Mice deficient in Dmrt7 show infertility with spermatogenic arrest at pachytene stage

Genes including DM domain regulate sexual development in diverse metazoan phyla. One of these genes, Dmrt7, was expressed only in testes of adult mice. To determine the role of Dmrt7 in mice, we generated Dmrt7-knockout mice (Dmrt7-/-). Although the Dmrt7-/- showed normal growth, null males were infertile. No sperm was detected in the epididymis of Dmrt7-/- adult males. Absence of spermatids in a histological analysis, decreased expression of Ccna1 mRNA and the accumulation of SCP3-positive spermatocytes showed the arrest of spermatogenesis at the pachytene stage in the Dmrt7-knockout mice.     

The fine structural localization of testicular phosphatases in man: The control testis

Summary Electron microscopic cytochemistry was used to determine the localization of five phosphatase enzymes—glucose-6-phosphatase, inosine diphosphatase, thiamine pyrophosphatase, acid phosphatase, and adenosine triphosphatase—in control human testes. Glucose-6-phosphatase occurred in the endoplasmic reticulum and nuclear envelope of Sertoli cells, Leydig cells and primitive spermatogonia, but was not observed in more advanced spermatogenic cells. The presence of glucose-6-phosphatase activity paralleled the presence of glycogen in spermatogenic cells, i.e., both occurred in type AL and AD spermatogonia but not in type AP or B spermatogonia or in more advanced spermatogenic cells. Inosine diphosphatase activity was found in the endoplasmic reticulum, nuclear envelope, and Golgi complex of Sertoli cells and all spermatogenic cells except late spermatids. Additionally, inosine diphosphatase activity was localized at the junctions between Sertoli cells and late spermatids, but was not associated with any other plasma membrane. Thiamine pyrophosphatase reaction product was found in the Golgi bodies of Sertoli cells and in spermatogenic cells through immature spermatids. Neither inosine diphosphatase nor thiamine pyrophosphatase was observed in the Golgi bodies of spermatids during acrosomal formation. Acid phosphatase activity was found in lysosomes of spermatogonia, spermatocytes, and spermatids, in lysosomes of Leydig cells, and in lysosomes, lipofuscin bodies, and Golgi cisternae of Sertoli cells. It is thought that Sertoli lysosomes play a role in the phagocytosis of degenerating germ cells; however, the role of spermatogenic or Leydig lysosomes is unknown. Adenosine triphosphatase activity occurred at the interfaces between two spermatogonia, and between Sertoli cells and spermatogonia, but was not observed in the spaces between two Sertoli cells, two spermatocytes, two spermatids, or between Sertoli cells and spermatocytes, or between Sertoli cells and spermatids.Supported in part by a grant from the U.S. Atomic Energy Commission (AT-(40-1)-4002).     

Critical function of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in mouse spermatogenesis

Small GTPase Rap1 has been implicated in the proper differentiation of testicular germ cells. In the present study, we investigated the functional significance of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in testicular differentiation using mice lacking RA-GEF-2. RA-GEF-2 was expressed predominantly on the luminal side of the seminiferous tubules in wild-type mice. No significant differences were observed in the body weights or hormonal parameters of RA-GEF-2^−/− and wild-type mice. However, the testes of RA-GEF-2^−/− male mice were significantly smaller than those of wild-type mice and were markedly atrophied as well as hypospermatogenic. The concentration and motility of epididymal sperm were also markedly reduced and frequently had an abnormal shape. The pregnancy rate and number of fetuses were markedly lower in wild-type females after they mated with RA-GEF-2^−/− males than with wild-type males, which demonstrated the male infertility phenotype of RA-GEF-2^−/− mice. Furthermore, a significant reduction and alteration were observed in the expression level and cell junctional localization of N-cadherin, respectively, in RA-GEF-2^−/− testes, which may, at least in part, account for the defects in testicular differentiation and spermatogenesis in these mice.     

Alpha-lipoic acid rebalances redox and immune-testicular milieu in septic rats

In the present study, lipopolysaccharide (LPS), as an immune modulator in male adult rats and alpha-lipoic acid (ALA), as a powerful biological antioxidant and anti-inflammatory, are examined to help understanding the role of the immune and redox perturbation in testicular dysfunction with a possible protection. A total of 60 male Swiss albino rats were divided into 5 groups (10/group) respectively as follows Saline, ALA-vehicle, ALA (200 mg/kg), LPS (5 mg/kg) started with 20 rats and LPS + ALA. Obtained data from previously reported study, in our laboratory, and from the present one revealed that LPS induced marked reductions in sperm's count, motility and resulted in deterioration of the testicular histological features. In addition, LPS decreased testicular reduced glutathione (GSH) level and lactate dehydrogenase isoenzyme-x (LDH-x) activity. However, it increased testicular levels of malondialdehyde (MDA), nitric oxide (NO) and 8-hydroxydeoxyguanosine (8-HDG) in testicular DNA, along with increased serum IL-2 level. In contrast, rats pretreated with ALA showed almost complete normalization of all the tested parameters. In conclusion, LPS induced perturbation of the immune-testicular barrier as a result of redox imbalance with a subsequent testicular dysfunction. Pretreatment with ALA ameliorated all these effects by its immune-modulator and antioxidant mechanisms suggesting a protective role against male infertility in septic or severely infected patients.     

Poly (A) binding protein is bound to both stored and polysomal mRNAs in the mammalian testis

RNA-binding proteins that bind to the 3′ untranslated region of mRNAs play important roles in regulating gene expression. Here we examine the association between the 70 kDa poly (A) binding protein (PABP) and stored (RNP) and polysomal mRNAs during mammalian male germ cell development. PABP mRNA levels increase as germ cells enter meiosis, reaching a maximum in the early postmeiotic stages, and decreasing to a nearly nondetectable level towards the end of spermatogenesis. Most of the PABP mRNA is found in the nonpolysomal fractions of postmitochondrial extracts, suggesting that PABP mRNA is either inefficiently translated or stored as RNPs during spermatogenesis. Virtually all of the testicular PABP is bound to either polysomal or nonpolysomal mRNAs, with little, if any, free PABP detectable. Analysis of several specific mRNAs reveals PABP is bound to both stored (RNP) and translated forms of the mRNAs. Western blot analysis and immunocytochemistry indicate PABP is widespread in the mammalian testis, with maximal amounts detected in postmeiotic round spermatids. The presence of PABP in elongating spermatids, a cell type in which PABP mRNA is nearly absent, suggests that PABP is a stable protein in the later stages of male germ cell development. The high level of testicular PABP in round spermatids and in mRNPs suggests a role for PABP in the storage as well as in the subsequent translation of developmentally regulated mRNAs in the mammalian testis. © 1995 Wiley-Liss, Inc.     

Expression of the soluble adenylyl cyclase during rat spermatogenesis: evidence for cytoplasmic sites of cAMP production in germ cells

To gain insight into the mechanisms of cAMP signaling in germ cells, the expression and subcellular localization of the full-length form of the soluble adenylyl cyclase (sAC) was investigated during rat spermatogenesis and in spermatozoa. A full-length sAC-specific antibody was generated by using a glutathione S-transferase (GST)-sAC carboxyl-terminal region (1399aa-1608aa) fusion protein as the antigen. The selectivity of the purified antibody was confirmed by immunoblotting with lysates from HEK293 cells overexpressing full-length sAC or truncated sAC. Western blot analysis demonstrated that full-length sAC protein appeared on day 25 during testis development. The expression levels increased progressively on days 30 and 35 and remained elevated in adult testis. Full-length sAC protein is retained in spermatozoa from the cauda epididymis. Consistent with the timing of the appearance of the Western blot signal, immunohistochemistry with testis sections at different stages of development detected sAC in late pachytene spermatocytes as well as round and elongating spermatids. Further experiments on the subcellular localization of native or recombinant enzymes revealed that full-length sAC is not only recovered in soluble fractions but also in particulate fractions of testis extracts. Immunofluorescence detection showed localization of the protein in the cytoplasm as well as in organelles of pachytene spermatocytes and spermatids. These findings indicate that cAMP production in spermatids and spermatozoa may occur at sites other than the plasma membrane and suggest that full-length sAC may play a role during spermatid differentiation.     

Site-specific phosphorylation of Tau protein is associated with deacetylation of microtubules in mouse spermatogenic cells during meiosis

Tau is one of the microtubule-associated proteins and a major component of paired helical filaments, a hallmark of Alzheimer’s disease. Its expression has also been indicated in the testis. However, its function and modification in the testis have not been established. Here, we analyzed the dynamics of phosphorylation patterns during spermatogenesis. The expression of Tau protein and its phosphorylation were shown in the mouse testis. Immunohistochemistry revealed that the phosphorylation was strongly detected during meiosis. Correspondingly, the expression of acetylated tubulin was inversely weakened during meiosis. These results suggest that phosphorylation of Tau protein contributes to spermatogenesis, especially in meiosis.     

蛋白质组学在哺乳动物睾丸和精子发生中的应用

“蛋白质组学”一词由Wilkins在1994年提出,被称作后基因组时代一个新兴的研究手段.它从整体水平上对组织或者细胞的蛋白质表达、功能、相互作用进行研究,现在成为生命科学未来发展的主要分支之一.睾丸是哺乳动物雄性生殖系统中的一个重要的器官,由曲精小管和间质细胞组成.蛋白质组学在睾丸和精子发生研究上的应用及其技术手段的不断创新,对睾丸功能、生殖机理、生殖疾病的研究起到了极其重要的作用.所以,从蛋白质水平对睾丸和精子发生进行研究,为更好地理解雄性哺乳动物的生殖机理和疾病提供了一个新思路.     

Peroxisomes are present in murine spermatogonia and disappear during the course of spermatogenesis

Peroxisomes are organelles that are almost ubiquitous in eukaryotic cells. They have, however, never been described in germ cells within the testis. Since some peroxisomal diseases like Adrenoleukodystrophy are associated with reduced fertility, we have re-investigated the peroxisomal compartment of the germinal epithelium of mice using in situ hybridization, immunohistochemistry, Western blotting and immunoelectron microscopy. Within the seminiferous tubules, peroxisomes are present in Sertoli cells and in germ cells. We could show that small-sized peroxisomes of typical ultrastructure are concentrated in spermatogonia and disappear during the course of spermatogenesis. Peroxisomes of spermatogonia differ in their relative protein composition from previously described peroxisomes of interstitial cells of Leydig. Since germ cells differentiate in mouse testis in a synchronized fashion, the disappearence of peroxisomes could be a suitable model system to investigate the degradation of an organelle as part of a physiological differentiation process in higher eukaryotes.