bound sperm are mature. Nonbinding sperm exhibited cytoplasmic and nuclear properties of diminished maturity. The acrosomal status of HA-bound sperm was either unreacted or slightly capacitated, but not acrosome reacted. Only viable sperm exhibited HA binding. CONCLUSION(S) Sperm that are able to bind to HA are mature and have completed the spermiogenetic processes of sperm plasma membrane remodeling, cytoplasmic extrusion, and nuclear histone-protamine replacement. Hyaluronic acid-bound sperm show unreacted acrosomes. These studies provide further insights into the relationship between spermiogenesis and sperm function.

Putative Creatine Kinase M-Isoform in Human Sperm Is Identifiedas the 70-Kilodalton Heat Shock Protein HspA2

Abstract We previously described a putative creatine kinase M isoform in human sperm that is developmentally regulated and expressed during late spermiogenesis, simultaneous with cytoplasmic extrusion. We have now identified this protein as the testis-expressed 70-kDa heat shock protein chaperone known as HspA2 (the human homologue of mouse Hsp70-2). We have isolated and characterized HspA2 (formerly CK-M) by amino acid sequencing and have localized it by immunocytochemistry to spermatocytes at low levels, to spermatids, and in the tail of mature sperm. The specificity of the CK-M/HspA2 antiserum to HspA2 was demonstrated on immunoblots of one- and two-dimensional SDS-PAGE. In agreement with our earlier biochemical data, immunocytochemistry of testicular tissue indicated that HspA2 is selectively expressed in mature spermatids and in sperm about to be released in the seminiferous tubuli. The identity of HspA2 has been further confirmed by cross-absorption of the mouse HSP70-2 antibody by the HspA2/CK-M fraction, and by identical immunostaining patterns of human testicular tissue using either the anti-CK-M/HspA2 or an anti-mouse Hsp70-2 antisera. During spermiogenesis, both cytoplasmic extrusion and plasma membrane remodeling, which facilitate the formation of the zona pellucida binding site, involve major intrasperm protein transport, which may be chaperoned by HspA2. Accordingly, in immature human sperm, which fail to express HspA2, there is cytoplasmic retention and lack of zona pellucida binding. The present findings provide the biological rationale for the role of the human HspA2 as an objective biochemical marker of sperm function and male fertility, which we have established in earlier clinical studies.

Creatine kinase immunocytochemistry of human sperm-hemizona complexes : selective binding of sperm with mature creatine kinase-staining pattern

Objective To examine the clinical significance of the increased sperm cytoplasmic content that is due to a fault of spermatogenesis, we have further studied the relationship between increased sperm creatine kinase (CK) concentrations and diminished fertilizing potential in men. In the present work, we used CK immunocytochemistry of human sperm-hemizona (HZ) complexes to examine whether the distribution of mature (clear heads), intermediate (sperm heads with light stippling), and immature (heads with heavy stippling or with solid CK staining) spermatozoa bound to the HZ would follow the incidence of these sperm in the samples tested, or if there is a preferential binding by the mature sperm. Design Two pairs of HZ were exposed to washed semen and to their swim-up sperm fractions. The sperm and sperm-HZ complexes were treated with a CK antibody followed by horseradish peroxidase immunostaining, and the sperm were evaluated for maturity. Setting Men presenting for fertility evaluation were studied in two university-based andrology laboratories. Results The binding of the HZ was selective for mature sperm as indicated by the incidence of intermediate and immature sperm in washed semen versus bound to the HZ (intermediate: 20.0% versus 1.4%; immature: 7.6% versus 0.5% [mean ± SEM]) or in swim-up sperm fractions versus the HZ (intermediate: 18.7% versus 3.4%; immature: 2.5% versus 0.2%). The binding was almost exclusive to normal sperm (96.4% to 98.1%) whether the HZ were exposed to washed semen or swim-up fractions in spite of the five to ten times higher incidence of intermediate and immature sperm. Conclusions Mature sperm selectively bind to the zona. We suggest that spermatozoa with immature CK-staining patterns are deficient in the site(s) of oocyte recognition and binding.

Selectivity of hyaluronic acid binding for spermatozoa with normal Tygerberg strict morphology

During spermiogenesis, a plasma membrane remodelling step facilitates formation of sperm zona pellucida and hyaluronic acid (HA) binding sites. Enrichment of Tygerberg normal spermatozoa in HA-bound versus semen sperm fractions was postulated. Semen was placed on the uncoated A side and HA-coated B side of a semen chamber. After 15 min, the HA binding score (proportion of HA-bound motile spermatozoa) was assessed on the B side, and unbound spermatozoa were removed by gentle rinsing. Following Diff-Quick staining, sperm morphology of A and B sides was evaluated by three blinded investigators at Yale and Tygerberg. The proportion of Tygerberg normal spermatozoa was higher in HA-bound versus semen spermatozoa (n = 63 subjects) with a 3.04-fold improvement (95% confidence limits: 1.9-4.7) in 37 teratozoospermic men, comparable with a 4.2-fold enrichment in zona pellucida-bound spermatozoa reported earlier by the Tygerberg group. The morphology scores of three investigators were different but related, indicating that the variations reflect individual-to-individual differences in the perception of shape normality. The selection power of HA and zona pellucida for normal spermatozoa are similar. The sperm biomarkers of creatine phosphokinase (reflecting retained cytoplasm in arrested maturity spermatozoa) and chaperone protein HspA2 (heat shock protein) were proportional with sperm HA binding. As HA binding reflects sperm maturity and function, the combination of Tygerberg morphology and HA binding is likely to improve male infertility management.

Biochemical markers of early and late spermatogenesis: Relationship between the lactate dehydrogenase‐X and creatine kinase‐M isoform concentrations in human spermatozoa

As part of our research program on biochemical markers of sperm maturity, we have studied sperm creatine kinase (CK) and lactate dehydrogenase (LDH) concentrations and the ratios of the CK‐M [ ÷ CK‐M/ (CCU‐M + CU‐B)] and LDH‐×[÷ LDH‐X/(LDH‐X + LDH‐a)] isoforms in 50 oligospermic and 95 normospermic men. Because the synthesis of LDH‐X is initiated in early spermatogenesis, and that of CK‐M commences in late spermiogenesis simultaneously with cytoplasmic extrusion, we proposed two working hypotheses: (1) LDH and CK concentrations reflect the retained cytoplasm in sperm, thus the activities of both enzymes will be related and will be higher in oligospermic specimens, which have a higher incidence of immature spermatozoa; and (2) because in normally developed sperm both LDH‐X and CK‐M are present, there will be a correlation between LDH‐X and CK‐M ratios in the mature sperm populations. However, among men with immature sperm samples with late spermiogenetic defect and diminished CK‐M ratios, there will be two groups: one which completed spermatogenesis prior to spermiogenetic failure (normal LDH‐X and diminished CK‐M ratios), and another group with defects in both spermatogenesis and spermiogenesis (low LDH‐X and diminished CK‐M ratios). Because of this heterogeneity, LDH‐X ratios will be a poor predictor of sperm maturity. The data support the hypotheses: (1) LDH and CK concentrations were higher in oligospermic vs. normospermic men (P < 0.001). (2) The LDH and CK concentrations were related (r = 0.65, P < 0.001, N = 145), and there were inverse correlations between CK, LDH, LDH‐X, or CK‐M ratios vs. sperm concentrations (P < 0.001 in all four). (3) The CK‐M and LDH‐X ratios were different between the oligospermic and normospermic groups (P < 0.001), although the means of the LDH‐X ratios were narrower (LDH‐X:1:1.3; CK‐M:1:1.9). (4) Dividing the 145 samples by the cut‐off value of mean minus 1 SD of the CK‐M and LDH‐X ratios (11% and 32%, respectively) demonstrated that the CK‐M ratios discriminated better than LDH‐X ratios between the samples with mature and immature sperm. These data on the biochemical markers of early and late spermatogenesis support the studies in which CK better reflected sperm quality than LDH or LDH‐X (Orlando et al., 1994: Int J Androl 17:13–18) and the >10% sperm CK‐M ratio predicted with a rate of 30.4% per cycle in the occurrence of pregnancies in a blinded study of 84 IVF couples (Huszar et al., 1992: Fertil Steril 57:882–888).