SLX2 interacting with BLOS2 is differentially expressed during mouse oocyte meiotic maturation.

Gametogenesis is a complex biological process of producing cells for sexual reproduction. Xlr super family members containing a conserved COR1 domain play essential roles in gametogenesis. In the present study, we identified that Slx2, a novel member of Xlr super family, is specifically expressed in the meiotic oocytes, which is demonstrated by western blotting and immunohistochemistry studies. In the first meiotic prophase, SLX2 is unevenly distributed in the nuclei of oocytes, during which phase SLX2 is partly co-localized with SYCP3 in synaptonemal complex and γH2AX in the nucleus of oocytes. Interestingly, the localization of SLX2 was found to be switched into the cytoplasm of oocytes after prometaphase I during oocyte maturation. Furthermore, yeast two-hybrid and coimmunoprecipitation studies demonstrated that SLX2 interacts with BLOS2, which is a novel centrosome-associated protein, and co-localized with γ-Tubulin, which is a protein marker of chromosome segregation in meiosis. These results indicated that SLX2 might get involved in chromosomes segregation during meiosis by interaction with BLOS2. In conclusion, SLX2 might be a novel gametogenesis-related protein that could play multiple roles in regulation of meiotic processes including synaptonemal complex assembly and chromosome segregation.

SYCP3-like X-linked 2 is expressed in meiotic germ cells and interacts with synaptonemal complex central element protein 2 and histone acetyltransferase TIP60.

Meiosis is the process by which diploid germ cells produce haploid gametes. A key event is the formation of the synaptonemal complex. In the pachytene stage, the unpaired regions of X and Y chromosomes form a specialized structure, the XY body, within which gene expression is mostly silenced. In the present study, we showed that SYCP3-like X-linked 2 (SLX2, 1700013H16Rik), a novel member of XLR (X-linked Lymphocyte-Regulated) family, was specifically expressed in meiotic germ cells. In the spermatocyte SLX2 was distributed in the nucleus of germ cells at the preleptotene, leptotene and zygotene stages and is then restricted to the XY body at the pachytene stage. This localization change is coincident with that of phosphorylated histone H2AX (γH2AX), a well-known component of the sex body. Through yeast two-hybrid screening and coimmunoprecipitation assays, we demonstrated that SLX2 interacts with synaptonemal complex central element protein 2 (SYCE2), an important component of synaptonemal complex, and histone acetyltransferase TIP60, which has been implicated in remodeling phospho-H2AX-containing nucleosomes at sites of DNA damage. These results suggest that SLX2 might be involved in DNA recombination, synaptonemal complex formation as well as sex body maintenance during meiosis.

Mouse Fem1b interacts with and induces ubiquitin-mediated degradation of Ankrd37.

Ankyrin repeat domain 37 (Ankrd37), a protein containing ankyrin repeats (ARs) and a putative nuclear localization signal (NLS), is highly conserved from zebrafish to humans. In mouse testes, Ankrd37 protein was initially present in the cytoplasm of elongating spermatids, and finally restricted to the nuclei of spermatozoa during spermatogenesis. Ankrd37 bound to feminization 1 homolog b (Fem1b) as indicated by yeast two-hybrid screening and co-immunoprecipitation assays. Ankrd37 facilitated the transport of Fem1b protein from cytoplasm to nuclei in co-transfected CHO cells. In addition, the protein level of Ankrd37 was decreased in a Fem1b dose-dependent manner as shown by the transfection experiments, and Ankrd37 was ubiquitinated in the presence of Fem1b. As the nematode Fem-1 has been shown to target its downstream effector TRA-1 for ubiquitin-mediated degradation, we report in the present study that mouse Fem1b targets Ankrd37 for degradation in the same manner.

Male germ cell-specific protein Trs4 binds to multiple proteins.

Temperature-related sequence 4 (Trs4) has been identified as a testis-specific gene with expression sensitive to the abdominal temperature changes induced by artificial cryptorchidism. In murine testes, Trs4 mRNA was detected in round spermatids and its protein was localized mainly in the elongating spermatids as well as in the acrosomes and tails of mature spermatozoa. Using a yeast two-hybrid screening system, we identified Rshl-2, Gstmu1, and Ddc8 as putative binding partners of the Trs4 protein in mouse testes. Their interactions were confirmed by in vivo and in vitro binding assays. Further studies demonstrated that Ddc8, a newly identified gene with unknown functions, displayed a similar expression pattern with Trs4 in mouse testes. In particular, Trs4, Ddc8, and Rshl-2 proteins were co-localized to the tails of mature spermatozoa. These results suggested that Trs4 might be involved in diverse processes of spermiogenesis and/or fertilization through interactions with its multiple binding partners.

Ankrd7, a novel gene specifically expressed in Sertoli cells and its potential roles in Sertoli cell maturation.

The somatic Sertoli cells play an essential role in testis determination and spermatogenesis by providing nutrition and structural support. In the current study, we report on the novel Ankrd7 gene that contains five ankyrin repeat domains. This gene was specifically expressed in Sertoli cells and was regulated in a maturation-dependent manner. Its expression was restricted to testicular tissue, and its mRNA could be detected in testes at as early as 14 dpp (days post partum) using RT-PCR analysis. In both testicular tissue sections and in vitro cultured Sertoli cells, the Ankrd7 protein was localized to the nucleus of the Sertoli cell. Immuno-histochemistry and immunocytochemistry investigations showed that the protein was detectable in testicular tissues at 20 dpp, at which time Sertoli cells were gradually differentiating into their mature cellular form. These results suggest that Ankrd7 is probably involved in the process of Sertoli cell maturation and in spermatogenesis.

WITHDRAWN: Fem1b interacts with Ankrd37 in mouse testis and induces its degradation by ubiquitin-mediated proteolysis pathway.

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Effect of 43 degrees treatment on expression of heat shock proteins 105, 70 and 60 in cultured monkey Sertoli cells.

AIM: To examine the possible effect of heat treatment on expression of heat shock proteins (Hsps) 105, 70, and 60 in primary monkey Sertoli cells and to evaluate the possible signal pathways. METHODS: Western blot analysis, real-time polymerase chain reaction (PCR), and confocal immunohistochemistry were used to analyze mRNA and protein levels of the Hsps in response to 43 degrees treatment of Sertoli cells isolated from pubertal monkey testes. RESULTS: Staining with Hoechst 33342 indicated Sertoli cells did not undergo apoptosis after heat treatment. Hsp105 was expressed in cytoplasm of untreated Sertoli cells. Both Hsp105 mRNA and protein levels were increased approximately 20-fold compared to those of the untreated controls at 12 h after heat treatment. Untreated Sertoli cells did not express Hsp70, but heat stress induced its expression in the cell cytoplasm. The time-course of changes in Hsp70 was similar to that of Hsp105. In contrast to Hsp105 and Hsp70, the change in Hsp60 expression was much less obvious. The protein level between 12 h and 48 h after heat treatment was only approximately 1.5-fold that of the untreated control. Extracellular regulated kinase (ERK) 1/2 inhibitor (U0126) or phosphoinositide kinase-3 (PI3K) inhibitor (LY294002) could partially block the response of Hsp105 and Hsp70 induced by heat treatment. CONCLUSION: These results indicate that the heat-induced expression of the three types of Hsp in monkey Sertoli cells might be regulated by ERK and/or PI3K signal pathways, but the profile of their expression is different, suggesting that they might have different regulatory functions in Sertoli cells.

Testosterone upregulation of tissue type plasminogen activator expression in Sertoli cells : tPA expression in Sertoli cells.

Our previous studies have demonstrated that tissue type plasminogen activator (tPA) might be involved in matrix degradation of blood-testis barrier in rat. In this study, we have further investigated the effect of testosterone (T) on tPA production in rat Sertoli cells. Our results showed that Sertoli cells isolated from rat testes at various ages in vitro secreted tPA in an age-dependent manner. The tPA activity was detected on day 20 after birth, and reached maximum on day 60. The Sertoli cells isolated from the testes on day 20 were then cultured in the presence or absence of testosterone, FSH, and forskolin, the tPA activities were upregulated by T, FSH and forskolin. Addition of H89 or U0126, both inhibited the testosterone-, FSH-, and forskolin-induced tPA expression. It is suggested that FSH- and testosterone-stimulated tPA expression in Sertoli cells may be via PKA and ERK signal transduction. Furthermore, we have observed that testosterone stimulated tPA secretion at all the stages of spermatogenesis (II-VI, VII-VIII, IX-XII and XIII-I), the highest stimulation of tPA activity was observed at stages VII-VIII. This study further suggests that testosterone-induced tPA activity in the Sertoli cells might be related to the function of blood-testis barrier opening and/or closing.

Heat treatment induces liver receptor homolog-1 expression in monkey and rat sertoli cells.

We demonstrated in this study that liver receptor homolog-1 (LRH-1) was expressed in the round spermatids in normal monkey testis, and no LRH-1 signal was observed in the Sertoli cells. After local warming (43 C) the monkey testis, however, LRH-1 expression was induced in the Sertoli cells in coincidence with activation of cytokeratin 18 (CK-18), a Sertoli cell dedifferentiated marker. Furthermore, we isolated rat primary Sertoli cells from testes at various stages of development and treated with 43 C water in vitro. The changes in LRH-1 as well as CK-18 expression were analyzed by confocal immunohistochemistry and Western blot. The results showed that LRH-1 was stage-dependently expressed in the Sertoli cells; no LRH-1-positive signal was detected in the cells obtained from the testes of adult rat on d 60 after birth when mature spermatozoa in the testis was completed. However, the mature Sertoli cells were warmed at the 43 C water bath for 15 min, and the LRH-1 signal was remarkably induced in a time-dependent manner, just like the changes of CK-18 expression in the Sertoli cells, suggesting that the heat-induced dedifferentiation of the mature Sertoli cells might be related to LRH-1 regulation. LRH-1 expression induced by the heat treatment was completely inhibited by the addition of ERK inhibitor U0126 in the culture, indicating that the heat-induced LRH-1 expression in the Sertoli cells may be regulated via ERK1/2 activation pathway. Testosterone was found to have no such effect on LRH-1 expression in the monkey and rat Sertoli cells.

In vitro propagation of spermatogonial stem cells from KM mice.

Spermatogonial stem cells (SSCs) are a unique type of stem cells in that they transmit genetic information to the next generation by producing sperms. Studies of SSC proliferation and differentiation have been hampered by the inability of reconstructing these processes in vitro, particularly in a serum-free culture system. Several groups have reported the long term culture of SSCs during which SSCs self-renew and restore spermatogenesis when transplanted back to recipient testes. However, different protocols and mice with particular genetic background have been used by different laboratories, and the techniques have not been adopted widely. In the present study, we first established a SSC isolation and culture system composed of differential adherence selection of SSCs, serum-free medium and mouse embryonic fibroblast (MEF) feeder cells. SSCs from KM pups could be cultured on MEF feeders in StemPro-34 SFM Medium supplemented with glial cell line-derived neurotrophic factor (GDNF), soluble GDNF family receptor alpha-1 (GFRa1) and basic fibroblast growth factor (bFGF) for 1 month. These SSCs were characterized morphologically and by examining the expression of marker genes. Expression of Oct4 and Sox2, which are crucial factors in embryonic stem cell (ESC) self-renewal, were detected in our cultured SSCs, suggesting that SSCs may share with ESCs some common mechanisms in self-renewal regulation. We also found that LIF had no effect on the proliferation of cultured SSCs derived from KM mice.

Cloning and characterization of a novel spermiogenesis-related gene, T6441, in rat testis.

We report in the present study the cloning and characterization of a novel gene, named T6441, initially derived by the suppressive subtracted hybridization (SSH) cDNA library. The full-length T6441cDNA was 664 bp long, containing a complete open-reading frame for a protein of 149 amino acids (aa). The protein bears no homology to any reported genes. It is predicted that the molecular mass was about 16.7 kDa. Northern blot analysis showed that the T6441 gene had about 4 transcripts in adult rat testis and was temporally regulated in a stage-dependent manner in the testis. In situ hybridization showed that T6441 mRNA was specifically localized in spermatids, and its expression level varied in the cells at different stages of the testicular development, with the highest level at steps 7-14. RT-PCR results showed that the T6441 mRNA was transcribed in most of the tested tissues with its strongest signal in the testis. Recombinant T6441 protein was prepared, purified, and was used to raise rabbit. Western blot analysis using the antiserum revealed four possible testicular specific proteins with their molecular weights being about 22, 25, 50 and 55 kDa respectively. The T6441 protein was expressed mainly in the cytoplasm of spermatids with the maximal levels at steps 12-19. At step 19 spermatid, the T6441 was mainly localized in the residual bodies. The cytoplasm localization of T6441 protein was supported by transient over expression of GFP-fusion protein in Hela cells. Interestingly, the expression of T6441 caused death of transfected cells within 48 h. Our preliminary experimental results suggest that the T6441 gene may play a role in cytoplasm movement and removal during spermiogenesis.