Dinesh K. Saxena

Molecular Modifications of MC31/CE9, a Sperm Surface Molecule, During Sperm Capacitation and the Acrosome Reaction in the Rat: Is MC31/CE9 Required for Fertilization?

Abstract We examined the modification of the MC31 molecule during capacitation, the acrosome reaction, and studied its role in fertilization. These studies revealed that the molecular mass of MC31 in cauda spermatozoa was approximately 28 000–26 000 Dalton (28–26 kDa). A limited change in molecular mass was seen in capacitated spermatozoa. Treatment of sperm extracts with peptide-N-glycosidase (PN glycosidase) reduced the molecular mass of MC31 in both cauda and capacitated spermatozoa from 28–26 kDa to 23–20 kDa, suggesting that MC31 from both cauda and capacitated spermatozoa is glycosylated, and indicating that capacitation induces minor posttranslational modifications in the structure of the MC31 antigen. The MC31 antigen was redistributed from the midpiece of cauda epididymal spermatozoa to the head and equatorial segment after capacitation and acrosome reaction, respectively, when traced by indirect immunofluorescence under in vitro fertilization (IVF) conditions. Some spermatozoa did not stain for the MC31 antigen and might represent spermatozoa that have shed the antigen. IVF experiments conducted to assess the effect of an anti-MC31 monoclonal antibody (mMC31) revealed that this antibody significantly (P < 0.01) inhibited fertilization of cumulus-invested zona pellucida-intact and the zona pellucida-free oocytes in a dose-dependent manner. However, sperm-oolemma binding was not affected. These findings suggest the MC31 antigen facilitates sperm-oocyte interactions.

IMMUNOCYTOCHEMICAL ALTERATIONS IN THE INTRA-ACROSOMAL ANTIGEN MN7 DURING EPIDIDYMAL MATURATION OF GUINEA PIG SPERMATOZOA

Abstract We have previously shown that a 90-kDa intra-acrosomal antigen, MN7, is restricted to the anterior acrosomal region of mouse, rat, and hamster spermatozoa. The present study has examined the localization and the behavior of MN7 during sperm maturation in the epididymis of the guinea pig by immunoelectron microscopy. MN7 showed not only a specific localization in the apical segment of the guinea pig sperm acrosome, but also a distinct alteration during maturation, as follows. MN7 was exclusively found both at the dorsal matrix and on the outer acrosome membrane (OAM)/matrix-associated materials in the apical segment. MN7 was initially distributed throughout the electron-lucent dorsal matrix in immature sperm but, during maturation, became more restricted to the spherical bodies within the electron-lucent area. MN7 on OAM/matrix-associated materials was first distributed along the ventral margin and the small area posterior to the dorsal matrix but, during maturation, disappeared from the ventral margin and became restricted to the dorsal region. These results indicate that MN7 is a good tool for studying the stepwise maturation of epididymal spermatozoa.

Behaviour and role of an intra-acrosomal antigenic molecule, acrin 3, during mouse fertilisation in vitro

In this study we examined the behaviour and role of an intra-acrosomal antigenic molecule, acrin 3, during mouse fertilisation in vitro by assessing the effect of its pertinent monoclonal antibody mMC101. Experiments were designed to assess the effect of mMC101 on sperm-zona pellucida binding, the acrosome reaction, zona pellucida penetration, sperm-egg fusion, and fertilisation in vitro. mMC101 did not affect sperm motility or primary and secondary binding to the zona pellucida, but significantly inhibited fertilisation of zona-pellucida-intact oocytes in a dose-dependent manner. In the presence of mMC101 at 100 microg/ml concentration in TYH medium, none of the oocytes developed to pronuclear stage by 5 h after co-incubation of the gametes, but the pronucleus formation rate recovered to some extent (45.3%) after 8 h, indicating a delay of early embryonic development. mMC101 also delayed and significantly suppressed zona pellucida penetration by sperm. Acrin 3 dispersed and did not remain on completely acrosome-reacted sperm. Although mMC101 did not influence the zona-pellucida-induced acrosome reaction, it significantly inhibited fertilisation when acrosome-reacted sperm in the presence of mMC101 inseminated zona-pellucida-free oocytes. However, fertilisation remained unaffected when acrosome-reacted sperm in the absence of mMC101 inseminated zona-pellucida-free oocytes even in its presence. Thus, acrin 3 appears to facilitate zona pellucida penetration and is also likely to be involved in sperm-oocyte fusion by modifying the sperm plasma membrane during the acrosome reaction.

The involvement of immunoglobulin superfamily proteins in spermatogenesis and sperm-egg interaction: Immunoglobulin superfamily proteins

The immunoglobulin superfamily (IgSF) proteins are expressed on the plasma membrane between Sertoli cells and germ cells in the testis. IgSF proteins are specifically present at the apical Sertoli‐germ cell junction, that is, ectoplasmic specialization and are involved in germ cell differentiation. Some IgSF proteins are present on the surface of germ cells and undergo further biochemical modifications during sperm maturation. These IgSF proteins undergo final modifications during capacitation and/or the acrosome reaction. The function and expression of IgSF proteins in the testis and spermatozoa, as they relate to spermatogenesis and sperm–egg interaction, are discussed. (Reprod Med Biol 2006; 5: 87–93)

Changes in the activity of sperm nitric oxide synthase in the oviductal reservoir during ovulation

BackgroundThe oviductal isthmus is known to act as a sperm reservoir in several mammalian species including mice, but it is still unclear how sperm are released from the reservoir after ovulation. Recently, nitric oxide (NO) was reported to have important roles as a mediator in various sperm functions, including hyperactivation and capacitation. Therefore, we have investigated the change of the activity of nitric oxide synthase (NOS) of sperm of the isthmus in relation to ovulation under in vivo fertilization conditions.Methods and ResultsThe sperm were collected from the isthmus and uterus of the female mated before or after ovulation. The NOS activity change was evaluated by using the β-nicotinamide adenine dinucleotide phosphate-diaphorase staining method, and sperm NOS activity was quantified by using NIH image software. The results showed that, in the reservoir, the peak intensity of sperm NOS activity was higher after ovulation (135.5 ± 22.4) than before ovulation (102.7 ± 15.5; P < - 0.05). After ovulation, the number of free sperm in the isthmus increased, and these sperm expressed strong NOS activity.ConclusionThe change of sperm NOS activity is related to their release from the epithelium of the oviductal reservoir.