Richard Bronson

Loss of Nectin-2 at Sertoli-Spermatid Junctions Leads to Male Infertility and Correlates with Severe Spermatozoan Head and Midpiece Malformation, Impaired Binding to the Zona Pellucida, and Oocyte Penetration

Abstract The members of the nectin/CD155 gene family represent a growing class of novel cell adhesion molecules of the immunoglobulin superfamily. In the present study, we describe the generation of a mouse line lacking a functional nectin-2 gene (nectin-2LacZ/LacZ) and analyze the resulting male-specific infertility phenotype. Although nectin-2LacZ/LacZ males produced normal amounts of motile spermatozoa, scanning electron microscopy revealed severe malformations of the spermatozoan head and midpiece. Besides a 4-fold reduction in migration of nectin-2LacZ/LacZ spermatozoa to the oviducts, in vitro binding to zona-intact mouse oocytes was reduced 6-fold. On the other hand, nectin-2LacZ/LacZ spermatozoa bound to zona-free hamster oocytes at near-wild type levels but, remarkably, failed to penetrate. In addition to the previously reported expression of nectin-2 and nectin-3 at Sertoli-spermatid junctions and of nectin-2 at inter-Sertoli cell junctions, we also found nectin-2 to localize at apical cell-cell junctions of the epididymal epithelium. Expression analysis of a LacZ knockin gene into the defunct nectin-2 gene in nectin-2LacZ/LacZ mice provided additional support for our earlier conjecture that in normal testis, nectin-2 is produced exclusively by Sertoli cells. Finally, we found Sertoli-spermatid junctions in nectin-2LacZ/LacZ mice to be virtually devoid of the actin-bundling protein espin, suggesting that ectoplasmic specializations fail to form in the absence of nectin-2. Our functional analyses indicate that the infertility phenotype of nectin-2-deficient male mice is caused by a combination of reduced migration to the oviduct, spermatozoa-zona binding, and sperm-oocyte fusion. We corroborate our previous description of a heterotypic adhesion complex between Sertoli cells and elongated spermatids that is maintained by nectin-2 and nectin-3, respectively.

Sperm-oolemmal interaction: role of the Arg-Gly-Asp (RGD) adhesion peptide.

The Arg-Gly-Asp tripeptide (RGD) plays a widespread role in cell-to-cell and cell-to-matrix recognition. We demonstrated that an RGD-containing peptide (Arg-Gly-Asp-Val, RGDV) inhibits both oolemmal binding and penetration of zona-free hamster eggs by human spermatozoa in vitro when added to the incubation medium. These results suggest that RGD-containing proteins may play a role in sperm-oolemmal interactions required for fertilization.

Identification of an Oolemmal IgG Fc Receptor: Its Role in Promoting Binding of Antibody-Labelled Human Sperm to Zona-Free Hamster Eggs

ABSTRACT: Antisperm antibodies (ASAs) present in sera of infertile men and women have been shown either to promote or inhibit penetration of zona‐free hamster eggs by antibody‐labelled human spermatozoa. Increased numbers of oolemmal‐bound sperm have been noted in association with increased sperm penetration frequencies, following antibody labelling, when compared with antibody‐free sperm. The promotion of adherence of ASA‐labelled sperm to the oolemma could be mediated through the binding of antibodies to common epitopes present on the sperm and egg surfaces or through Fc‐mediated binding to an oolemmal Fc receptor. In support of the latter hypothesis, we report that zona‐free hamster eggs bind aggregated human IgG and IgG Fc fragments. The presence of an oolemmal IgG Fc receptor has been confirmed using a rat monoclonal antibody (2.4G2) directed against a murine IgG Fc receptor (Fc γ RII) as judged both by indirect immunofluorescence and by immunobead binding. In addition, the pre‐incubation of zona‐free hamster eggs with IgG Fc diminished both adhesion to and penetration of the oolemma by human spermatozoa.

Monoclonal antibodies identify Fcγ receptors on unfertilized human oocytes but not spermatozoa

Abstract Oolemmal Fc receptors have previously been shown to play a role in the promotion of adhesion by antibody labeled human spermatozoa to zona-free hamster eggs. In this work, we demonstrated the presence of FcγRI, FcγRII and FcγRIII on the oolemma of unfertilized human oocytes by means of monoclonal antibodies directed against these receptors, detected both by immunobead rosetting and indirect immunofluorescence. These receptors were also functionally active in that they were able to bind human aggregated IgG, human IgG-Fc, mouse IgG 1 and IgG 2a . While the presence of oolemmal IgG-Fc receptors might play a role in reproductive failure, by their promotion of polyspermic fertilization, in cases where antisperm antibodies bound to the spermatozoan surface, their role in the normal physiology of fertilization or in other events unrelated to sperm incorporation remains to be determined. In contrast, Fcγ receptors were not present on human spermatozoa, irrespective of their functional state (fresh ejaculated, capacitated or acrosome reacted).

Laparoscopic gonadectomy for gonadal dysgenesis

Females with a 46,XY karyotype have approximately a 25% chance of developing a malignancy in the dysgenetic gonad. For this reason, it has been recommended that all patients with Y-chromosomal material and dysgenetic gonads undergo exploratory laparotomy and gonadectomy. This report describes laparoscopic adnexectomy in a patient with gonadal dysgenesis

Surface Expression of Complement Receptor gC1q-R/p33 Is Increased on the Plasma Membrane of Human Spermatozoa after Capacitation

Abstract Evidence is increasing that complement components might play a role in fertilization. C1q, the first component of the classical complement cascade, has the ability to promote sperm agglutination in a capacitation-dependent manner as well as an effect on sperm-oolemma binding and fusion. We have previously detected gC1qR, the receptor for the globular head portion of C1q, on the surface of capacitated sperm. In this study, we examined the expression of gC1qR in both fresh and capacitated human spermatozoa. We performed immunoprecipitation for gC1qR and analyzed biotinylated sperm membrane by Western blot to illustrate an increase in receptor density after overnight capacitation. These results were confirmed by flow cytometric analysis of spermatozoa using fluorescein isothiocyanate-labeled monoclonal anti-gC1qR antibody. Confocal, indirect immunofluorescence microscopy revealed an increase in receptor expression over the rostral portion of the sperm head after capacitation. In addition, the ability of live spermatozoa to bind to monoclonal anti-gC1qR antibody-coated microtiter wells was also increased after capacitation. These results suggest that gC1qR may play a role in human fertilization.

Biology of the male reproductive tract: its cellular and morphological considerations.

Citation u2028Bronson R. Biology of the male reproductive tract: Its cellular and morphological considerations. Am J Reprod Immunol 2011; 65: 212–219

Acrosin antibodies and infertility. I. Detection of antibodies towards proacrosin/acrosin in women consulting for infertility and evaluation of their effects upon the sperm protease activities

OBJECTIVEnTo detect the presence of antibodies to the proacrosin/acrosin system and to evaluate their effect on the sperm acrosomal protein activities in women consulting for infertility.nnnDESIGNnRetrospective study.nnnSETTINGnBasic research laboratory.nnnPATIENT(S)nRecombinant proteins derived from human proacrosin (Rec-40, Rec-30, Rec-20, Rec-10) and recombinant human zona pellucida (ZP) glycoprotein A( *) (rec-hZPA).nnnINTERVENTION(S)nDevelopment of an ELISA-Acro to test for antiacrosin antibodies using Rec-40 and truncated acrosin proteins as antigens.nnnMAIN OUTCOME MEASURE(S)nEvaluation of: 1) the presence of antiacrosin antibodies; 2) the protein regions recognized by the antibodies; 3) the relationship between antiacrosin antibodies and surface antisperm antibodies (ASA) identified by the immunobead binding test (IBT); and 4) the effect of antiacrosin antibodies upon proacrosin/acrosin binding activity to ZPA and acrosin amidase activity.nnnRESULT(S)nAntiacrosin antibodies were detected in sera from 34 of 179 women (19%). Detection of ASA by the IBT resulted in a similar incidence (36 of 179, 20%), although only six of them showed correspondence between both assays; five of these six sera were IBT-positive IgGs to the sperm head. Antiacrosin antibodies directed toward different protein regions inhibited proacrosin binding activity to rec-hZPA as well as its activation and acrosin amidase activity in protein sperm extracts.nnnCONCLUSION(S)nAntiacrosin antibodies are present in sera of women consulting for infertility in both IBT-positive and IBT-negative samples, and they affect proacrosin/acrosin activities.

Detection of complement C1q receptors on human spermatozoa

Clq, the first component of the classical complement pathway, is known to play roles in promoting phagocytic events, in addition to its role in activation of complement. Although the molecular events in fertilization leading to the entrance of the spermatozoan into the egg are not well understood, ultrastructural observations suggest that the process is quasi-phagocytic in nature. There is increasing evidence that complement components might play roles in fertilization. Previously, we have shown that C1q promoted the agglutination of capacitated human sperm as well as their adhesion to zona-free hamster eggs. In the present experiments, human spermatozoa were solubilized and, following their phase separation in Triton X-114, subject to 1-D polyacrylamide gel electrophoresis and immunoblotting for the presence of C1q receptors. Both gC1q-R and cC1q-R were detected. In addition, the ability of C1q to promote sperm agglutination was shown to be dependent upon capacitation, suggesting the increased expression of C1q receptors during this process.

An investigation of the latency period between sperm oolemmal adhesion and oocyte penetration

In clinical studies of the ability of capacitated human sperm to penetrate zona‐free hamster eggs, we have previously observed that the ratio of oolemmal adherent to penetrating sperm varied between men. Sperm incorporation did not occur immediately following gamete adhesion and not all adherent sperm penetrated the egg. To further investigate this phenomenon, comparisons were made of the kinetics of gamete adhesion, membrane fusion, and sperm incorporation of capacitated mouse and human spermatozoa by zona‐free hamster eggs and of mouse sperm by zona‐free mouse and hamster eggs. Eggs were inseminated with either capacitated human or mouse sperm or combinations of both, washed out of sperm suspension after initial gamete adherence, and further incubated in sperm‐free medium. Gamete membrane fusion was judged by dye transfer of Hoechst 33342 and sperm entry of the cortical ooplasm by observation of expanded sperm heads within acridine orange stained eggs. Oolemmal adherent mouse and human sperm fused with and penetrated zona‐free hamster eggs at different times whether eggs were inseminated in parallel or with combinations of sperm of both species. Oolemmal adherent mouse sperm penetrated zona‐free hamster eggs prior to their penetration of zona‐free mouse eggs. Ultrastructural studies of zona‐free human eggs inseminated with human sperm confirmed prior observations with hamster eggs that only acrosome‐reacted human sperm adhere to the oolemma. These results have lead us to postulate that sperm entry into the egg may occur through a “zipper” mechanism involving the ligation of local gamete receptors similar to the incorporation of target particles by phagocytes and suggest that not all oolemmal adherent human sperm are capable of being incorporated although they have undergone an acrosome reaction. Mol. Reprod. Dev. 52:319–327, 1999.