Rossella Puglisi

The mammalian homologues of frog Bv8 are mainly expressed in spermatocytes.

Bv8, a protein from skin secretions of Bombina variegata, reacts with receptors present in mammalian brain and intestine (Mollay et al. (1999) Eur. J. Pharmacol. 374, 189–196). As deduced from cloned cDNAs, the murine and human Bv8 homologues have identical amino‐terminal sequences and also contain 10 cysteines. From mouse testes, two forms of Bv8 mRNA have been characterized, of which one contains an additional exon which codes for 21 mostly basic amino acids. The mouse Bv8 gene is most active in mid‐late pachytene spermatocytes. In mouse testes, Bv8 mRNA can first be detected at the end of the second week post partum.

Selenium, a Key Element in Spermatogenesis and Male Fertility

Selenium is essential for normal spermatogenesis of mammals and its critical role is mainly mediated by two selenoproteins, namely phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4) and Selenoprotein P. PHGPx/GPx4 is the major selenoprotein expressed by germ cells in the testis, having multiple functions and representing the pivotal link between selenium, sperm quality and male fertility. Selenoprotein P is a plasma protein that is required for selenium supply to the testis. In the last years, nutritional studies and experimental animal models lacking/overexpressing a specific PHGPx isoform and selenoprotein P have highly expanded our understanding on how the male reproductive system depends on selenium. The focus of this review, is to report and discuss the most relevant and recent findings in this field. Clinical data have pointed to a correlation between abnormal PHGPx content in sperm and disturbance of human male fertility. However, additional evidence is still required to draw any definitive conclusions about therapeutical strategies for improving fertility by selenium administration.

Differential Splicing of the Phospholipid Hydroperoxide Glutathione Peroxidase Gene in Diploid and Haploid Male Germ Cells in the Rat

Abstract Phospholipid hydroperoxide glutathione peroxidase (PHGPx, 20 kDa) and sperm nuclei glutathione peroxidase (snGPx, 34 kDa) are two selenoproteins present in mammalian testis and epididymal spermatozoa. They originate from the differential splicing of the PHGPx gene and appear to play important roles in sperm physiology. To determine the stages of spermatogenesis in which they are present, we compared the expression pattern of these two enzymes in highly purified populations of germ cells during specific phases of differentiation. In Northern and Western blotting experiments, both PHGPx transcript and protein were markedly expressed in pachytene spermatocytes and round spermatids. In contrast, the testis-specific snGPx was detected at both the mRNA and protein level only in haploid round spermatids. Accordingly, the developmental analysis of testicular RNAs from rats of different ages first revealed the appearance of PHGPx and snGPx transcripts at Day 20 and Day 30, respectively. Furthermore, both meiotic and postmeiotic cells contained catalytically active PHGPx/snGPx, with higher activity in the haploid cells. The intracellular distribution of PHGPx in mitochondria and nuclei of meiotic cells was demonstrated by immunocytochemical electron microscopy and Western blotting. These findings provide evidence that the PHGPx gene is differentially spliced during the meiotic prophase and haploid cell phases of spermatogenesis.

The nuclear form of Glutathione Peroxidase 4 is associated with sperm nuclear matrix and is required for proper paternal chromatin decondensation at fertilization

The nuclear isoform of the selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase (nGPx4) is expressed in haploid male germ cells, contains several cysteines and is able to oxidize protein thiols, besides glutathione. In this study we have investigated the subnuclear localization of this isoform in isolated mouse male germ cells at different steps of maturation. Immunoblotting and confocal microscopy analyses of subnuclear fractions showed that nGPx4 is localized to the nuclear matrix together with well known markers of this subnuclear compartment like lamin B and topoisomerase IIβ at all stages of germ cell differentiation. The peculiar nGPx4 distribution was confirmed by both biochemical and morphological analyses of COS‐1 cells overexpressing Flag‐tagged nGPx4. To test the functional role of nGPx4 in the process of chromatin assembly, sperm isolated from the caput and the cauda epididymides of wild‐type (WT) and genetically deficient in nGPx4 (nGPx4‐KO) mice were analyzed in an in vitro chromatin decondensation assay. Results showed that sperm from nGPx4‐KO mice were more prone to decondense than those from WT mice at all stages of epididymal maturation, providing conclusive evidence that nGPx4 is required for a correct sperm chromatin compaction. We next addressed the issue of whether the lack of nGPx4 impacts on early events occurring at fertilization. Indeed, in vitro fertilization experiments showed an acceleration of sperm chromatin dispersion in oocytes fertilized by nGpx4‐KO sperm compared with control. Overall these data indicate that the absence of nGPx4 leads to sperm nuclear matrix/chromatin instability that may negatively affect the embryo development. J. Cell. Physiol. 227: 1420–1427, 2012.

Ryanodine receptors are expressed and functionally active in mouse spermatogenic cells and their inhibition interferes with spermatogonial differentiation

Ryanodine receptors (RyRs) are intracellular calcium release channels that are highly expressed in striated muscle and neurons but are also detected in several non-excitable cells. We have studied the expression of the three RyR isoforms in male germ cells at different stages of maturation by western blot and RT-PCR. RyR1 was expressed in spermatogonia, pachytene spermatocytes and round spermatids whereas RyR2 was found only in 5- to 10-day-old testis but not in germ cells. RyR3 was not revealed at the protein level, although its mRNA was detected in mixed populations of germ cells. Caffeine, a known agonist of RyRs, was able to induce release of Ca2+ from intracellular stores in spermatogonia, pachytene spermatocytes and round spermatids, but not spermatozoa. Treatment with high doses of ryanodine, which are known to block RyR channel activity, reduced spermatogonial proliferation and induced meiosis in in vitro organ cultures of testis from 7-day-old mice. In conclusion, the results presented here indicate that RyRs are present in germ cells and that calcium mobilization through RyR channels could participate to the regulation of male germ maturation.

The Nuclear Genes Mtfr1 and Dufd1 Regulate Mitochondrial Dynamic and Cellular Respiration

Dufd1 (DUF729 domain containing 1) is related to Mtfr1 (mitochondrial fission regulator 1), a gene involved in the regulation of antioxidant activity in the mouse testis. The present study was undertaken to better understand their role in regulating mitochondrial architecture and function in the mouse. We show that Dufd1 is expressed as a 2 kb mRNA and has a more specific tissue pattern compared to Mtfr1, with highest level of expression in testes, lower level in spleen, and negligible levels in other organs and/or tissues. In the male gonad, Dufd1 mRNA expression increases during postnatal development, similarly to Mtfr1. In situ hybridization and real‐time PCR analyses show that Dufd1 is expressed in the seminiferous tubules by middle‐late pachytene spermatocytes and spermatids. In transfected cells, the Dufd1‐tagged protein is located in mitochondria, associated with the tips of mitochondrial tubules and to tubules constrictions, and induces mitochondrial fission although with a lesser efficiency than Mtfr1. We also found that both endogenous Dufd1 and Mtfr1 proteins are associated with membrane‐enriched subcellular fractions, including mitochondria. Inhibition of Mtfr1 and/or Dufd1 expression, in a testicular germ cells line, severely impairs O2 consumption and indicates that both genes are required for mitochondrial respiration. Accordingly, analysis of testes mitochondria from Mtfr1‐deficient mice reveals severely reduced O2 consumption and ATP synthesis compared to wt animals. These data show that, in murine testis, Dufd1 and Mtfr1 have redundant functions related to mitochondrial physiology and represent genes with a potential role in testicular function. J. Cell. Physiol. 225: 767–776, 2010.

Impaired expression of genes coding for reactive oxygen species scavenging enzymes in testes of Mtfr1/Chppr-deficient mice

Mtfr1/Chppr is a nuclear gene coding for a mitochondrial protein capable of inducing fission of this organelle in a sequence-specific manner. Here we show that in mice, Mtfr1/Chppr is ubiquitously expressed and displays the highest level of expression in pubertal and adult testes and in particular in spermatids and Leydig cells. To investigate Mtfr1 function in vivo, we analyzed homozygous mice null for this gene obtained through a gene trap approach. We show that these mice fail to express Mtfr1 and that in their testes several genes coding for enzymes involved in the defense against oxidative stress are downregulated. Among these, we studied in particular glutathione peroxidase 3 and show its expression in selected testis cell types. Furthermore, we demonstrate oxidative DNA damage specifically in testes of Mtfr1-deficient mice likely resulting from a reduced antioxidant activity. As a whole, these data suggest that Mtfr1 protects the male gonads against oxidative stress.

The nuclear form of glutathione peroxidase 4 colocalizes and directly interacts with protamines in the nuclear matrix during mouse sperm chromatin assembly.

The testis-specific nuclear form of Phospholipid Hydroperoxide Glutathione Peroxidase (nGPx4) is associated with the nuclear matrix during spermiogenesis and is implicated in sperm chromatin condensation. In this study, we have addressed the question whether nGPx4 directly interacts with protamines by transiently sharing a nuclear matrix localization. We first expressed tagged protamine 1-myc and protamine 2-V5 in HeLa and COS-1 cells and showed by both confocal microscopy and immunoblotting analyses that protamines were produced in vitro and colocalized correctly to the nucleus. Co-transfection experiments demonstrated that protamine 1 was physically associated with flag-nGPx4 specifically at the level of nuclear matrix. The peculiar presence of protamines together with nGPx4 in this subnuclear compartment was also confirmed in mouse elongated spermatids by immunofluorescence, suggesting that nGPx4 is a physiological component of a novel protein complex relevant to chromatin assembly in condensing haploid cells. Also, in epididymal sperm, nGPx4 and protamine 1 co-immunoprecipitated, indicating that nGPx4, although localized to a subnuclear compartment different from that of protamines, represents a constant link between nuclear matrix and chromatin in mammalian male gamete.

Involvement of sperm acetylated histones and the nuclear isoform of Glutathione peroxidase 4 in fertilization

We previously demonstrated that the nuclear form of Glutathione peroxidase 4 (nGPx4) has a peculiar distribution in sperm head, being localized to nuclear matrix and acrosome and that sperm lacking nGPx4 are more prone to decondensation in vitro. In this study we have hypothesized that sperm retained acetylated histones and nGPx4 are implicated in paternal chromatin decondensation and male pronucleus formation at fertilization. Indeed, significant higher amounts of acetylated histone H4 and acetylated histone H3 were observed by both immunofluorescence and western blotting in nGPx4‐KO sperm vs WT ones. In vitro fertilization of zona pellucida‐deprived oocytes by WT sperm in the presence of trichostatin (TSA) also demonstrated that paternal histone acetylation was inversely related to the timing of sperm nucleus decondensation at fertilization. In contrast, TSA had no effect on nGPx4‐KO sperm, indicating they had a maximal level of histone acetylation. Moreover the paternally imprinted gene Igf2/H19 was hypomethylated in KO sperm compared to WT ones. The lack of nGPx4 negatively affected male fertility, causing a marked decrease in total pups and pregnancies with delivery, a significant reduction in pronuclei (PN) embryos in in vitro fertilization assays and an approximately 2 h delay in egg fertilization in vivo. Because the zona pellucida binding and fusion to oolemma of nGPx4‐KO and WT sperm were similar, the subfertility of nGPx4 sperm reflected a decreased sperm progression through egg cumulus/zona pellucida, pinpointing a defective acrosome in line with acrosomal nGPx4 localization. We conclude that paternal acetylated histones and acrosomal nGPx4 are directly involved in fertilization.