Susan Benoff

Cadmium concentrations in blood and seminal plasma: correlations with sperm number and motility in three male populations (infertility patients, artificial insemination donors, and unselected volunteers).

To investigate a possible common environmental exposure that may partially explain the observed decrease in human semen quality, we correlated seminal plasma and blood cadmium levels with sperm concentration and sperm motility. We studied three separate human populations: group 1, infertility patients (Long Island, NY, USA); group 2, artificial insemination donors (AID) (Rochester, NY, USA); and group 3, general population volunteers (Rochester, NY, USA). Information about confounding factors was collected by questionnaire. Seminal plasma cadmium did not correlate with blood cadmium (Spearman correlation, n = 91, r = −0.092, P = 0.386, NS). Both blood and seminal plasma cadmium were significantly higher among infertility patients than the other subjects studied (for example, median seminal plasma cadmium was 0.282 µg/L in infertility patients versus 0.091 µg/L in AID and 0.092 µg/L in general population volunteers; Kruskal-Wallis test, P < 0.001). The percentage of motile sperm and sperm concentration correlated inversely with seminal plasma cadmium among the infertility patients (r = −0.201, P < 0.036 and r = −189, P < 0.05, respectively), but not in the other two groups. Age (among infertility patients) was the only positive confounder correlating with seminal plasma cadmium. To validate our human findings in an animal model, we chronically exposed adolescent male Wistar rats to low-moderate cadmium in drinking water. Though otherwise healthy, the rats exhibited decreases in epididymal sperm count and sperm motility associated with cadmium dose and time of exposure. Our human and rat study results are consistent with the hypothesis that environmental cadmium exposures may contribute significantly to reduced human male sperm concentration and sperm motility.

Voltage dependent calcium channels in mammalian spermatozoa.

Calcium influx is an absolute requirement for the physiological acrosome reaction in sperm from all sources examined, both invertebrate and mammalian. Pharmacological studies suggest that the major channel in the sperm head plasma membrane responsible for modulating calcium entry and intracellular ionized calcium levels could be either an L-type (a class of high voltage-activated) or a T-type (low voltage-activated) voltage-dependent calcium channel. Patch clamp analysis of calcium currents in immature spermatogenic cells demonstrates the presence of T-type currents. Therefore, an argument has been put forth that the acrosome reaction of ejaculated sperm is regulated by a T-type calcium channel. However, indirect analysis of calcium currents in mature sperm after transfer of ion channels to planar lipid bilayers detects three current types, including that similar, but not identical, to an L-type channel, but no T-type currents. Molecular cloning of the alpha-1 pore forming subunit of calcium channels expressed in the male reproductive tract and in ejaculated sperm has resolved this controversy, demonstrating the existence of only high voltage-activated channels. Further analysis of the alpha-1 subunit isoform from rat and human testis and sperm suggests that, as a result of alternate splicing, this L-type alpha-1 subunit could produce calcium currents that were T-like, e.g., transient, rapidly inactivating with slow deactivation. Multiple splice variants of this isoform were detected in human testis, suggesting a correlation with intra-individual variation in the ability of sperm to undergo an induced acrosome reaction and with male infertility. These variants could be developed as useful biomarkers for susceptibility to environmental and occupational toxicants. Knowledge of calcium channels structure will also contribute to design of new male contraceptives based on existing calcium channel antagonists.

Antisperm antibody binding to human sperm inhibits capacitation induced changes in the levels of plasma membrane sterols

PROBLEM: Human spermatozoa express mannose ligand receptors (MLRs) over the entire head when incubated under conditions that promote loss of free cholesterol. Binding of IgA, IgG. and/or IgM head‐directed anti‐sperm antibodies (ASAs) to freshly isolated sperm blocks MLR expression in a dose dependent manner.

Co-Expression of Mannose-Ligand and Non-Nuclear Progesterone Receptors on Motile Human Sperm Identifies an Acrosome-Reaction Inducible Subpopulation

PROBLEM: To determine whether surface expression of receptors for progesterone and mannose can be used to identify spermatozoa likely to undergo an acrosome reaction after zona binding and to compare the reactivity of these receptors with naturally occurring sperm head‐directed anti‐sperm antibodies (ASAs).

Molecular and other predictors for infertility in patients with varicoceles.

Varicoceles are a treatable cause of male infertility, but very clinically diverse. Both histologic and molecular changes occur in the testes of men with varicocele. Physical measurements (scrotal temperature, testicular volume, pressure within the pampiniform plexus, basal lamina thickness) correlate with prognosis, but these correlations have not been accepted as predictors of successful repair because of variation within patient populations. Conventional semen parameters similarly correlate, but these correlations apply only to men with >5 x106 sperm/ejaculate. Levels of toxicants (e.g. norepinephrine, cadmium), reactive oxygen species byproducts, and hormones, their receptors and modulators have been evaluated as predictors in small-scale studies. Medical therapies (antoxidants, anti-inflammatories and hormones) have been applied empirically to small groups of patients with positive results that have not been verified in large-scale trials. Thus, urologists still face a challenge to determine which patients will benefit from varicocelectomies and/or medical interventions. In this review we summarize our current understanding of the pathophysiology of varicoceles, and discuss some of the new findings that may be applicable to specific clinical situations.

Voltage-dependent calcium channels in mammalian spermatozoa revisited.

The last few years have seen an explosion in the number of voltage-dependent ion channel sequences detected in sperm and testes. The complex structural paradigm of these channels is now known to include a pore-forming alpha1 subunit(s) whose electrophysiological properties are modulated by an intracellular beta subunit, a disulfide-linked complex of a membrane-spanning delta subunit with an extracellular alpha2 subunit, and a transmembrane gamma subunit. Many of these are alternatively spliced. Furthermore, the known number of genes coding each subtype has expanded significantly (10 alpha1, 4 beta, 4 alpha2delta, 8 gamma). Recently, the CatSper gene family has been characterized based on similarity to the voltage-dependent calcium channel alpha1 subunit. From among this multiplicity, a wide cross-section is active in sperm, including many splice variants. For example, expression of the various alpha1 subunits appears strictly localized in discrete domains of mature sperm, and seems to control distinct physiological roles such as cellular signaling pathways. These include alpha1 alternative splicing variants that are regulated by ions passed by channels in developing sperm. Various combinations of ion channel sequence variants have been studies in research models and in a variety of human diseases, including male infertility. For example, rats that are genetically resistant to testes damage by lead seem to respond to lead ions by increasing alpha1 alternative splicing. In contrast, in varicocele-associated male infertility, the outcome from surgical correction correlates with suppression of alpha1 alternative splicing, Ion channel blockers remain attractive model contraceptive drugs because of their ability to modulate cholesterol levels. However, the large number of sperm ion channel variants shared with other cell types make ion channels less attractive targets for male contraceptive development than a few years ago. In this review, the genetics, structure and function of voltage-dependent calcium channels and related CatSper molecules will be discussed, and several practical clinical applications associated with these channels will be reported.

The effect of calcium ion channel blockers on sperm fertilization potential*†*Supported in part by an office based research grant from the American Foundation for Urologic Disease with funds contributed by Searle.†The Society for Assisted Reproductive Technology Prize Paper presented at the 49th Annual Meeting of The American Fertility Society, Montreal, Quebec, Canada, October 11 to 14, 1993.

OBJECTIVE To evaluate the effects of calcium ion (Ca2+) channel blockers on male fertility potential. DESIGN A case comparison of the surface expression of mannose-ligand receptors on motile spermatozoa from 10 known fertile males and from 10 normospermic men taking Ca2+ channel blockers who were seeking infertility treatment. Examination of the effects of in vitro exposure of sperm from fertile donors (n = 14) to antihypertensive medications. SETTING Patients from a successful university hospital-based IVF-assisted reproductive technology program and from a male urology private practice. INTERVENTIONS Prescription of alternate hypotensive medications for four male patients; cholesterol loading and unloading in vitro of fertile donor sperm. MAIN OUTCOME MEASURES Motile sperm were tested for their ability to bind fluorescein isothiocyanate-labeled, mannosylated bovine serum albumin as an index of the surface expression of mannose-ligand receptors associated with fertility potential. Acrosome status was simultaneously evaluated by fluorescence microscopy with rhodamine-labeled Pisum sativum lectin. Sperm were assayed before and after an 18-hour or 3-day incubation under capacitating conditions in vitro. RESULTS Motile spermatozoa of normospermic men taking calcium antagonists for hypertension control do not express head-directed mannose-ligand receptors at high frequency, nor do they undergo spontaneous acrosome loss. Unexpectedly, mannose-ligand receptor translocation from the subplasmalemmal space over the acrosome to the sperm surface and aggregation over the equatorial-postacrosomal regions occurred in acrosome-intact sperm. This differs from fertile controls in whom receptor translocation to the equatorial-postacrosomal segment is coupled with the acrosome reaction (AR). Discontinuation of calcium antagonists results in complete recovery of parameters associated with sperm fertilizing potential: time-dependent increases in the percentages of spermatozoa exhibiting surface mannose-ligand binding and spontaneous ARs in vitro. The effects of in vivo administration of calcium antagonists is mimicked in control fertile donor sperm by inclusion of a Ca2+ channel blocker in the media employed during capacitating incubations. CONCLUSIONS Therapeutic administrations of calcium antagonists for hypertension control cause reversible male infertility associated with an IVF failure. A mechanism of inhibition of sperm fertilizing potential through insertion of lipophilic calcium ion antagonists into the lipid bilayer of the sperm plasma membrane is consistent with our in vitro studies.

Human sperm fertilizing potential in vitro is correlated with differential expression of a head-specific mannose-ligand receptor**Supported in part by grant RR05924 from the National Center for Research Resources Biomedical Research Support Program, National Institutes of Health, Bethesda, Maryland.††Presented in part at the 39th Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 18 to 21, 1992.

OBJECTIVE To determine whether different patterns of surface expression of mannose-ligand binding sites are correlated with capacitation and predictive of the ability of human sperm to recognize and fertilize eggs in vitro. DESIGN Analysis of motile sperm populations (from fertile donors, males presenting for routine semen analysis, and men undergoing IVF) before and after incubation in capacitating media. SETTING Patients from an infertility practice at a major university hospital. INTERVENTIONS None. MAIN OUTCOME MEASURES The mannose ligand-binding capacity of sperm populations were initially assayed by solid-phase mannosylated polyacrylamide bead binding studies. Sperm surface D-mannose binding sites thus detected were localized and visualized by fluorescence microscopy after reaction with a mannosylated fluorescein isothiocyanate-labeled neoglycoprotein probe. Results were correlated with acrosomal status, reproductive histories, and IVF outcomes. RESULTS The percent of sperm with head-directed surface expression of a mannose-specific receptor was increased in fertile donors and males exhibiting normal fertilization in IVF after incubation in albumin-supplemented Hams F-10 medium (GIBCO Laboratories, Grand Island, NY). In normospermic males exhibiting zona binding failure in IVF, mannose-specific receptor was observed over the head surface of few incubated sperm. CONCLUSIONS The appearance of D-mannose-ligand binding sites on the surface of heads of human spermatozoa is associated with zona binding ability in IVF and is a putative determinant in human gamete recognition and fertilization.