Influence of antifertility agents Dutasteride and Nifedipine on CatSper gene level in epididymis during sperm maturation in BALB/c mice.

Calcium (Ca2+) signaling is critical for successful fertilization. In spermatozoa, capacitation, hyperactivation of motility and the acrosome reaction are all mediated by increases in intracellular Ca2+ through CatSper (sperm-specific cation channel). The CatSper channel complex contains four pore-forming α subunits (CatSper1-4) and five accessory subunits called ß, δ, ε, γ and ζ. Genetic deletion of any of the four CatSper genes in mice results in loss of hyperactivated motility and male infertility. Despite their vital role in male fertility, almost very little is known about influence of antifertility agents on CatSper gene expression in epididymis and epididymal spermatozoa. Therefore, we performed quantitative real-time qPCR analysis for CatSper expression in the epididymis and epididymal sperm of BALB/c mice after treatment with Dutasteride (DS), a dual 5-α reductase inhibitor and Nifedipine (NF) a calcium channel blocker as positive control. We observed that treatment with antifertility agents Dutasteride and Nifedipine induced significant decreases in the caput and cauda epididymal sperm counts, motility and fertility which could partly be attributed to alteration in the normal morphology of the sperm associated with downregulation/upregulation of CatSper mRNAs in epididymis and epididymal spermatozoa of male BALB/c mice. These can be explained on the basis of interference with mechanisms affecting calcium ion signaling resulting in changes in intracellular calcium required for sperm activity, finally affecting sperm maturation and fertility of male BALB/c mice. These studies provide some novel avenues for developing new male contraceptives in future.

Inhibition of hyaluronidase activity of human and rat spermatozoa in vitro and antispermatogenic activity in rats in vivo by Terminalia chebula, a flavonoid rich plant.

Our interest in development of hyaluronidase inhibitors as male antifertility agents led to identification of Terminalia chebula (T. chebula) plant with hyaluronidase (HAase) inhibitory activity of human spermatozoa ( approximately 93% inhibition) and rat caudal epididymal spermatozoa ( approximately 86% inhibition) in vitro at 30 mg/ml. We further demonstrated inhibition of hyaluronidase activity of testis and epididymal spermatozoa in vivo coincident with antispermatogenic activity and contraceptive efficacy of TC extract administered at 50 and 100mg/kg/day orally for 60 days in male albino rats. The significant decrease in motility, count and increase in morphological abnormalities of epididymal spermatozoa and severe reduction in fertility (-100%) of male rats treated with T. chebula fruit extract at 100mg/kg dose could be attributed to either direct effect on testis or direct or indirect interference with sperm maturation in epididymis, and/or inhibition of testicular and epididymal sperm hyaluronidase enzyme in vivo probably caused by flavonoids like tannins present in T. chebula.

Post-translational modifications in glycosylation status during epididymal passage and significance in fertility of a 33 kDa glycoprotein (MEF3) of rhesus monkey (Macaca mulatta).

The present study reports data on post-translational modifications in the glycosylation status during epididymal passage and significance in fertility of a 33 kDa glycoprotein of rhesus monkey (Macaca mulatta), designated as MEF3 (monkey epididymal fluid protein 3). MEF3 exhibited strong affinity for N-linked alpha-D-mannose groups and O-linked N-Ac-galactosamine linkages in epididymal fluids and exhibited moderate affinity for N-Ac-glucosaminylated (wheat germ agglutinin), fucosylated (Tetragonolotus purpurea), and N-Ac-galactosamine (peanut agglutinin) residues on more mature corpus and caudal spermatozoa in a maturation-dependent manner on Western blots probed with specific biotinylated lectins. Polyclonal antiserum raised against affinity-purified MEF3 from caudal epididymal fluid (CEF) cross-reacted specifically with CEF and caudal sperm membrane of macaque and with Triton X-100 extract of ejaculated human spermatozoa, suggesting the existence of antigenically related components in both species. The tangled agglutination caused by anti-33 kDa serum of human spermatozoa, along with localization of MEF3 on entire sperm surface of epididymal and testicular sperm of monkey and human spermatozoa, suggest the significance of MEF3 in sperm function. The 100% inhibition of fertility of immunized female rabbits with this protein in vivo and inhibition of human sperm penetration in zona-free hamster eggs in vitro suggests the functional significance of MEF3 in fertility. Together, these results clearly indicate that MEF3 has potential significance as a target for antibodies that inhibit sperm function and fertility.

Partial characterization, sperm association and significance of N- and O-linked glycoproteins in epididymal fluid of rhesus monkeys (Macaca mulatta).

The present study investigated regional modifications of glycosylation status, sperm association and functional significance of N- and O-linked glycoproteins in epididymal luminal fluid of the rhesus monkey (Macaca mulatta). The predominant glycoproteins of the epididymal luminal fluid that increase in the extent of glycosylation or unmasking of exposed epitopes in a region-specific, maturation-dependent manner, included those of 150, 116, 68, 64, 58 (N- and O-linked) and 170 kDa (O-linked). The higher expression of 40 (N-linked), 38 (N- and O-linked) and 60, 56 and 33 kDa (O-linked) glycoproteins in the proximal caput epididymal fluid was followed by alteration or reorganization of 60, 38 and 33 kDa (O-linked) glycoproteins in the distal segments of the epididymis. The association of epididymal fluid glycoproteins with maturing spermatozoa was identified by generating polyclonal antiserum against monkey caudal sperm membrane in female albino rabbits. The antiserum crossreacted strongly with 58 and 33 kDa epididymal fluid glycoproteins of monkeys and also reacted with 116, 68, 58, 56 and 33 kDa glycoproteins from Triton X-100 extracts of human spermatozoa, indicating the presence of antigenically related components in both species. The functional significance of epididymal fluid glycoproteins in sperm functions was investigated by raising antiserum against a heavily glycosylated 58 kDa glycoprotein (MEF1) of caudal epididymal fluid, which crossreacted with the Triton X-100 extracts of epididymal spermatozoa of monkey and ejaculated human spermatozoa on immunoblots. In an in vitro micro-sperm agglutination assay, anti-MEF1 serum agglutinated both rat caudal epididymal spermatozoa and human spermatozoa. MEF1 seemed to be involved in fertilization as demonstrated by inhibition of fertility (100%) in female albino rabbits and rats immunized with this protein. A sperm-agglutinating 58 kDa glycoprotein of rhesus monkey epididymis with functional significance in fertility was identified, thus indicating that it is a potential candidate for contraceptive vaccine development.