Role In Germ Cell Development Research Articles

The Potential Role of the Piwi Gene in the Development and Reproduction of Plutella xylostella.

Piwi proteins play a significant role in germ cell development and the silencing of transposons in animals by associating with small non-coding RNAs known as Piwi-interacting RNAs (piRNAs). While the Piwi gene has been well characterized in various insect species, the role of the Piwi (PxPiwi) gene in the diamondback moth (Plutella xylostella), a globally distributed pest of cruciferous crops, remains unclear. Expression analysis demonstrated the upregulation of PxPiwi in pupae and testes. Furthermore, we generated a PxPiwi-knockout mutant using CRISPR/Cas9 technology, which resulted in a significantly prolonged pupal stage and the failure of pupae to develop into adults. Additionally, the knockdown of PxPiwi, through RNA interference (RNAi), led to a substantial decrease in the oviposition and hatchability of P. xylostella. These findings indicate that PxPiwi is specifically expressed and essential for the development and reproduction of P. xylostella. This is the first report indicating the involvement of the Piwi gene in the development of lepidopteran insects, except for reproduction and germ cell development, which provides a foundation for future investigations into the functions of PxPiwi.

Mitochondria Quality Control and Male Fertility.

Mitochondria are pivotal to cellular homeostasis, performing vital functions such as bioenergetics, biosynthesis, and cell signalling. Proper maintenance of these processes is crucial to prevent disease development and ensure optimal cell function. Mitochondrial dynamics, including fission, fusion, biogenesis, mitophagy, and apoptosis, maintain mitochondrial quality control, which is essential for overall cell health. In male reproduction, mitochondria play a pivotal role in germ cell development and any defects in mitochondrial quality can have serious consequences on male fertility. Reactive oxygen species (ROS) also play a crucial role in sperm capacitation, but excessive ROS levels can trigger oxidative damage. Any imbalance between ROS and sperm quality control, caused by non-communicable diseases or environmental factors, can lead to an increase in oxidative stress, cell damage, and apoptosis, which in turn affect sperm concentration, quality, and motility. Therefore, assessing mitochondrial functionality and quality control is essential to gain valuable insights into male infertility. In sum, proper mitochondrial functionality is essential for overall health, and particularly important for male fertility. The assessment of mitochondrial functionality and quality control can provide crucial information for the study and management of male infertility and may lead to the development of new strategies for its management.

Identification and functional characterization of developmental-stage-dependent piRNAs in Tibetan sheep testes.

The core function of the testes is to produce sperms, which is the prerequisite for maintaining male fertility. PIWI-interacting RNAs (piRNAs) are a class of non-coding small RNAs that are mainly enriched in the reproductive organ and play a key role in germ cell development and spermatogenesis. However, the expression and function of piRNAs in the testes of Tibetan sheep, a domestic animal endemic to the Tibetan Plateau, remain unknown. In this study, we evaluated the sequence structure, expression profile, and potential function of piRNAs in testicular tissues from Tibetan sheep at different developmental stages (3 months, 1 year, and 3 years of age, respectively) by small RNA sequencing. Of the identified piRNAs, the sequence lengths of 24-26 nt and 29 nt dominate. Most piRNA sequences begin with uracil and have a distinct ping-pong structure which mainly distributes in exons, repeat regions, introns, and other unannotated regions of the genome. The piRNAs in the repeat region are primarily derived from the retrotransposons: long terminal repeats, long interspersed nuclear elements, and short interspersed elements. These piRNAs constitute 2,568 piRNA clusters, which mainly distribute on chromosomes 1, 2, 3, 5, 11, 13, 14, and 24, and of these clusters, a total of 529 piRNA clusters were differentially expressed in at least two age groups. Most of the piRNAs were expressed in a low abundance in the testes of developing Tibetan sheep. A total of 41,552 and 2,529 differential piRNAs were identified in testes from 3 months vs. 1 year, and 1 year vs. 3 years, respectively, presenting significantly increased abundance for most piRNAs in 1 year and 3 years compared with 3 months. The functional evaluation of the target genes showed that the differential piRNAs are mainly involved in regulating gene expression, transcription, protein modification, and cell development during spermatogenesis and testicular development. In conclusion, this study focused on the sequence structure and expression characteristics of piRNAs in the testis of Tibetan sheep and provided new insights into the functional mechanism of piRNAs in testicular development and spermatogenesis of sheep.

SERBP1 Promotes Stress Granule Clearance by Regulating 26S Proteasome Activity and G3BP1 Ubiquitination and Protects Male Germ Cells from Thermostimuli Damage.

Stress granules (SGs) are membraneless cytoplasmic condensates that dynamically assemble in response to various stressors and reversibly disassemble after stimulus removal; however, the mechanisms underlying SG dynamics and their physiological roles in germ cell development are elusive. Here, we show that SERBP1 (SERPINE1 mRNA binding protein 1) is a universal SG component and conserved regulator of SG clearance in somatic and male germ cells. SERBP1 interacts with the SG core component G3BP1 and 26S proteasome proteins PSMD10 and PSMA3 and recruits them to SGs. In the absence of SERBP1, reduced 20S proteasome activity, mislocalized valosin containing protein (VCP) and Fas associated factor family member 2 (FAF2), and diminished K63-linked polyubiquitination of G3BP1 during the SG recovery period were observed. Interestingly, the depletion of SERBP1 in testicular cells invivo causes increased germ cell apoptosis upon scrotal heat stress. Accordingly, we propose that a SERBP1-mediated mechanism regulates 26S proteasome activity and G3BP1 ubiquitination to facilitate SG clearance in both somatic and germ cell lines.

Vasa Is a Potential Germ Cell Marker in Leopard Coral Grouper (Plectropomus leopardus).

Vasa (Ddx4, DEAD box polypeptide 4), an extremely specific marker of germ cells in vivo, is an ATP-dependent RNA helicase that plays an essential role in germ cell development and gametogenesis. However, the expression and function information about this gene in groupers remains lacking. Here, vasa homolog termed Plvasa was isolated and identified Plvasa as a putative germ cell marker in the leopard coral grouper, (Plectropomus leopardus). Results indicated that Plvasa contained 17 exons in the genomic sequence and 9 conserved motifs of the DEAD-box protein by sequence analysis. The sequence comparison, phylogenetic analyses and synteny analyses showed that Plvasa was homologous with other teleosts. Additionally, the expression of Plvasa was significantly higher in gonads than in other tissues in adult individuals (p < 0.05). Further, the distribution of Plvasa revealed that it was only expressed in the germ cells, such as spermatids, germline stem cells and oocytes at different stages, and could not be detected in the somatic cells of gonads. The current study verified that the Plvasa gene is a valuable molecular marker of germ cells in leopard coral grouper, which potentially plays an important role in investigating the genesis and development of teleost germ cells.

Pou5f1 and Nanog Are Reliable Germ Cell-Specific Genes in Gonad of a Protogynous Hermaphroditic Fish, Orange-Spotted Grouper (Epinephelus coioides).

Pluripotency markers Pou5f1 and Nanog are core transcription factors regulating early embryonic development and maintaining the pluripotency and self-renewal of stem cells. Pou5f1 and Nanog also play important roles in germ cell development and gametogenesis. In this study, Pou5f1 (EcPou5f1) and Nanog (EcNanog) were cloned from orange-spotted grouper, Epinephelus coioides. The full-length cDNAs of EcPou5f1 and EcNanog were 2790 and 1820 bp, and encoded 475 and 432 amino acids, respectively. EcPou5f1 exhibited a specific expression in gonads, whereas EcNanog was expressed highly in gonads and weakly in some somatic tissues. In situ hybridization analyses showed that the mRNA signals of EcNanog and EcPou5f1 were exclusively restricted to germ cells in gonads. Likewise, immunohistofluorescence staining revealed that EcNanog protein was limited to germ cells. Moreover, both EcPou5f1 and EcNanog mRNAs were discovered to be co-localized with Vasa mRNA, a well-known germ cell maker, in male and female germ cells. These results implied that EcPou5f1 and EcNanog could be also regarded as reliable germ cell marker genes. Therefore, the findings of this study would pave the way for elucidating the mechanism whereby EcPou5f1 and EcNanog regulate germ cell development and gametogenesis in grouper fish, and even in other protogynous hermaphroditic species.

Regulation of GDF9 and CDKN1B expression in Tibetan sheep testes during different stages of maturity

Normal spermatogenesis is heavily dependent on the balance of germ cell proliferation, differentiation and apoptosis. Growth differentiation factor 9 (GDF9) and cyclin-dependent kinase inhibitor 1 B (CDKN1B) are strongly associated with cell cycle transition from G0/G1 to S and G2/M phase and hence regulating the growth and development of testicular germ cells and somatic cells. The current study was aimed at seeking out scientific evidence to determine if GDF9 and CDKN1B gene expression functions in the development of Tibetan sheep testes. To this end, developmental testes were derived from three-month-old (pre-puberty), one-year-old (sexual maturity), and three-year-old (adult) Tibetan sheep and then the expression and localization patterns of GDF9 and CDKN1B in these testes were evaluated using quantitative real-time PCR (qRT-PCR), Western blot and immunofluorescence. qRT-PCR and Western blot results showed that GDF9 and CDKN1B were detected in the testes throughout the different developmental stages. The abundance of GDF9 mRNA and protein in the testes of one- and three-year-old Tibetan sheep were higher than that in the testes of three-month-old Tibetan sheep; the mRNA and protein abundance of the CDKN1B gene in three-month-old Tibetan sheep testes were higher than that in the testes of the one-and three-year-old sheep. Moreover, immunofluorescence results suggested that the GDF9 protein was expressed in spermatogonia and Leydig cells, and that the CDKN1B protein was localized mainly in Leydig cells with some in the seminiferous epithelium throughout developmental stages. This indicated a novel role of the GDF9 and CDKN1B genes in Leydig cell development over and above their known roles in germ cell development. These findings have significant implications for our understanding of the molecular mechanisms of GDF9 and CDKN1B genes in Tibetan sheep spermatogenesis.

The Dynamic Regulation of mRNA Translation and Ribosome Biogenesis During Germ Cell Development and Reproductive Aging.

The regulation of mRNA translation, both globally and at the level of individual transcripts, plays a central role in the development and function of germ cells across species. Genetic studies using flies, worms, zebrafish and mice have highlighted the importance of specific RNA binding proteins in driving various aspects of germ cell formation and function. Many of these mRNA binding proteins, including Pumilio, Nanos, Vasa and Dazl have been conserved through evolution, specifically mark germ cells, and carry out similar functions across species. These proteins typically influence mRNA translation by binding to specific elements within the 3′ untranslated region (UTR) of target messages. Emerging evidence indicates that the global regulation of mRNA translation also plays an important role in germ cell development. For example, ribosome biogenesis is often regulated in a stage specific manner during gametogenesis. Moreover, oocytes need to produce and store a sufficient number of ribosomes to support the development of the early embryo until the initiation of zygotic transcription. Accumulating evidence indicates that disruption of mRNA translation regulatory mechanisms likely contributes to infertility and reproductive aging in humans. These findings highlight the importance of gaining further insights into the mechanisms that control mRNA translation within germ cells. Future work in this area will likely have important impacts beyond germ cell biology.

Germline development during embryogenesis of the larvacean, Oikopleura dioica

Germ cells develop into eggs and sperms and represent a lineage that survives through multiple generations. Germ cell specification during embryogenesis proceeds through one of two basic modes: either the cell-autonomous mode or the inductive mode. In the cell-autonomous mode, specification of germ cell fate involves asymmetric partitioning of the specialized maternal cytoplasm, known as the germplasm. Oikopleura dioica is a larvacean (class Appendicularia) and a chordate. It is regarded as a promising animal model for studying chordate development because of its short life cycle (5 days) and small genome size (∼60 ​Mb). We show that their embryos possess germplasm, as observed in ascidians (class Ascidiacea). The vegetal cytoplasm shifted towards the future posterior pole before the first cleavage occurred. A bilateral pair of primordial germ cells (PGC, B11 ​cells) was formed at the posterior pole at the 32-cell stage through two rounds of unequal cleavage. These B11 ​cells did not undergo further division before hatching of the tadpole-shaped larvae. The centrosome-attracting body (CAB) is a subcellular structure that contains the germplasm and plays crucial roles in germ cell development in ascidians. The presence of CAB with germplasm was observed in the germline lineage cells of larvaceans via electron microscopy and using extracted embryos. The CAB appeared at the 8-cell stage and persisted until the middle stage of embryogenesis. The antigen for the phosphorylated histone 3 antibody was localized to the CAB and persisted in the PGC until hatching after the CAB disappeared. Maternal snail mRNA, which encodes a transcription factor, was co-localized with the antigen for the H3S28p antibody. Furthermore, we found a novel PGC-specific subcellular structure that we call the germ body (GB). This study thus highlights the conserved and non-conserved features of germline development between ascidians and larvaceans. The rapid development and short life cycle (five days) of O. dioica would open the way to genetically analyze germ cell development in the future.

Vasa expression is associated with sex differentiation in the Asian yellow pond turtle, Mauremys mutica.

Vasa, one of the best-studied germ cell markers plays a critical role in germ cell development and differentiation in animals. Vasa deficiency would lead to male-specific sterility in most vertebrates, but female sterility in the fly. However, the role of the vasa gene involved in germ cell differentiation is largely elusive. Here, we first characterized the expression profile of vasa products in the Asian yellow pond turtle by quantitative reverse-transcription polymerase chain reaction and fluorescence immunostaining. The results showed that vasa messenger RNA (mRNA) is initially detected in embryos at stage 16, and then dramatically increased in embryos at stage 19. In particular, like the sex-related genes, vasa mRNA exhibited differential expression in embryos between the male-producing temperature (MPT, 25°C) and the female-producing temperature (FPT, 33°C), whereas there was no difference in methylation levels of vasa promoter detected between FPT and MPT. In contrast, in the adult Asian yellow pond, the level of vasa mRNA was much higher in the testis than ovary. Moreover, the immunostaining on testicular sections and cells showed that Vasa protein was exclusively expressed in germ cells: Weak but detectable in spermatogonia, highest in spermatocytes, moderate and concentrated in chromatid bodies in spermatids and spermatozoa, and bare in somatic cells. The expression profile of Vasa protein is similar in turtle species studied so far but distinct from those in fish species in this study. The findings of this study would provide new insights into our understanding of the conservation and divergence of the vasa gene, even other germ cell genes across phyla.

Investigation of piwil2 expression as a biomarker in solid tumors of childhood

Aim: PIWIL 2 belongs to a subgroup of argonaute proteins, which has an essential role in germ cell development and repair. Recent studies have demonstrated PIWIL 2 as a stem cell marker in some solid tumors. In the present study, we aimed to evaluate the expression of PIWIL 2 in childhood solid tumors and to show being a potential marker for a therapeutic drug.Materials and Methods: The study included 150 patients who were diagnosed with pediatric solid tumor and followed between 2000 and 2011. Solid tumor preparations were stained with polyclonal PIWIL 2 antibody by using immunohistochemical methods.Results: Assessment of the tumor sub-groups showed that, among 30 patients with neuroblastoma, there was a strong staining in 5 (16.70%) (p=0.020). PIWIL 2 was showed strong staining in 3 (21.40%) osteosarcoma patients (p=0.030). Among 34 patients with a germ cell tumor, there was a strong staining in 6 (17.60%) patients (p=0.040).Conclusion: Significant PIWIL 2 positivity was found in neuroblastoma, osteosarcoma and germ cell tumors and it can be a potential marker for these tumors. Because of non-tumor tissue involvement in Wilms’ tumor it can be particularly useful in determining surgical margins.

PRMT7 is involved in regulation of germ cell proliferation during embryonic stage

Arginine methylation is one of the most important post-translational modifications which is catalyzed by protein arginine methyltransferases (PRMTs). Previous studies have demonstrated that Prmt5 plays important role in germ cell development. Prmt7 is the only family member responsible for mono-methylation of arginine residue. However, whether Prmt7 is also involved in germ cell development remains unclear. In this study, we find that PRMT7 is abundantly expressed in the male germ cells during embryonic stage (from E10.5). Depletion of Prmt7 results in the defect of germ cell proliferation during embryonic stage and the number of primordial germ cells is significantly reduced in Prmt7−/− mice at E11.5. We also find that the size of testes is reduced in Prmt7−/− mice at P5 with reduced germ cell number and the diameter of seminiferous tubules. Further study reveals that the expression of BMPs and TGF-β singling pathway is significantly changed in germ cells of Prmt7−/− mice at E12.5. However, no defect of testes development is observed in adult Prmt7-/flox; Mvh-Cre mice. Collectively, this study demonstrates that Prmt7 plays roles in male germ cell proliferation during embryonic stages and it is not required for germ cell development postnatally.

Homologues of Piwi control transposable elements and development of male germline in Penaeus monodon

PIWI belongs to the Argonaute protein family, which is a major protein component in RNA silencing pathway. Piwi proteins play roles in the control of transposons and germline development. They have been widely studied in vertebrates and flies, while very little is known in crustacean so far. We have previously identified and characterized a cDNA encoding Piwi protein (PmPiwi1) in the black tiger shrimp Penaeus monodon. In this study, a cDNA encoding another Piwi protein namely PmPiwi2 was identified by rapid amplification of cDNA ends (RACEs). PmPiwi2 was expressed solely in shrimp testis and ovary, indicating its potential role in germ cell development. Similar to PmPiwi1, PmPiwi2 also plays a part in the control of transposons as PmPiwi2-knockdown shrimp showed a significant increase in the expression of gypsy2 retrotransposon and mariner element in the testis. In addition, a reduction of sperm numbers in the spermatophore of PmPiwi2-knockdown shrimp suggests that PmPiwi2 is required for spermatogenesis similar to PmPiwi1. This study further demonstrated that apoptotic cell death was strongly detected in spermatogonia and spermatocyte cells of both PmPiwi-knockdown shrimp and thus, could be the cause of reduced sperm count. Investigation of sperm morphology showed a remarkably high proportion of abnormal sperms in the spermatophore of the PmPiwi1-knockdown shrimp, while PmPiwi2-knockdown shrimp had comparable percentage of abnormal sperms to the control shrimp. Consistently, the expression of KIFC1, a gene that is necessary for spermiogenesis was significantly reduced upon PmPiwi1 silencing, but not in the PmPiwi2-knockdown shrimp. Our results suggested that while both PmPiwis are required for the development of spermatid, only PmPiwi1 is possibly involved in the final stage of sperm maturation.

Growth Factors, and Cytokines; Understanding the Role of Tyrosine Phosphatase SHP2 in Gametogenesis and Early Embryo Development.

Growth factors and cytokines have vital roles in germ cell development, gamete maturation, and early embryo development. Cell surface receptors are present for growth factors and cytokines to integrate with and trigger protein signaling in the germ and embryo intracellular milieu. Src-homology-2-containing phosphotyrosine phosphatase (SHP2) is a ubiquitously expressed, multifunctional protein that plays a central role in the signaling pathways involved in growth factor receptors, cytokine receptors, integrins, and G protein-coupled receptors. Over recent decades, researchers have recapitulated the protein signaling networks that influence gamete progenitor specification as well as gamete differentiation and maturation. SHP2 plays an indispensable role in cellular growth, survival, proliferation, differentiation, and migration, as well as the basic events in gametogenesis and early embryo development. SHP2, a classic cytosolic protein and a key regulator of signal transduction, displays unconventional nuclear expression in the genital organs. Several observations provided shreds of evidence that this behavior is essential for fertility. The growth factor and cytokine-dependent roles of SHP2 and its nuclear/cytoplasmic presence during gamete maturation, early embryonic development and embryo implantation are fascinating and complex subjects. This review is intended to summarize the previous and recent knowledge about the SHP2 functions in gametogenesis and early embryo development.

BRG1 Is Dispensable for Sertoli Cell Development and Functions in Mice.

Sertoli cells are somatic supporting cells in spermatogenic niche and play critical roles in germ cell development, but it is yet to be understood how epigenetic modifiers regulate Sertoli cell development and contribution to spermatogenesis. BRG1 (Brahma related gene 1) is a catalytic subunit of the mammalian SWI/SNF chromatin remodeling complex and participates in transcriptional regulation. The present study aimed to define the functions of BRG1 in mouse Sertoli cells during mouse spermatogenesis. We found that BRG1 protein was localized in the nuclei of both Sertoli cells and germ cells in seminiferous tubules. We further examined the requirement of BRG1 in Sertoli cell development using a Brg1 conditional knockout mouse model and two Amh-Cre mouse strains to specifically delete Brg1 gene from Sertoli cells. We found that the Amh-Cre mice from Jackson Laboratory had inefficient recombinase activities in Sertoli cells, while the other Amh-Cre strain from the European Mouse Mutant Archive achieved complete Brg1 deletion in Sertoli cells. Nevertheless, the conditional knockout of Brg1 from Sertoli cells by neither of Amh-Cre strains led to any detectable abnormalities in the development of either Sertoli cells or germ cells, suggesting that BRG1-SWI/SNF complex is dispensable to the functions of Sertoli cells in spermatogenesis.

Expression pattern and potential role of Nanos3 in regulating testosterone biosynthesis in Leydig cells of sheep

Nanos3 is from a highly conserved family of genes with putative RNA-binding activity and plays an important role in germ cell development. Yet, its function in Leydig cells (LCs) for supporting germ cell development, is still poorly understood. In this study, we investigated the expression pattern and function of Nanos3 in the sheep testis. The Nanos3 mRNA and protein were highly expressed in the ram testis. The sheep Nanos3 amino acid sequence was 93.6%–88.6% homologous with those of other mammalian species. The expression of Nanos3 in the ram testis was significantly lower in 9 and 24-month-old sheep than in at 3-month-old sheep (P < 0.05). Immunohistochemistry analysis revealed that sheep Nanos3 was not only localized at spermatogenesis cell but also at LCs. Thus the functions of Nanos3 in LCs were further investigated by using siRNA-Nanos3 in vitro. The secretion of T hormone and expression of testosterone synthesis-related enzymes in LCs were significantly inhibited (P < 0.05) by siRNA-Nanos3. Moreover, the BAX/Bcl-2 mRNA and protein levels were significantly increased (P < 0.05) by siRNA-Nanos3. In addition, we examined the expression levels of Nanos3 mRNA and protein in energy-restricted(ER) rams and in vitro ER LCs. Their levels were also significantly decreased (P < 0.05) and were accompanied by an increased BAX/Bcl-2 mRNA expression compared with the control group (P < 0.05). To the best of our knowledge, our study is the first to show that Nanos3 is an important regulator of testosterone biosynthesis in rams for supporting germ cell development.

Survival Motor Neuron Protein Participates in Mouse Germ Cell Development and Spermatogonium Maintenance.

The defective human survival motor neuron 1 (SMN1) gene leads to spinal muscular atrophy (SMA), the most common genetic cause of infant mortality. We previously reported that loss of SMN results in rapid differentiation of Drosophila germline stem cells and mouse embryonic stem cells (ESCs), indicating that SMN also plays important roles in germ cell development and stem cell biology. Here, we show that in healthy mice, SMN is highly expressed in the gonadal tissues, prepubertal spermatogonia, and adult spermatocytes, whereas low SMN expression is found in differentiated spermatid and sperm. In SMA-like mice, the growth of testis tissues is retarded, accompanied with gamete development abnormalities and loss of the spermatogonia-specific marker. Consistently, knockdown of Smn1 in spermatogonial stem cells (SSCs) leads to a compromised regeneration capacity in vitro and in vivo in transplantation experiments. In SMA-like mice, apoptosis and accumulation of the R-loop structure were significantly elevated, indicating that SMN plays a critical role in the survival of male germ cells. The present work demonstrates that SMN, in addition to its critical roles in neuronal development, participates in mouse germ cell and spermatogonium maintenance.

Identification and characterization of germ cell genes vasa and dazl in a protogynous hermaphrodite fish, orange-spotted grouper (Epinephelus coioides)

Vasa and dazl genes have been reported to play pivotal roles in germ cell development and differentiation both in vertebrates and invertebrates; however, little is known about their functions in germ cell differentiation during gametogenesis and sex reversal in hermaphroditic fish. In the present study, vasa (Ecvasa) and dazl (Ecdazl) cDNA were cloned from orange-spotted grouper (Epinephelus coioides). The full-length cDNA sequences of Ecvasa and Ecdazl were 2162 and 2101 bp, and encoded 646 and 214 amino acid residues, respectively. Reversal transcription PCR showed that Ecvasa and Ecdazl mRNA were highly expressed in the gonads. Further, in situ hybridization revealed that Ecvasa and Ecdazl RNA were dynamically expressed in germ cells at different stages during oogenesis, sex reversal, and spermatogenesis in orange-spotted grouper. Intriguingly, the signals for Ecvasa and Ecdazl mRNA became weaker in oocytes of ovo-testes gonads, indicating that the expression of germ cell genes could be suppressed in oocytes during sex reversal in the orange-spotted grouper. Our study is the first time to describe the expression profiles of vasa and dazl mRNA in germ cells during gametogenesis and sex reversal in the orange-spotted grouper. These findings will provide new insights into understanding the mechanisms through which vasa and dazl regulate germ cell differentiation in hermaphrodite fish species.

Identification and characterization of DAZ family genes in Chinese soft-shell turtle (Pelodiscus sinensis).

The DAZ family genes, including boule, dazl, and daz, play pivotal roles in germ cell development and differentiation during gametogenesis in organisms, which have been widely studied in mammals, reptiles, or fishes. Dazl was bisexual expressed in both mitotic and meiotic germ cells, daz was male premeiotic expressed, whereas boule exhibits largely in unisexual meiotic germ cells but bisexual expression in several fishes, however, there is lack of report on boule gene and the evolutionary conservation and divergence of dazl and boule in reptile. Here, both boule and dazl genes were characterized in Pelodiscus sinensis. The quantitative real-time polymerase chain reaction analysis showed that boule and dazl were abundantly expressed in adult ovary and testis but barely in somatic tissues, such as heart, brain, liver, spleen, and kidney. Moreover, through fluorescent in situ hybridization, bisexual and germline-specific expression profiles of boule and dazl messenger RNAs (mRNAs) were demonstrated. Boule mRNA exhibited a maximal meiotic expression in spermatocytes, and a relatively low, but distinct expression in oocytes at meiotic stages in P. sinensis, similar to the expression profile of human boule in ovary. However, dazl mRNA was richly distributed in male germ cells at almost all stages during spermatogenesis, and predominantly expressed in most of stages of oocytes including premeiotic and meiotic stages. These findings imply that boule and dazl would play distinct roles in the sexual differentiation of germ cells during turtle gametogenesis, and the major functions of daz family members involved in germ cell differentiation would be conserved across species including P. sinensis.

Abnormal Meiosis Initiation in Germ Cell Caused by Aberrant Differentiation of Gonad Somatic Cell.

The interaction between germ cell and somatic cell plays important roles in germ cell development. However, the exact function of gonad somatic cell in germ cell differentiation is unclear. In the present study, the function of gonad somatic cell in germ cell meiosis was examined by using mouse models with aberrant somatic cell differentiation. In Wt1R394W/R394W mice, the genital ridge is absent due to the apoptosis of coelomic epithelial cells. Interestingly, in both male and female Wt1R394W/R394W germ cells, STRA8 was detected at E12.5 and the scattered SYCP3 foci were observed at E13.5 which was consistent with control females. In Wt1-/flox; Cre-ERTM mice, Wt1 was inactivated by the injection of tamoxifen at E9.5 and the differentiation of Sertoli and granulosa cells was completely blocked. We found that most germ cells were located outside of genital ridge after Wt1 inactivation. STRA8, SYCP3, and γH2AX proteins were detected in germ cells of both male and female Wt1-/flox; Cre-ERTM gonads, whereas no thread-like SYCP3 signal was observed. Our study demonstrates that aberrant development of gonad somatic cells leads to ectopic expression of meiosis-associated genes in germ cells, but meiosis was arrested before prophase I. These results suggest that the proper differentiation of gonad somatic cells is essential for germ cell meiosis.