Dicer1 depletion in male germ cells leads to infertility due to cumulative meiotic and spermiogenic defects.

Background: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice.Principal Findings: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.Conclusions/Significance: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis.

Early Development and Putative Primordial Germ Cells Characterization in Dogs

Contents Previously, three distinct populations of putative primordial germ cells (PGCs), namely gonocytes, intermediate cells and pre-spermatogonia, have been described in the human foetal testis. According to our knowledge, these PGCs have not been studied in any other species. The aim of our study was to identify similar PGC populations in canine embryos. First, we develop a protocol for canine embryo isolation. Following our protocol, 15 canine embryos at 21-25 days of pregnancy were isolated by ovaryhysterectomy surgery. Our data indicate that dramatic changes occur in canine embryo development and PGCs specification between 21 to 25 days of gestation. At that moment, only two PGC populations with distinct morphology can be identified by histological analyses. Cell population 1 presented round nuclei with prominent nucleolus and a high nuclear to cytoplasm ratio, showing gonocyte morphology. Cell population 2 was often localized at the periphery of the testicular cords and presented typical features of PGC. Both germ cell populations were positively immunostained with anti-human OCT-4 antibody. However, at day 25, all cells of population 1 reacted positively with OCT-4, whereas in population 2, fewer cells were positive for this marker. These two PGCs populations present morphological features similar to gonocytes and intermediate cells from human foetal testis. It is expected that a population of pre-spermatogonia would be observed at later stages of canine foetus development. We also showed that anti-human OCT-4 antibody can be useful to identify canine PGC in vivo.

Testosterone-immunopositive primordial germ cells in the testis of the bullfrog, Rana catesbeiana

In amphibia, steroidogenesis remains quiescent in distinct seasonal periods, but the mechanism by which spermatogenesis is maintained under low steroidogenic conditions is not clear. In the present study, testosterone location in the testes of Rana catesbeiana was investigated immunohistochemically during breeding (summer) and nonbreeding (winter) periods. In winter, the scarce interstitial tissue exhibited occasional testosterone immunopositivity in the interstitial cells but the cytoplasm of primordial germ cells (PG cells) was clearly immunopositive. By contrast, in summer, PG cells contained little or no immunoreactivity whereas strong immunolabelling was present in the well-developed interstitial tissue. These results suggest that PG cells could retain testosterone during winter. This androgen reservoir could be involved in the control of early spermatogenesis in winter and/or to guarantee spermiogenesis and spermiation in the next spring/summer. The weak or negative immunoreaction in the summer PG cells might reflect consumption of androgen reservoir by the intense spermatogenic activity from spring to summer. Thus, besides acting as stem cells, PG cells of R. catesbeiana could exert an androgen regulatory role during seasonal spermatogenesis.

Cytotoxic effects of permethrin in oocytes of Rhipicephalus sanguineus (Acari: Ixodidae) fully engorged females: I. Direct or indirect action of the acaricide in germ cells?

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ultrastructural aspects of the intercellular bridges between female bee germ cells

No ovário das abelhas as células germinativas e as células foliculares são interconectadas por pontes intercelulares mantidas abertas por reforços do citoesqueleto na membrana plasmática. As pontes entre as células germinativas têm comportamento dinâmico e provavelmente atuam na determinação do ovócito entre as células do clone formado pelas mitoses pré meióticas formando posteriormente uma via de transporte para que os produtos sintetizados pelas células nutridoras atinjam o ovócito durante sua maturação. Os elementos do citoesqueleto presentes nas pontes intercelulares das gônadas das abelhas são basicamente microfilamentos e microtúbulos, mas nas pontes entre os cistócitos pré-meióticos outro tipo de filamento (espesso de natureza não definida, associado a elementos do retículo endoplasmático) está presente, atravessando a ponte e prendendo-se através dos microfilamentos à membrana plasmática. Estes filamentos aparentemente controlam o vão da ponte. Terminada a fase de proliferação os cistócitos tomam a forma de uma roseta e um fusoma, formado pela convergência das pontes, aparece no centro desta. Nesta conformação os filamentos grossos não estão presentes. Nova mudança ocorre com a diferenciação do ovócito e das células nutridoras, com a reorientação de todas as pontes de maneira a canalizar o conteúdo das futuras células nutridoras para o ovócito.

Radioprotection of beta-carotene evaluated on mouse somatic and germ cells

In the present paper, the protective effect of beta-carotene was evaluated after whole body exposure of mice to 2 Gy of X-rays. Splenocytes, reticulocytes, bone marrow cells and spermatids were evaluated for the frequency of micronuclei (MN) induced by X-rays. Mice were treated (gavage) with beta-carotene (10, 25 and 50 mg/kg b.w.) for 5 consecutive days and, 4 h after the last treatment, the animals were irradiated. The results obtained showed different frequencies of X-ray-induced-MN between different cell populations analysed and also different response of these cells to the beta-carotene treatment. The radioprotective effect of beta-carotene was observed in splenocytes, reticulocytes, and spermatids but not in bone marrow cells. No dose-response relationship for beta-carotene was detected. The time of sampling, the sensitivity of the cells as well as the antioxidant activity of beta-carotene are discussed as important factors for the radioprotective action of this provitamin.

Early Development and Putative Primordial Germ Cells Characterization in Dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A New and Fast Technique to Generate Offspring after Germ Cells Transplantation in Adult Fish: The Nile Tilapia (Oreochromis niloticus) Model

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Enhanced ERbeta immunoexpression and apoptosis in the germ cells of cimetidine-treated rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytoplasmic bridges, intercellular junctions, and individualization of germ cells during spermatogenesis in Dermatobia hominis (Diptera: Cuterebridae)

During mitotic and meiotic divisions in Dermatobia hominis spermatogenesis, the germ cells stay interlinked by cytoplasm, bridges as a result of incomplete cytokinesis. By the end of each division, cytoplasmic bridges flow to the center of the cyst, forming a complex, called the fusoma. During meiotic prophase I, spermatocytes I present desmosome-like junctions and meiotic cytoplasmic bridges. At the beginning of spermiogenesis, the fusoma moves to the future caudal end of the cyst, and at this time the early spermatids are linked by desmosome-like junctions. Throughout spermiogensis, new and sometimes broad cytoplasmic bridges are formed among spermatids at times making them share cytoplasm. In this case the individualization of cells is assured by the presence of smooth cisternae that outline then structures The more differentiated spermatids have in addition to narrow cytoplasmic bridges, plasmic membranes junctions. By the end of spermiogenesis the excess cytoplasmic mass is eliminated leading to spermatid individualization. Desmosome-like junctions of spermatocytes I and early spermatids appear during the fusoma readjustment and segregations; on the other hand, plasmic membrane junctions appear in differentiating spermatids and are eliminated along with the cytoplasmic excess. These circumstances suggest that belt desmosome-like and plasmic membrane junctions are involved in the maintenance of the relative positions of male germ cells in D. hominis while they are inside the cysts. © 1996 Wiley-Liss, Inc.

Automatic classification of fish germ cells through optimum-path forest

The spermatogenesis is crucial to the species reproduction, and its monitoring may shed light over some important information of such process. Thus, the germ cells quantification can provide useful tools to improve the reproduction cycle. In this paper, we present the first work that address this problem in fishes with machine learning techniques. We show here how to obtain high recognition accuracies in order to identify fish germ cells with several state-of-the-art supervised pattern recognition techniques. © 2011 IEEE.

Primordial germ cells (spermatogonial stem cells) of bullfrogs express sex hormone-binding globulin and steroid receptors during seasonal spermatogenesis

In vertebrate species, testosterone seems to inhibit spermatogonial differentiation and proliferation. However, this androgen can also be converted, via aromatase, into estrogen which stimulates spermatogonial differentiation and mitotic activity. During seasonal spermatogenesis of adult bullfrogs Lithobates catesbeianus, primordial germ cells (PGCs) show enhanced testosterone cytoplasm immunoexpression in winter; however, in summer, weak or no testosterone immunolabelling was observed. The aim of this study was to confirm if PGCs express stem cell markers-alkaline phosphatase (AP) activity and GFRα1 (glial-cell-line-derived neurotrophic factor)-and verify whether testosterone is maintained in these cells by androgen receptors (ARs) and/or sex hormone-binding globulin (SHBG) in winter. Furthermore, regarding the possibility that testosterone is converted into estrogen by PGCs in summer, the immunoexpression of estrogen receptor (ER)β was investigated. Bullfrog testes were collected in winter and in summer and were embedded in glycol methacrylate for morphological analyses or in paraffin for the histochemical detection of AP activity. GFRα1, AR, SHBG and ERβ expression were detected by Western blot and immunohistochemical analyses. The expression of AP activity and GFRα1 in the PGCs suggest that these cells are spermatogonial stem cells. In winter, the cytoplasmic immunoexpression of ARs and SHBG in the PGCs indicates that testosterone is maintained by these proteins in these cells. The cytoplasmic immunoexpression of ERβ, in summer, also points to an ER-mediated action of estrogen in PGCs. The results indicate a participation of testosterone and estrogen in the control of the primordial spermatogonia during the seasonal spermatogenesis of L. catesbeianus. © 2012 S. Karger AG, Basel.