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Progesterone (P4) exerts its effects by binding to specific genomic (nPR-A/B) and non-genomic (mPRalpha/beta, PGRMC1/2) receptors. P4 has a role in the regulation of the ovulatory cycle, but its participation in oocyte maturation in mammals has not yet been clarified. Therefore, the aim of the present study was to characterize the protein expression of P4 receptors (PRs) in bovine oocytes and cumulus cells during in vitro maturation (IVM) and to study the effect of P4 and its receptors on oocyte developmental competence. Cumulus-oocyte complexes (COCs) were subjected to IVM, in vitro fertilization, and in vitro culture. IVM was performed for 24 h in the presence or absence of P4, luteinizing hormone (LH), follicle-stimulating hormone (FSH), trilostane, promegestone (R5020), mifepristone (RU 486), or antibodies against mPRalpha or mPRbeta. Protein expression of PRs was studied by Western blotting and immunofluorescence. The results demonstrate the presence of both genomic and nongenomic PRs in bovine COCs. The dynamic changes observed in the protein expression of PRs following IVM or in response to supplementation with LH, FSH, or P4 suggest an important role during bovine oocyte maturation. Inhibition of P4 synthesis by cumulus cells or blocking of nPR and mPR alpha activity produced a decrease in bovine embryo development, indicating that P4 intracellular signaling is mediated by its interaction with nuclear and membrane PRs and is important for oocyte developmental competence.

Macroautophagy (hereafter autophagy) is an evolutionarily highly conserved cellular process that participates in the maintenance of intracellular homeostasis through the degradation of most long-lived proteins and entire organelles. Autophagy participates in some reproductive events; however, there are not reports regarding the role of autophagy in the regulation of sperm physiology. Hence, the aim of this study was to investigate whether autophagy-related proteins are present and functionally active in human spermatozoa. Proteins related to autophagy/mitophagy process (LC3, Atg5, Atg16, Beclin 1, p62, m-TOR, AMPKα 1/2, and PINK1) were present in human spermatozoa. LC3 colocalized with p62 in the middle piece of the spermatozoa. Autophagy activation induced a significant increase in motility and a decrease in PINK1, TOM20 expression and caspase 3/7 activation. In contrast, autophagy inhibition resulted in decreased motility, viability, ATP and intracellular calcium concentration whereas PINK1, TOM20 expression, AMPK phosphorylation and caspase 3/7 activation were significantly increased. In conclusion our results show that autophagy related proteins and upstream regulators are present and functional in human spermatozoa. Modification of mitochondrial proteins expression after autophagy activation/inhibition may be indicating that a specialized form of autophagy named mitophagy may be regulating sperm function such as motility and viability and may be cooperating with apoptosis.

Sperm functions are critically controlled through the phosphorylation state of specific proteins. Glycogen synthase kinase-3 (GSK3) is a serine/threonine kinase with two different isoforms (a and b), the enzyme activity of which is inhibited by serine phosphorylation. Recent studies suggest that GSK3 is involved in the control of bovine sperm motility. Our aim was to investigate whether GSK3 is present in porcine spermatozoa and its role in the function of these cells. This work shows that both isoforms of GSK3 are present in whole cell lysates of porcine sperm and are phosphorylated on serine in spermatozoa stimulated with the cAMP analog, 8Br-cAMP. A parallel increase in serine phosphorylation of the isoform GSK3a, but not in the isoform GSK3b, is observed after treatments that also induce a significant increase in porcine sperm velocity parameters. Therefore, a significant positive correlation among straight-line velocity, circular velocity, average velocity, rapid-speed spermatozoa, and GSK3a serine phosphorylation levels exists. Inhibition of GSK3 activity by alsterpaullone leads to a significant increase in the percentage of rapid-and medium-speed spermatozoa as well as in all sperm velocity parameters and coefficients. Moreover, pretreatment of porcine spermatozoa with alsterpaullone significantly increased the percentage of capacitated porcine spermatozoa and presents no effect in the number of acrosome-reacted porcine spermatozoa. Our work suggests that the isoform GSK3a plays a negative role in the regulation of porcine sperm motility and points out the possibility that sperm motile quality might be modulated according the activity state of GSK3a. Reproduction (2007) 134 435-444

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