Spermatogenesis is a complex process that generates spermatozoa; its molecular mechanisms are not completely understood. Here we focused on the functions of three testis-specific serine proteases: Prss42/Tessp-2, Prss43/Tessp-3, and Prss44/Tessp-4. These protease genes, which constitute a gene cluster on chromosome 9F2-F3, were presumed to be paralogs and were expressed only in the testis. By investigating their mRNA distribution, we found that all three genes were expressed in primary and secondary spermatocytes. However, interestingly, the translated proteins were produced at different locations. Prss42/Tessp-2 was found in the membranes and cytoplasm of secondary spermatocytes and spermatids, whereas Prss43/Tessp-3 was present only in the membranes of spermatocytes and spermatids. Prss44/Tessp-4 was detected in the cytoplasm of spermatocytes and spermatids. To assess the roles of these proteases in spermatogenesis, we used organ culture of mouse testis fragments. Adding antibodies against Prss42/Tessp-2 and Prss43/Tessp-3 resulted in meiotic arrest at the stage when each protease was beginning to be translated. Furthermore, the number of apoptotic cells dramatically increased after the addition of these antibodies. These results strongly suggest that the three paralogous Prss/Tessp proteases play different roles in spermatogenesis and that Prss42/Tessp-2 and Prss43/Tessp-3 are required for germ cell survival during meiosis.

BackgroundTranslocated in LipoSarcoma (TLS, also known as FUsed in Sarcoma) is an RNA/DNA binding protein whose mutation cause amyotrophic lateral sclerosis. In previous study, we demonstrated that TLS binds to long noncoding RNA, promoter-associated ncRNA-D (pncRNA-D), transcribed from the 5′ upstream region of cyclin D1 (CCND1), and inhibits the expression of CCND1.ResultsIn order to elucidate the binding specificity between TLS and pncRNA-D, we divided pncRNA-D into seven fragments and examined the binding with full-length TLS, TLS–RGG2–zinc finger–RGG3, and TLS–RGG3 by RNA pull down assay. As a result, TLS was able to bind to all the seven fragments, but the fragments containing reported recognition motifs (GGUG and GGU) tend to bind more solidly. The full-length TLS and TLS–RGG2–zinc finger–RGG3 showed a similar interaction with pncRNA-D, but the binding specificity of TLS–RGG3 was lower compared to the full-length TLS and TLS–RGG2–zinc finger–RGG3. Mutation in GGUG and GGU motifs dramatically decreased the binding, and unexpectedly, we could only detect weak interaction with the RNA sequence with stem loop structure.ConclusionThe binding of TLS and pncRNA-D was affected by the presence of GGUG and GGU sequences, and the C terminal domains of TLS function in the interaction with pncRNA-D.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-016-0068-8) contains supplementary material, which is available to authorized users.

Translocated in liposarcoma (TLS) is an RNA-binding protein and a transcription-regulatory sensor of DNA damage. TLS binds promoter-associated noncoding RNA (pncRNA) and inhibits histone acetyltransferase (HAT) activity of CREB-binding protein (CBP)/E1A-binding protein P300 (p300) on the cyclin D1 () gene. Although post-translational modifications of TLS, such as arginine methylation, are known to regulate TLS's nucleocytoplasmic shuttling and assembly in stress granules, its interactions with RNAs remain poorly characterized. Herein, using various biochemical assays, we confirmed the earlier observations that TLS is methylated by protein arginine methyltransferase 1 (PRMT1) The arginine methylation of TLS disrupted binding to pncRNA and also prevented binding of TLS to and inhibition of CBP/p300. This result indicated that arginine methylation of TLS abrogates both binding to pncRNA and TLS-mediated inhibition of CBP/p300 HAT activities. We also report that an arginine residue within the Arg-Gly-Gly domain of TLS, Arg-476, serves as the major determinant for binding to pncRNA. Either methylation or mutation of Arg-476 of TLS significantly decreased pncRNA binding and thereby prevented a pncRNA-induced allosteric alteration in TLS that is required for its interaction with CBP/p300. Moreover, unlike WT TLS, an R476A TLS mutant did not inhibit promoter activity in luciferase reporter assays. Taken together, we propose the hypothesis that arginine methylation of TLS regulates both TLS-nucleic acid and TLS-protein interactions and thereby participates in transcriptional regulation.

Anti-Müllerian hormone (AMH) is critical to the regression of Müllerian ducts during mammalian male differentiation and targets ovarian granulosa cells and testicular Sertoli and Leydig cells of adults. Specific effects of AMH are exerted via its receptor, AMH type II receptor (Amhr2), but the mechanism by which the Amhr2 gene is specifically activated is not fully understood. To see whether a proximal promoter was sufficient for Amhr2 gene activation, we generated transgenic mice that bore the enhanced green fluorescent protein (EGFP) gene driven by a 500-bp mouse Amhr2 gene promoter. None of the established 10 lines, however, showed appropriate EGFP expression, indicating that the 500-bp promoter was insufficient for Amhr2 gene activation. As a regulatory element, we found a long noncoding RNA, lncRNA-Amhr2, transcribed from upstream of the Amhr2 gene in ovarian granulosa cells and testicular Sertoli cells. In primary granulosa cells, knockdown of lncRNA-Amhr2 resulted in a decrease of Amhr2 messnger RNA level, and a transient reporter gene assay showed that lncRNA-Amhr2 activation increased Amhr2 promoter activity. The activity was correlated with lncRNA-Amhr2 transcription in stably transfected OV3121 cells derived from mouse granulosa cells. Moreover, by the Tet-on system, the induction of lncRNA-Amhr2 transcription dramatically increased Amhr2 promoter activity in OV3121 cells. These results indicate that lncRNA-Amhr2 plays a role in Amhr2 gene activation in ovarian granulosa cells by enhancing promoter activity, providing insight into Amhr2 gene regulation underlying the AMH signaling in the female reproductive system.

SUMMARYSpermatogenesis is precisely regulated by many meiotic stage-specific genes, but their regulatory mechanisms are not fully understood. The Prss/Tessp gene cluster is located on the mouse chromosome 9F2-F3, and the three genes, Prss42/Tessp-2, Prss43/Tessp-3, and Prss44/Tessp-4 on the cluster, are specifically activated in pachytene spermatocytes during meiosis. To elucidate a mechanism of their activation, we searched for DNase I hypersensitive sites (HSs) and long noncoding RNAs (lncRNAs) at the Prss/Tessp locus. We found eight DNase I HSs, three of which were testicular germ cell-specific at or close to the Prss42/Tessp-2 promoter, and a testis-specific lncRNA, lncRNA-HSVIII, which was transcribed from 3' to the Prss42/Tessp-2 gene. By in situ hybridization, lncRNA-HSVIII was localized in nuclei of most pachytene spermatocytes and in cytosols of pachytene spermatocytes at stage X and spermatids. Chromosome conformation capture assay showed that the chromatin at lncRNA-HSVIII specifically interacted with the Prss42/Tessp-2 promoter in primary and secondary spermatocytes. By reporter gene assay, a 5.8-kb genome sequence, encompassing the entire lncRNA-HSVIII sequence and its flanking regions, significantly increased Prss42/Tessp-2 promoter activity, but transfection of this construct did not change the lncRNA-HSVIII expression, which indicated that the increased promoter activity was likely to be dependent on enhancer activity. Indeed, we found that both upstream and downstream regions of the lncRNA-HSVIII sequence significantly increased Prss42/Tessp-2 promoter activity. Our data indicate the direct interaction of a genomic region at lncRNA-HSVIII with the Prss42/Tessp-2 promoter in spermatocytes and suggest that adjacent sequences to the lncRNA function as enhancers for the Prss42/Tessp-2 gene.4