In mammals, DNA methylation (DNAme) erasure and reinstatement during embryo development and germline establishment are sensitive to the intrauterine environment. Maternal intake of a high-fat diet (HFD), associated with excessive gestational weight gain, has transgenerational effects on offspring health, which may be mediated by changes in DNAme in the germline. Here, we tested the impact of a maternal HFD on embryonic germline DNAme erasure using a rat strain that expresses green fluorescent protein specifically in germ cells. DNAme was analysed by methyl-seq capture in germ cells collected from male and female F1 gonads at gestational day 16. Our data show that although HFD induced global hypomethylation in both sexes, DNAme erasure in female germ cells was more advanced compared to male germ cells. The delay in DNAme erasure in males and the greater impact of HFD suggest that male germ cells are more vulnerable to alterations by exogenous factors.

Spermatogenesis is a highly complex process through which mature sperms are produced, and it requires three important stages; mitosis, meiosis and sperm formation. The expression of genes regulated by transcription factors at specific stages exerts important regulatory effects on the development process of germ cells. Male mice with overexpressed programmed death ligand 1 (PD-L1) (B7 homolog1) in the testis have infertility and abnormal sperm development, thereby exhibiting severe malformation and sloughing throughout spermatid maturation and collapsed and disorganized seminiferous epithelium structure. Furthermore, PD-L1 overexpression causes overexpression of cysteine-rich secretory protein 1 (CRISP1) in the epididymis and adversely affects or precludes sperm energization, sperm-pellucida binding and sperm-oocyte fusion. These findings suggest that CRISP1 and PD-L1 can interact with each other to induce male infertility and germ-cell dissociation.

Spermatogonial stem cells (SSCs) are the basis of male spermatogenesis and fertility. SSCs are distinguished by their ability to self-renew and differentiate into spermatozoa throughout the male reproductive life and pass genetic information to the next generation. Immunohistochemistry (IHC), immunocytochemistry (ICC) and Fluidigm reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyze the expression of PLZF and VASA in mice testis tissue. In this experimental study, whereas undifferentiated spermatogonial cells sharply expressed PLZF, other types of germ cells located in the seminiferous tubule were negative for this marker. Conversely, the germ cells near the basal membrane of the seminiferous tubule showed VASA expression, whereas the undifferentiated germ cells located on the basal membrane were negative. The ICC analysis indicated higher expression of PLZF in the isolated undifferentiated cells compared with differentiated germ cells. Fluidigm real-time RT-PCR results demonstrated a significant expression (P < 0.05) of VASA in the SSCs compared with differentiated cells and also showed expression of PLZF in undifferentiated spermatogonia. These results clearly proved the role of PLZF as a specific marker for SSCs, and can be beneficial for advanced research on in vitro differentiation of SSCs to functional sperms.

Spermatogenesis is a complex and elaborate differentiation process and is vital for male fertility. Sertoli cells play a major role in fertility and induce spermatogenesis by protecting, nourishing, and supporting germ cells. It has been speculated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could directly affect the male reproductive system, and therefore heredity and fertility. The similarity of SARS-CoV-2 to SARS-CoV could confirm this hypothesis because both viruses use angiotensin-converting enzyme (ACE2) as the receptor to enter human cells. ACE2 is expressed by Sertoli cells and other testicular cells, therefore COVID-19 has the potential to impair fertility by destroying Sertoli cells. This hypothesis should be evaluated and confirmed by monitoring fertility in patients with COVID-19.

Avian testes have been used in the study of germ cell transfer, importantly for understanding the preservation and control of birds. For this purpose, we use light microscopy, electron microscopy and immunohistochemistry to understand the reproductive efficiency of dove testes. The tunica albuginea was thin and septula testes were not observed. The testicular parenchyma was formed mainly of closely packed convoluted seminiferous tubules with little interstitial area. Three types of spermatogonia were distinguished. The primary spermatocyte appeared as the largest spermatogenic cell and was identified at different stages of meiosis. Different morphological stages of the spermatid were categorized. Various cellular associations were described within the seminiferous epithelium. The cytoplasm of Sertoli cells was pale and ill defined due to its close relationship to the germinal epithelium. The spermatid attached to the luminal border of Sertoli cells and germ cells were closely associated. A single layer of myoid cells surrounded the seminiferous tubule. Testicular telocytes of doves were located in the peritubular region and near the blood vessels. Telopods appeared as long cytoplasmic processes arising from the cell body. Leydig cells were distributed singly or in small groups and cords. The intensity of androgen receptor (AR) immunostaining in the testes of the dove was established for the first time and is described in this paper.

The decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.

This study aimed to understand how germ cell development occurs in females of Devario aequipinnatus, by morphologically describing oogenesis and the reproductive phases. Sexually mature females of D. aequipinnatus (n = 70) were obtained from commercial fisheries and delivered to the Laboratório de Ictiologia Neotropical, UNESP, Ilha Solteira, SP, Brazil. The ovaries were removed, fragmented and fixed following the usual techniques for light microscopy. The stages of ovarian development in D. aequipinnatus begin with the oogonia, which proliferate into new cells or differentiate into prophasic oocytes that, at the end of this process, form the ovarian follicle and end folliculogenesis. In the previtellogenic stage, the oocytes were characterized mainly by the gradual loss of basophilia and an increase in oocyte diameter. Vitellogenesis was marked mainly by the incorporation of yolk granules. Mature oocytes were defined by their migration from the nucleus to the micropyle. Postovulatory follicles and atresic oocytes were also observed. The reproductive phases were classified as: immature, early and final developing, spawning capable, regressing and regenerating. Therefore, the development of an understanding of cell modifications that occurs up to oogenesis is a basic step that is essential for the description of the reproductive biology of D. aequipinnatus, given the lack of information about the reproductive aspects of this species.

This study aimed to examine the gonadal morphology of diploid and triploid fish through stereological analysis. Triploid individuals were obtained after temperature shock (40°C for 2 min) at 2 min post-fertilization and reared until 175 days post-fertilization (dpf). Intact eggs were used to obtain the diploids. Gonads were collected for histological analysis at 83, 114, 144 and 175 dpf. Diploid females and males presented normal oogenesis and spermatogenesis through all the experimental period. Conversely, stereological analysis revealed that triploid females were sterile and oogonia were the prevalent cell type in the ovaries. Triploid males presented increased amounts of spermatocyte cysts and a large area of lumen when compared with diploids and in addition the amount of spermatozoa was lower than that observed for diploids. However, some triploid males presented spermatogenesis similar to diploids. Therefore, we concluded that triploidization is an interesting alternative to produce sterile individuals in A. altiparanae.

In a previous study, we have identified a set of conserved spermatogenic genes whose expression is restricted to testis and ovary and that are developmentally regulated. One of these genes, the transcription factor Mael, has been reported to play an essential role in mouse spermatogenesis. Nevertheless, the role of Mael in mouse oogenesis has not been defined. In order to analyse the role of Mael in mouse oogenesis, the expression of this gene was blocked during early oogenesis in mouse in vitro using RNAi technology. In addition, the role of Mael during differentiation of embryonic stem cells (ESC) into germ cells in vitro was analysed. Results show that downregulation of Mael by a specific short interfering RNA disrupted fetal oocyte growth and differentiation in fetal ovary explants in culture and the expression of several germ-cell markers in ESC during their differentiation. These results suggest that there is an important role for Mael in early oogenesis and during germ-cell differentiation from embryonic stem cells in mouse in vitro.