Immunolocalization of Vasa,PIWI, and TDRKH proteins in male germ cells during spermatogenesis of the teleost fish Poecilia reticulata

Vasa, PIWI and TDRKH are conserved components of germ granules that in metazoans are involved in germline specification and differentiation, as documented by mutational experiments in some model animals. So far, investigations on PIWI during spermatogenesis of fish has been limited to a few species, and no information is available for TDRKH, another protein involved in the piRNA pathway. In this study, the immunolocalization of these three germline determinants was analyzed in male gonads of the teleost fish Poecilia reticulata to document their localization pattern in the different stages of germ cell differentiation.To analyze their distribution pattern during the different stages of spermatogenesis we performed immunohistochemistry (IHC) and immunofluorescence (IF) assays using primary polyclonal antibodies after testing their specificity with Western Blot. Moreover, sections of testis stained with haematoxylin and eosin clarified the structural organization of P. reticulata testis, while the use of the confocal microscope and the nuclear staining clarified the different stages of germ cell differentiation during spermatogenesis.The results showed that Vasa, PIWI and TDRKH were specifically immunolocalized in the germ cells of P. reticulata, with no specific signal detected in Sertoli cells and in other somatic cells of the gonad. These markers were detected in all stages of differentiation from early spermatogonia to advanced spermatids. Vasa staining was the strongest in spermatogonia, and then decreases throughout differentiation. Instead, both PIWI and TDRKH staining increases during differentiation, and their distribution pattern, similar to what observed in the mouse, suggests their concerted participation in the piRNA pathway also in this fish.     

OMA-1 is a P granules-associated protein that is required for germline specification in Caenorhabditis elegans embryos

In Caenorhabditis elegans, CCCH-type zinc-finger proteins have been shown to be involved in the differentiation of germ cells during embryonic development. Previously, we and others have identified novel redundant CCCH-type zinc-finger proteins, OMA-1 and OMA-2, that are involved in oocyte maturation. In this study, we report that the cytoplasmic expression level of OMA-1 protein was largely reduced after fertilization. In contrast to its cytoplasmic degradation, OMA-1 was found to accumulate exclusively on P granules in germline blastomeres during embryogenesis. A notable finding is that embryos with partially suppressed oma-1; oma-2 expression showed inappropriate germline specification, including abnormal distributions of PGL-1, MEX-1 and PIE-1 proteins. Thus, our results suggest that oma gene products are novel multifunctional proteins that participate in crucial processes for germline specification during embryonic development.     

MAGE-A1, GAGE and NY-ESO-1 cancer/testis antigen expression during human gonadal development

BACKGROUND: Cancer/testis antigens (CTAs) are expressed in several cancers and during normal adult male germ cell differentiation. Little is known about their role in fetal development of human germ cells. METHODS: We examined expression of the CTAs MAGE-A1, GAGE and NY-ESO-1 in fetal gonads by single and double immunohistochemical staining. RESULTS: We found that GAGE was expressed in the primordial germ cells of the gonadal primordium, whereas MAGE-A1 and NY-ESO-1 were first detected in germ cells of both testis and ovary after sexual differentiation was initiated. The number of positive germ cells and the staining intensity of all three CTAs peaked during the second trimester and gradually decreased towards birth in both male and female germ cells. In oocytes, MAGE-A1 expression terminated around birth, whereas NY-ESO-1 expression persisted through the neonatal stage and GAGE expression was maintained until adulthood. The population of GAGE-expressing male and female germ cells partially overlapped the population of OCT4-positive cells, whereas MAGE-A1 and NY-ESO-1 were clearly expressed only by OCT4-negative cells. CONCLUSIONS: Our results suggest that MAGE-A1 and NY-ESO-1 are associated with highly proliferating germ cells, whereas GAGE proteins have a more general function in germ cells unrelated to any specific developmental stage. The recognition of differential cellular expression of GAGE, MAGE-A1, NY-ESO-1 and OCT4 may help define biologically distinct germ cell subpopulations.     

Generation of biallelic F0 mutants in medaka using the CRISPR/Cas9 system

Several animal models generated by genome editing methods develop somatic mosaic mutations including wild‐type genome sequence in F0 generation because it is difficult to use editing tools at the one‐cell stage. Producing complete knockout animals quickly is a great advantage in determining the function of target genes. This study investigated the generation of F0 knockout medaka using the CRISPR/Cas9 system. To determine whether this editing system induced mutations in the medaka genome at the one‐cell stage, recombinant Cas9 protein, tracrRNA and crRNA for dead end (dnd), which is essential for germ cell development, were injected into one‐cell stage embryos of olvas‐DsRedExpress transgenic medaka. This allowed germ cells to be visualized by DsRed fluorescence. Genomic DNA extracted from embryos at the one‐cell stage was analyzed by sequencing. Predictably, biallelic mutated sequence patterns in the target sites of dnd were found in the injected embryos. To investigate the phenotypes of the mutated fish, fluorescent and histological observations of germ cells were carried out using fry and adults. The mutations resulted in a complete loss of germ cells, suggesting loss of function of dnd in the injected embryos. Therefore, this system appears to be extremely effective for the production of F0 knockout medaka.     

Involvement of E-cadherin and beta-catenin in germ cell tumours and in normal male fetal germ cell development

Intercellular contacts, mediated by E-cadherin, are essential for germ cell migration and maturation. Furthermore, it has been suggested that decrease or loss of E-cadherin correlates with tumour progression and invasive behaviour. beta-catenin is involved in a number of different processes, including cell--cell interaction when bound to cadherins, and determination of cell fate in pluripotent cells when activated via the Wnt signal-transduction pathway. To shed more light on the role of these factors in normal fetal germ cell development and the pathogenesis of germ cell tumours (GCTs), the present study investigated the presence and localization of E-cadherin and beta-catenin by immunohistochemistry. E-cadherin was only weakly expressed in or absent from fetal germ cells of the second and third trimesters, and was not expressed in carcinoma in situ/intratubular germ cell neoplasia unclassified (CIS/ITGCNU) and gonadoblastoma, the precursor of an invasive GCT in dysgenetic gonads. In GCTs, it was generally not expressed in seminoma and dysgerminoma, but was found in the vast majority of non-seminoma cells. beta-catenin was found in the cytoplasm of fetal germ cells at all gestational ages and in spermatogenesis in post-pubertal testes. It was also present in CIS/ITGCNU and gonadoblastoma. Whereas seminomas and dysgerminoma were negative, non-seminoma cells were frequently found to express beta-catenin. Expression of both factors therefore reflects the degree of differentiation of these tumours. No differences for either E-cadherin or beta-catenin were observed between samples of tumours resistant or sensitive to chemotherapy, and E-cadherin expression did not correlate with vascular invasion. E-cadherin and beta-catenin therefore play a role in both normal and malignant germ cell development and differentiation that warrants further investigation, but they seem to be of limited value as predictive or prognostic factors in GCTs.     

Differential expression of anti-Müllerian hormone (amh) and anti-Müllerian hormone receptor type II (amhrII) in the teleost medaka.

In mammals, the anti-Müllerian hormone (Amh) is responsible for the regression of the Müllerian ducts; therefore, Amh is an important factor of male sex differentiation. The amh gene has been cloned in various vertebrates, as well as in several teleost species. To date, all described species show a sexually dimorphic expression of amh during sex differentiation or at least in differentiated juvenile gonads. We have identified the medaka amh ortholog and examined its expression pattern. Medaka amh shows no sexually dimorphic expression pattern. It is expressed in both developing XY male and XX female gonads. In adult testes, amh is expressed in the Sertoli cells and in adult ovaries in granulosa cells surrounding the oocytes, like in mammals. To better understand the function of amh, we cloned the anti-Müllerian hormone receptor type II (amhrII) ortholog and compared its expression pattern with amh, aromatase (cyp19a1), and scp3. During gonad development, amhrII is coexpressed with medaka amh in somatic cells of the gonads and shows no sexually dimorphic expression. Only the expression level of the Amh type II receptor gene was decreased noticeably in adult female gonads. These results suggest that medaka Amh and AmhrII are involved in gonad formation and maintenance in both sexes.     

Restricted distribution of mrg‐1 mRNA in C. elegans primordial germ cells through germ granule‐independent regulation

The chromodomain protein MRG‐1 is an essential maternal factor for proper germline development that protects germ cells from cell death in C. elegans. Unlike germ granules, which are exclusively segregated to the germline blastomeres at each cell division from the first cleavage of the embryo, MRG‐1 is abundant in all cells in early embryos and is then gradually restricted to the primordial germ cells (PGCs) by the morphogenesis stage. Here, we show that this characteristic spatiotemporal expression pattern is dictated by the mrg‐1 3'UTR and is differentially regulated at the RNA level between germline and somatic cells. Asymmetric segregation of germ granules is not necessary to localize MRG‐1 to the PGCs. We found that MES‐4, an essential chromatin regulator in germ cells, also accumulates in the PGCs in a germ granule‐independent manner. We propose that C. elegans PGCs have a novel mechanism to accumulate at least some chromatin‐associated proteins that are essential for germline immortality.     

Proteomic analysis of murine Piwi proteins reveals a role for arginine methylation in specifying interaction with Tudor family members

In germ cells, Piwi proteins interact with a specific class of small noncoding RNAs, piwi-interacting RNAs (piRNAs). Together, these form a pathway that represses transposable elements, thus safeguarding germ cell genomes. Basic models describe the overall operation of piRNA pathways. However, the protein compositions of Piwi complexes, the critical protein–protein interactions that drive small RNA production and target recognition, and the precise molecular consequences of conserved localization to germline structures, call nuage, remains poorly understood. We purified the three murine Piwi family proteins, MILI, MIWI, and MIWI2, from mouse germ cells and characterized their interacting protein partners. Piwi proteins were found in complex with PRMT5/WDR77, an enzyme that dimethylates arginine residues. By immunoprecipitation with specific antibodies and by mass spectrometry, we found that Piwi proteins are arginine methylated at conserved positions in their N termini. These modifications are essential to direct complex formation with specific members of the Tudor protein family. Recognition of methylarginine marks by Tudor proteins can drive the localization of Piwi proteins to cytoplasmic foci in an artificial setting, supporting a role for this interaction in Piwi localization to nuage, a characteristic that correlates with proper operation of the piRNA pathway and transposon silencing in multiple organisms.     

Developmental expression of FOG-1/CPEB protein and its control in the Caenorhabditis elegans hermaphrodite germ line.

The specification of a germ cell as sperm or oocyte and determination of cell number remain unsolved questions in developmental biology. This paper examines Caenorhabditis elegans FOG-1, a CPEB-related RNA-binding protein that controls the sperm fate. We find that abundant FOG-1 protein is observed transiently in germ cells just prior to their expression of an early sperm-differentiation marker. As the germline tissue elongates, abundant FOG-1 appears more and more distally as sperm become specified, but disappears when the germ line switches to oogenesis. This dynamic pattern is controlled by both globally acting and germline-specific sex-determining regulators. Importantly, the extent of FOG-1 expression corresponds roughly to sperm number in wild-type and mutants, altering sperm number. By contrast, three other key regulators of the sperm/oocyte decision do not similarly correspond to sperm number. We suggest that FOG-1 is precisely modulated in both time and space to specify sperm fate and control sperm number.     

A zebrafish nanos-related gene is essential for the development of primordial germ cells

Asymmetrically distributed cytoplasmic determinants collectively termed germ plasm have been shown to play an essential role in the development of primordial germ cells (PGCs). Here, we report the identification of a nanos-like (nanos1) gene, which is expressed in the germ plasm and in the PGCs of the zebrafish. We find that several mechanisms act in concert to restrict the activity of Nanos1 to the germ cells including RNA localization and control over the stability and translatability of the RNA. Reducing the level of Nanos1 in zebrafish embryos revealed an essential role for the protein in ensuring proper migration and survival of PGCs in this vertebrate model organism.     

Ultrastructure of fetal human gonad before sexual differentiation and during early testicular and ovarian development

The development of the human gonad was investigated by light and transmission electron microscopy in 20 embryos and fetuses between 4.5 and 11.5 weeks of gestation, i.e. during the stages of sex-indifferent gonad, initial testicular and ovarian development. The gonadal blastema in 4.5-week-old embryos appeared formed by poorly differentiated somatic mesothelial cells, and by specialized germ cells (PGCs) with signs of ameboidism, cellular structures suggesting active protein biosynthesis and mitotic activity. The sexual differentiation of the gonads was clearly observed in 7-week-old embryos and involved at the same time the testis and the ovary. The former contained seminiferous cords formed by palisades of poorly differentiated Sertoli cells, which were segregated from the mesothelium by a rudimentary albuginea. The interstitial tissue at this age contained mesenchymal cells. Between 8 and 11.5 weeks of gestation, there was a synchronous cytodifferentiation of both Sertoli and Leydig cells. The latter acquired features of steroidogenic elements. The ovaries of 7-week-old fetuses contained packed ovigerous cords formed by somatic and germ cells (oogonia). The former embraced the oogonia with thin overlapping cytoplasm projections, and acquired features similar to those of cells in primary follicles, already at this early fetal age. At the same time the sexual differentiation of the gonads involved somatic and germ cells. In the female, the oogonia continued to show the main features they had during migration and colonization, including a high mitotic rate, signs of ameboidism and a developed apparatus for protein synthesis. On the contrary Gonocytes enclosed in the seminiferous cords progressively entered a quiescent phase characterized by a reduced mitotic rate, a decrease of endoplasmic reticulum and nucleolar complexity. The chronological relationship between the cytodifferentiation of Sertoli and Leydig cells, and changes of germ cells, suggest that somatic components of the testis may contribute to a male type of differentiation of germ cells from the very beginning of sexual differentiation.     

NANOS3 function in human germ cell development

Human infertility is common and frequently linked to poor germ cell development. Yet, human germ cell development is poorly understood, at least in part due to the inaccessibility of germ cells to study especially during fetal development. Here, we explored the function of a highly conserved family of genes, the NANOS genes, in the differentiation of human germ cells from human embryonic stem cells. We observed that NANOS-1, -2 and -3 mRNAs and proteins were expressed in human gonads. We also noted that NANOS3 was expressed in germ cells throughout spermatogenesis and oogenesis and thus, focused further efforts on this family member. NANOS3 expression was highest in human germ cell nuclei where the protein co-localized with chromosomal DNA during mitosis/meiosis. Reduced expression of NANOS3 (via morpholinos or short hairpin RNA) resulted in a reduction in germ cell numbers and decreased expression of germ cell-intrinsic genes required for the maintenance of pluripotency and meiotic initiation and progression. These data provide the first direct experimental evidence that NANOS3 functions in human germ cell development; indeed, NANOS3 is now one of just two genes that has been directly shown to function in germ cell development across diverse species from flies, worms, frogs and mice to humans [the other is BOULE, a member of the Deleted in Azoospermia (DAZ) gene family]. Findings may contribute to our understanding of the basic biology of human germ cell development and may provide clinical insights regarding infertility.     

Subcellular distribution of importins correlates with germ cell maturation.

Importin proteins regulate access to the nucleus by recognizing and transporting distinct cargo proteins. Building on studies in Drosophila and Caenorhabditis elegans, we hypothesized that regulated expression and subcellular localization of specific importins may be linked to mammalian gonadal differentiation. We identified distinct developmental and cellular localization patterns for importins beta1, alpha3, alpha4 and RanBP5 (importin beta3) in fetal and postnatal murine testes using Western blotting and immunohistochemistry. Importin beta1 protein is detected in selected germ and somatic cells in fetal gonads, with a striking perinuclear staining evident from embryonic day (E) 14.5 within testicular gonocytes. RanBP5 exhibits age- and gender-specific subcellular localization within fetal gonads. At E12.5, RanBP5 protein is cytoplasmic in gonocytes but predominantly nuclear in oogonia, but by E14.5 RanBP5 appears nuclear in gonocytes and cytoplasmic in oogonia. In postnatal testes, importin alpha3 and alpha4 in spermatocytes, spermatids, and Sertoli cells display cytoplasmic and nuclear localization, respectively.     

Identification and characterisation of mRif1: a mouse telomere-associated protein highly expressed in germ cells and embryo-derived pluripotent stem cells.

We have identified a mouse ortholog of the yeast Rif1 family of telomere-associated proteins on the basis of its high expression in primordial germ cells and embryo-derived pluripotent stem cell lines. mRif1 is also highly expressed in totipotent and pluripotent cells during early mouse development, and in male and female germ cells in adult mice. mRif1 expression is induced during derivation of embryonic stem cells and is rapidly down-regulated upon differentiation of embryonic stem cells in vitro. Furthermore, we show that mRif1 physically interacts with the telomere-associated protein mTrf2 and can be cross-linked to telomeric repeat DNA in mouse embryonic stem cells. mRif1 may be involved in the maintenance of telomere length or pluripotency in the germline and during early mouse development.     

Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes.

The recent discovery of the DMY gene (DM domain gene on Y chromosome and one of the DMRT1 family genes) as a key determinant of male development in the medaka (Oryzias latipes) has led to its designation as the prime candidate gene for sex-determination in this species. This study focused on the sites and pattern of expression of DMY and DMRT1 genes during gonadal differentiation of medaka to further determine their roles in testis development. DMY mRNA and protein are expressed specifically in the somatic cells surrounding primordial germ cells (PGCs) in the early gonadal primordium, before morphological sex differences are seen. However, somatic cells surrounding PGCs never express DMY during the early migratory period. Expression of DMY persists in Sertoli cell lineage cells, from PGC-supporting cells to Sertoli cells, indicating that only DMY-positive cells enclose PGCs during mitotic arrest after hatching. DMRT1 is expressed in spermatogonium-supporting cells after testicular differentiation (20-30 days after hatching), and its expression is much higher than that of DMY in mature testes. In XX sex-reversed testes, DMRT1 is expressed in the Sertoli cell lineage, similar to the expression of DMY in XY testes. These results suggest strongly that DMY regulates PGC proliferation and differentiation sex-specifically during early gonadal differentiation of XY individuals and that DMRT1 regulates spermatogonial differentiation.