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Vasa is the most studied germ cell marker that is indispensable for germ cell development in teleost fishes. Here, a vasa full-length cDNA from Oryzias celebensis was isolated. Analysis of gene expression by reversed transcription polymerase chain reaction and in situ hybridization showed the vasa transcript was maternally inherited and specifically expressed in germ cells during embryogenesis and in adult gonads. During embryogenesis, vasa mRNA was widely distributed in the embryos until the somitogenesis stage and then specifically expressed in primordial germ cells (PGCs). In the testis, vasa expression was highest in spermatogonia and gradually decreased during spermatogenesis. In ovary, vasa expression was present predominantly in immature oocytes and persisted throughout oogenesis. Constructs containing green or red fluorescence proteins and vasa 3' UTR or dnd 3' UTR, confirmed stable vasa expression in the PGCs of O. celebensis and co-expression of the two genes. In summary, the conservation of vasa expression in embryonic and adult germ cells of both sexes compared to other vertebrates suggests its function is also widely conserved., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mild testicular heating safely and reversibly suppresses spermatogenesis. In this study, we attempted to clarify the underlying molecular mechanism(s) involved in heat-induced spermatogenesis suppression in human testis. We conducted global proteomic analyses of human testicular biopsies before, and at 2 and 9 wk after heat treatment. Thirty-one and Twenty-six known proteins were identified with significant differential expression at 2 and 9 wk after heat treatment, respectively. These were used to characterize the cellular and molecular events in the testes when seminiferous epithelia became damaged (2 wk) and recovered (9 wk). At 2 wk post-treatment, the changed expression of a series of proteins could promote apoptosis or suppress proliferation and cell survival. At 9 wk post-treatment, the changed expression of proteins mainly promoted cell proliferation, differentiation and survival, but resisted cell apoptosis. Among those heat-regulated proteins, HNRNPH1 was selected for the further functional study. We found that HNRNPH1 was an anti-apoptosis protein that could regulate the expression of other heat-induced proteins. In conclusion, heat-induced reversible suppression of spermatogenesis occurred by modulating the expression of proteins related to proliferation, differentiation, apoptosis and cell survival pathways. These differentially expressed proteins were found to be key molecular targets affecting spermatogenesis after heat treatment.

The testis is the male gonad responsible for spermatogenesis and male hormone secretion. The complicated processes of spermatogenesis and steroidogenesis determine the complexity of protein expression control in the testis. In this study, the heterogeneity of human testis proteins was investigated using 2-dimensional gel electrophoresis. A total of 847 protein spots corresponding to 462 unique proteins were identified successfully by mass spectrometry. Notable heterogeneity was evidenced by the presence of more than 1 spot with different molecular weight and/or Isoelectric point values for each of 180 different proteins. Analysis of the detected peptides of these proteins indicated that this heterogeneity was partly the result of alternative splicing and/or proteolysis. SP_PIR_Keywords analysis suggested that alternative initiation sites and various forms of posttranslational modification may also contribute toward this heterogeneity. Using Pro-Q Diamond phosphostain, 68 spots representing 52 proteins were stained, confirming the presence of phosphorylated forms of these proteins in the human testis. These data were used to establish a proteome reference database, which can be accessed over the Internet (http://reprod.njmu.edu.cn/2d). This database provides an initial reference map of the human testis and serves as a useful resource for comparative proteomics studies of the human testis under normal and pathological states. The abundant protein heterogeneity observed in this study and further investigation of its biological significance will contribute toward understanding protein expression regulation in the human testis and will generate insight into the molecular mechanism of spermatogenesis.

The heterotrimeric G-protein pathway controls numerous cellular processes, including proliferation, differentiation, migration, membrane trafficking, and embryonic development. Regulator of G-protein signaling (RGS) proteins are known to function at the G-protein level. Here, the functional role of a novel RGS protein, regulator of G-protein signaling 22 (RGS22), in the testis was investigated at the mRNA and protein levels. Our results demonstrate that RGS22 is a testis-specific gene. However, significantly decreased expression of RGS22 was found in the testes of patients with azoospermia. RGS22 was translated or posttranslationally modified into multiple proteins of different molecular sizes in prokaryocytes as well as in the testes. Its protein (NP_056483) was localized in spermatogenic cells and Leydig cells and could interact with guanine nucleotide binding protein, alpha 12, 13, and 11 (GNA12, GNA13, and GNA11). Fragmental GFP-fusion protein tracking revealed that the N-terminal of RGS22 was localized in the nucleus. RGS22 and GNA13 were localized in the nucleus from the elongated spermatid stage onward. Indirect immunofluorescence studies revealed defective expression of GNA13 in macrocephalic and global nucleus spermatozoa. These findings suggest that their functions in this subcellular compartment are likely related to the postmeiotic developmental phase, spermiogenesis. RGS22 may also play a role in GNA13 translocation from the cytoplasm to the nucleus during spermiogenesis.

Treatment with injectable testosterone undecanoate (TU) alone or in combination with oral levonorgestrel (LNG) resulted in marked decreases in sperm concentrations. In this study, we used proteomic analyses to examine the cellular/molecular events occurring in the human testis after TU or TU + LNG treatment. We conducted a global proteomic analysis of the human testicular biopsies before and at 2 weeks after TU alone or TU + LNG treatment. Proteins showing significant changes in expression were identified and analyzed. As a result, 17 and 46 protein spots were found with significant differential expression after the treatment with TU alone and TU + LNG, respectively. TU treatment changed the expression of heterogeneous nuclear ribonucleoprotein K (hnRNP K), proteasome inhibitor PI31 subunit (PSMF1), and superoxide dismutase [Mn] mitochondrial precursor (SOD2). These proteins inhibit "assembly", induce cell death, and promote compensatory "cell survival" in the testis. After TU + LNG treatment, "proliferation/cell survival" and "apoptosis/death" were the predominant responses in the testis. TU + LNG treatment inhibited the expression of Prolyl 4-hydroxylase beta subunit (P4HB) and Annexin A2 (Annexin II). These proteins are involved in apoptosis and cell proliferation, respectively. TU + LNG treatment also enhanced the expression of SOD2 and Parvalbumin alpha (Pvalb). These two proteins may protect testicular cells against apoptosis/death and promote cell survival. In conclusion, TU and TU + LNG treatments suppress spermatogenesis through different pathways by changing the expression of different proteins. hnRNP K, PSMF1, SOD2, P4HB, Annexin II, and Pvalb, are key proteins that may be early molecular targets responsible for spermatogenesis suppression induced by hormone treatment.

Insulin-like growth factor-1 receptors (igf1rs) play important roles in regulating development, differentiation, and proliferation in diverse organisms. In the present study, subtypes of medaka igf1r, igf1ra, and igf1rb were isolated and characterized. RT-PCR results showed that igf1ra and igf1rb mRNA were expressed in all tissues and throughout embryogenesis. Using real-time PCR, the differential expression of igf1ra and igf1rb mRNA during folliculogenesis was observed. The results of in situ hybridization (ISH) revealed that both of them were expressed in ovarian follicles at different stages, and igf1rb was also expressed in theca cells and granulosa cells. In the testis, both igf1ra and igf1rb mRNA were highly expressed in sperm, while igf1rb mRNA was also obviously detected in spermatogonia. In addition, igf1ra mRNA was also present in Leydig cells in contrast to the distribution of igf1rb mRNA in Sertoli cells. Collectively, we demonstrated that differential igf1rs RNA expression identifies medaka meiotic germ cells and somatic cells of both sexes. These findings highlight the importance of the igf system in the development of fish gonads. [ABSTRACT FROM AUTHOR]

Insulin-like growth factors (IGFs) play significant roles in regulating cell proliferation, differentiation and reproduction in diverse organisms. Here, we report the identification of medaka igf1 and the expression pattern of igf1 RNA in adult medaka gonads. Using RT-PCR, igf1 RNA was easily detected in several somatic organs and adult gonads of both sexes. Using chromogenic in situ hybridization, igf1 RNA in the ovary was found in stage I-IV oocytes and in the somatic cells throughout oogenesis. In the testis, igf1 RNA was present in meiotic products and the somatic cells surrounding male germ cells throughout spermatogenesis. We performed fluorescent in situ hybridization and immunostaining for the Vasa protein as a germ cell marker on gonadal cryosections. We showed that igf1 RNA in the ovary was most abundant in theca cells and meiotic oocytes at stage I-IV, which was detectable in granulosa cells and infrequently occurred in the mature oocytes at stage V but was absent in oogonia. The igf1 RNA in the testis was most prominent in Leydig cells and Sertoli cells and meiotic spermatids and sperm as well as detectable in spermaotgonia and spermatocytes. We conclude that differential igf1 RNA expression identifies medaka somatic cells and meiotic germ cells of both sexes. [ABSTRACT FROM AUTHOR]