Hyper-activated motility in sperm capacitation is mediated by phospholipase D-dependent actin polymerization.

In order to fertilize the oocyte, sperm must undergo a series of biochemical changes in the female reproductive tract, known as capacitation. Once capacitated, spermatozoon can bind to the zona pellucida of the egg and undergo the acrosome reaction (AR), a process that enables its penetration and fertilization of the oocyte. Important processes that characterize sperm capacitation are actin polymerization and the development of hyper-activated motility (HAM). Previously, we showed that Phospholipase D (PLD)-dependent actin polymerization occurs during sperm capacitation, however the role of this process in sperm capacitation is not yet known. In the present study, we showed for the first time the involvement of PLD-dependent actin polymerization in sperm motility during mouse and human capacitation. Sperm incubated under capacitation conditions revealed a time dependent increase in actin polymerization and HAM. Inhibition of Phosphatidic Acid (PA) formation by PLD using butan-1-ol, inhibited actin polymerization and motility, as well as in vitro fertilization (IVF) and the ability of the sperm to undergo the AR. The inhibition of sperm HAM by low concentration of butan-1-ol is completely restored by adding PA, further indicating the involvement of PLD in these processes. Furthermore, exogenous PA enhanced rapid actin polymerization that was followed by a rise in the HAM, as well as an increased in IVF rate. In conclusion, our results demonstrate that PLD-dependent actin polymerization is a critical step needed for the development of HAM during mouse and human sperm capacitation.

Meig1 deficiency causes a severe defect in mouse spermatogenesis.

Meig1 is a mouse gene, abundantly expressed in the testis. It encodes two alternative transcripts that are expressed differentially in the somatic and germinal compartments of the testis. These transcripts share the same coding region but differ in their 5' un-translated regions, due to alternative promoters. Here we show that MEIG1 is a highly conserved short metazoan protein with a conserved core of 81 residues. It is present from chordates to radial symmetry animals, with an intriguing absence in insects and nematodes. It is also present in two earlier diverging protist lineages. To elucidate the role of MEIG1 during gamete production we established a knockout mouse line by eliminating the common coding region. Our results identified Meig1 as a critical spermatogenic gene, whose absence results in complete male infertility. Seminiferous tubules in Meig1-null males contained all early stages of spermatogenesis, up to elongating spermatids, but mature elongated spermatids were absent. Accordingly, the caudal epididymis was apparently missing spermatozoa, and the very few spermatozoa-like cells that were obtained were immotile and exhibited a wide range of severe morphological abnormalities. These results point at late spermiogenesis as the differentiative stage at which MEIG1's function is crucial. Nevertheless, delayed kinetics of earlier meiotic stages together with increased apoptosis of meiotic spermatocytes and haploid round spermadids in Meig1 knockout males, suggest involvement of MEIG1 in meiotic stages as well.

Effect of interleukin-1 receptor antagonist gene deletion on male mouse fertility.

Members of the IL-1 family are pleiotropic cytokines that are involved in inflammation, immunoregulation, and other homeostatic functions in the body. IL-1alpha, IL-1beta, and the IL-1 antagonistic molecule [IL-1 receptor antagonist (IL-1 Ra)] are present in the testis under normal homeostasis, and they further increase upon infection/inflammation. In the present study, we examined the effect of IL-1 Ra gene deletion on male mouse fertility. Male mice [wild type (WT) and IL-1 Ra knockout (KO)] were mated with WT females, and the birth and number of offspring were recorded 21-45 d after mating. Furthermore, the concentration, motility, and morphology of sperm isolated from the cauda of the epididymis were evaluated. The ability of the calcium ionophore (A23187) to induce acrosome reaction (AR) in the sperm of WT and IL-1 Ra KO mice was compared with their ability to fertilize in vitro oocytes from WT females. The direct effect of IL-1alpha and IL-1beta on AR and abnormal morphology in sperm from WT were evaluated. The levels of IL-1alpha and IL-1beta in the testes of WT and IL-1 Ra KO mice were examined by specific ELISA and real-time PCR. Our results show a significant reduction in the capacity of IL-1 Ra KO male mice to fertilize WT females (P < 0.05). Furthermore, the number of offspring in mice fertilized with IL-1 Ra KO male mice was significantly lower than with WT males (P < 0.05). Sperm concentration and the percentage of motile sperm from IL-1 Ra KO and WT were similar; however, the percentage of sperm with abnormal morphology (mainly in the head) and acrosome-reacted sperm cells were significantly higher in the IL-1 Ra KO, compared with that of WT males (P < 0.05). In vitro, the ability of sperm from IL-1 Ra KO male mice to fertilize oocytes from WT females was significantly lower than sperm from WT mice (P < 0.05). In addition, the percentage of reacted sperm from IL-1 Ra KO, spontaneously without ionophore induction, was significantly higher than from WT (P < 0.05). Sperm from WT underwent induction of AR only by ionophore; however, sperm from IL-1 Ra KO were unable to undergo the AR by ionophore, indicating that they are induced and, thus, are inactive in fertilization. Testicular IL-1alpha and IL-1beta levels were significantly higher in IL-1 Ra KO, compared with WT male mice (P < 0.05). The addition of recombinant IL-1alpha or IL-1beta to sperm from a WT mouse induced their AR, and significantly increased abnormal sperm morphology, as compared with controls (P < 0.05). This effect was neutralized by the addition of IL-1 Ra. Our results indicate the involvement of IL-1 in sperm physiology, affecting its morphology and fertilization ability. Higher than homeostatic levels of IL-1 in the testis, as observed in IL-1 Ra KO mice, impaired the ability of sperm to fertilize oocytes. Together, these results may explain some of the male infertility cases with an infection/inflammation background and may hint at the ability to use IL-1 Ra in future therapeutic strategies in these cases.