In the present study, the effects of seminal plasma (SP), cumulus-oocyte-complexes (COCs) conditioned medium (CCM) and hyaluronan (HA) on functional changes and in vitro fertilizing ability of porcine spermatozoa were examined. In in vitro fertilization (IVF) experiments, 10% (v/v) of exogenous SP in the fertilization medium prevented sperm penetration (using fresh-extended and frozen-thawed ejaculated spermatozoa). Analysis of frozen-thawed CCM revealed a HA content to levels of 30 ng/mL per incubated COC. Presence of frozen-thawed CCM did not, however, prove effective to increase (furthermore decreasing) oocyte penetration in vitro, and neither did supplementation with exogenous HA at the same concentration as that present in the CCM (secreted by COCs). Analysis of sperm capacitation using the chlortetracycline (CTC) assay showed that frozen-thawed CCM had no elevating effect on 'B-pattern' spermatozoa (implying capacitation-like changes) and that addition of 10% (v/v) SP held spermatozoa in the 'F-pattern' (intact) status. Dose of 500 microg/mL HA and freshly prepared CCM increased, however, the frequency of capacitated spermatozoa (B-pattern) without resulting in increased rates of 'AR-pattern' (acrosome-reacted) spermatozoa, compared with controls. The present results confirm the decapacitating effect of SP and suggest capacitating actions of HA (dose-related) and CCM (freshly prepared) on boar spermatozoa in vitro. The unclear effects of frozen-thawed CCM and a low dose of HA on penetration rates of boar spermatozoa call for further researches of their function in vivo.
The molecular mechanism by which sperm triggers Ca 2C oscillation, oocyte activation, and early embryonic development has not been clarified. Recently, oocyte activation has been shown to be induced by sperm-specific phospholipase Cz (PLCz). The ability of PLCz to induce oocyte activation is highly conserved across vertebrates. In the present study, porcine PLCz cDNA was identified and the nucleotide sequence was determined. The expression pattern of porcine PLCz mRNA during the period of postnatal testicular development was shown to be similar to that of mouse PLCz. PLCz mRNA expression in the pig and mouse was detected only in the testes when the elongated spermatids had differentiated, and was detected from day 96 after birth in the pig. Histological examination of porcine testis during the period of postnatal development revealed the presence of spermatozoa from day 110 after birth. These findings suggest that the synthesis of PLCz mRNA starts when spermiogenesis is initiated. Microinjection of porcine PLCz complementary RNA into porcine oocytes demonstrated that porcine PLCz has the ability to trigger repetitive Ca 2C transients in porcine oocytes similar to that observed during fertilization. It was also found that porcine PLCz cRNA has the potential to induce oocyte activation and initiate embryonic development up to the blastocyst stage.
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