Glass wool column filtration for stallion sperm selection: A comparative analysis with the single-layer colloid centrifugation.

Glass wool column filtration (GWCF) selects human, bull, boar, dog and buffalo spermatozoa, but reports in the horse are scarce. Single-layer colloid centrifugation with Androcoll-E™ is currently the standard procedure to select good-quality equine sperm. This study was designed to assess GWCF (50 and 75 mg columns; GWCF-50 and GWCF-75, respectively) efficacy to select good-quality sperm from fresh and frozen-thawed equine semen, and to compare its performance with Androcoll-E™ colloid centrifugation. Percentage total motile (TM), progressively motile (PM), morphologically normal (MN), osmotically competent (HOS+) and acrosome-intact/osmotically competent (AI/HOS+) sperm were determined. In studies done with fresh semen samples (n = 17), suspensions subjected to GWCF-50 showed an improvement (p < .05) in PM and HOS+ sperm after selection. With GWCF-75, an increase (p < .05) in PM, MN and HOS+ sperm was observed. Results with GWCF were comparable or better than with Androcoll-E™ selection. Sperm recovery was similar between procedures for all semen parameters. Total sperm count recovery was lower after GWCF-75 (GWCF-50 = 60.0; GWCF-75 = 51.0; Androcoll-E™ = 76.0 million sperm; median; p = .013), but results on total progressive sperm count were similar (GWCF-50 = 23.0; GWCF-75 = 27.0; Androcoll-E™ = 24.0 million sperm; median; p = .3850). Using frozen-thawed semen samples (n = 16), an improvement (p < .05) in TM, PM, NM, HOS+ and AI/HOS+ sperm was observed in GWCF-75 filtrates. Results were comparable to Androcoll-E™ centrifugation, except HOS+ that increased (p < .05) only after GWCF-75. Recovery was comparable for all parameters in frozen samples. GWCF is a simple and low-cost procedure that selects equine sperm with a quality comparable to colloid centrifugation with Androcoll-E™.

Capacitation-Induced Mitochondrial Activity Is Required for Sperm Fertilizing Ability in Mice by Modulating Hyperactivation.

To become fully competent to fertilize an egg, mammalian sperm undergo a series of functional changes within the female tract, known as capacitation, that require an adequate supply and management of energy. However, the contribution of each ATP generating pathway to sustain the capacitation-associated changes remains unclear. Based on this, we investigated the role of mitochondrial activity in the acquisition of sperm fertilizing ability during capacitation in mice. For this purpose, the dynamics of the mitochondrial membrane potential (MMP) was studied by flow cytometry with the probe tetramethylrhodamine ethyl ester (TMRE). We observed a time-dependent increase in MMP only in capacitated sperm as well as a specific staining with the probe in the flagellar region where mitochondria are confined. The MMP rise was prevented when sperm were exposed to the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) or the protein kinase A (PKA) inhibitor H89 during capacitation, indicating that MMP increase is dependent on capacitation and H89-sensitive events. Results showed that whereas nearly all motile sperm were TMRE positive, immotile cells were mostly TMRE negative, supporting an association between high MMP and sperm motility. Furthermore, CCCP treatment during capacitation did not affect PKA substrate and tyrosine phosphorylations but produced a decrease in hyperactivation measured by computer assisted sperm analysis (CASA), similar to that observed after H89 exposure. In addition, CCCP inhibited the in vitro sperm fertilizing ability without affecting cumulus penetration and gamete fusion, indicating that the hyperactivation supported by mitochondrial function is needed mainly for zona pellucida penetration. Finally, complementary in vivo fertilization experiments further demonstrated the fundamental role of mitochondrial activity for sperm function. Altogether, our results show the physiological relevance of mitochondrial functionality for sperm fertilization competence.

Low Doses of Glyphosate/Roundup Alter Blood-Testis Barrier Integrity in Juvenile Rats.

It has been postulated that glyphosate (G) or its commercial formulation Roundup (R) might lead to male fertility impairment. In this study, we investigated the possible effects of G or R treatment of juvenile male rats on blood-testis barrier function and on adult male sperm production. Pups were randomly assigned to the following groups: control group (C), receiving water; G2 and G50 groups, receiving 2 and 50 mg/kg/day G respectively; and R2 and R50 groups receiving 2 and 50 mg/kg/day R respectively. Treatments were performed orally from postnatal day (PND) 14 to 30, period of life that is essential to complete a functional blood-testis barrier. Evaluation was done on PND 31. No differences in body and testis weight were observed between groups. Testis histological analysis showed disorganized seminiferous epithelium, with apparent low cellular adhesion in treated animals. Blood-testis barrier permeability to a biotin tracer was examined. A significant increase in permeable tubules was observed in treated groups. To evaluate possible mechanisms that could explain the effects on blood-testis barrier permeability, intratesticular testosterone levels, androgen receptor expression, thiobarbituric acid reactive substances (TBARS) and the expression of intercellular junction proteins (claudin11, occludin, ZO-1, connexin43, 46, and 50 which are components of the blood-testis barrier) were examined. No modifications in the above-mentioned parameters were detected. To evaluate whether juvenile exposure to G and R could have consequences during adulthood, a set of animals of the R50 group was allowed to grow up until PND 90. Histological analysis showed that control and R50 groups had normal cellular associations and complete spermatogenesis. Also, blood-testis barrier function was recovered and testicular weight, daily sperm production, and epididymal sperm motility and morphology did not seem to be modified by juvenile treatment. In conclusion, the results presented herein show that continuous exposure to low doses of G or R alters blood-testis barrier permeability in juvenile rats. However, considering that adult animals treated during the juvenile stage showed no differences in daily sperm production compared with control animals, it is feasible to think that blood-testis barrier impairment is a reversible phenomenon. More studies are needed to determine possible damage in the reproductive function of human juvenile populations exposed to low doses of G or R.

Epithelial and Neural Cadherin in Mammalian Fertilization: Studies in the Mouse Model.

Successful mammalian fertilization requires a well-orchestrated sequence of molecular events leading to gamete fusion. Since this interaction involves Ca2+-dependent adhesion events, the participation of the Ca+2-dependent cell-cell adhesion proteins Epithelial (E-cad) and Neural (N-cad) cadherin is envisaged. We have previously reported the expression of E-cad and N-cad in human gametes and showed evidence of their involvement in sperm-oocyte adhesion events leading to fertilization. To overcome ethical limitations associated with the use of human gametes in fertilization-related studies, the mouse has been selected worldwide as the experimental model for over 4 decades. Herein, we report a detailed study aimed at characterizing the expression of E-cad and N-cad in murine gametes and their involvement in murine fertilization using specific antibodies and blocking peptides towards both adhesion proteins. E-cad and N-cad protein forms, as well as other members of the adhesion complex, specifically ß-catenin and actin, were identified in spermatozoa, cumulus cells and oocytes protein extracts by means of Western immunoblotting. In addition, subcellular localization of these proteins was determined in whole cells using optical fluorescent microscopy. Gamete pre-incubation with anti-E-cad (ECCD-1) or N-cad (H-63) antibodies resulted in decreased (p < 0.05) In Vitro Fertilization (IVF) rates, when using both cumulus-oocytes complexes and cumulus-free oocytes. Moreover, IVF assays done with denuded oocytes and either antibodies or blocking peptides against E-cad and N-cad led to lower (p < 0.05) fertilization rates. When assessing each step, penetration of the cumulus mass was lower (p < 0.05) when spermatozoa were pre-incubated with ECCD-1 or blocking peptides towards E-cad or towards both E- and N-cad. Moreover, sperm-oolemma binding was impaired (p < 0.0005) after sperm pre-incubation with E-cad antibody or blocking peptide towards E-cad, N-cad or both proteins. Finally, sperm-oocyte fusion was lower (p < 0.05) after sperm pre-incubation with either antibody or blocking peptide against E-cad or N-cad. Our studies demonstrate the expression of members of the adherent complex in the murine model, and the use of antibodies and specific peptides revealed E-cad and N-cad participation in mammalian fertilization.

Incidence of Sperm Surface Autoantibodies and Relationship with Routine Semen Parameters and Sperm Kinematics.

PROBLEM: Antisperm antibodies (ASA) are associated with male subfertility. However, results on sperm surface autoantibodies are controversial, the relationship between ASA and semen parameters (WHO, 2010) is unknown, and data on ASA and sperm kinematics are scarce. METHOD OF STUDY: A retrospective study carried out in men undergoing routine semen analysis (WHO 2010), ASA evaluation (direct SpermMAR(™) (IgG) test), and computer-assisted sperm analysis (CASA). RESULTS: A 2.6% and a 5.9% incidence of ASA-positive cases were found (cut-off 50% and 10%, respectively; n = 7492). ASA-positive samples had lower (P < 0.0001) sperm concentration, count, motility, and hypo-osmotic swelling (HOS) test score. HOS results did not correlate with sperm vitality in normozoospermic samples with high ASA levels. In unselected samples, ASA-positive samples (cut-off 50%) showed decreased sperm kinematics (VSL, VAP, LIN, ALH, STR, BCF, WOB), but in normozoospermic samples, ASA-positive and ASA-negative subgroups had similar CASA results. CONCLUSIONS: ASA evaluation is highly relevant in full semen assessment.

Epithelial and neural cadherin expression in the mammalian reproductive tract and gametes and their participation in fertilization-related events.

Mammalian fertilization involves a series of well-orchestrated cell-cell interaction steps between gametes, as well as among spermatozoa and somatic cells of both the male and female reproductive tracts. Cadherins are Ca(2+)-dependent glycoproteins that have been involved in cellular adhesion and signaling in somatic cells. Taking into account that Ca(2+) ions are required during fertilization, the involvement of these proteins in adhesion events during this process can be anticipated. This report presents an overview on two members of classical cadherins, Epithelial (E-) and Neural (N-) cadherin in reproductive biology. Its provides evidence of studies done by several research groups about the expression of E- and N-cadherin during spermatogenesis, oogenesis and folliculogenesis, and their involvement in gamete transport in the reproductive tracts. Moreover, it describes current knowledge of E- and N-cadherin presence in cells of the cumulus-oocyte complex and spermatozoa from several mammalian species, and shows gathered evidence on their participation in different steps of the fertilization process. A brief summary on general information of both proteins is also presented.