Function analysis and molecular characterization of cyclin A in ovary development of oriental river prawn, Macrobrachium nipponense.

Macrobrachium nipponense has the characteristics of fast ovarian development cycle, which leads to the coexistence of multiple generations, the reduction of commodity specifications and the low economic benefit. Therefore, the study on the mechanism of ovarian development is of great significance to the development of industry. Cyclin A (CycA)is a key gene regulating ovarian development in vertebrates, but little information was available for its function in crustaceans. In this study, the full-length cDNA of Mn-CycA was obtained from the ovary. The full-length cDNA (2033 bp) with an open reading frame of 1368 bp, encoded a 456-amino acid protein. qRT-PCR revealed tissue-specific expression pattern of Mn-CycA, with abundant expression in the ovary. Results in different developmental stages of ovary indicated that Mn-CycA expression is positively correlated with ovarian maturation. qRT-PCR In different developmental stages, the expression of Mn-CycA mRNA gradually increased during the embryonic stage and decreased significantly on the first day of the hatching stage. At the 25th day of the metamorphosis stage, the expression level of Mn-CycAmRNA in female shrimp was 3.5 times higher than that in male shrimp, which may be related to the proliferation of oogonia and the formation of oocytes. In situ hybridization (ISH) of ovary showed Mn-CycA was examined in all stages and was mainly located in oogonia and oocytes. Compared with the control group, the obvious change of gonad somatic index (GSI) proved that injection of Mn-CycA dsRNA could delay the ovarian development cycle, which provided strong evidence for the involvement of Mn-CycA in ovarian maturation and oogenesis, and expanded a new perspective for studying the fast ovarian development cycle in M. nipponense.

The DNA damage response is required for oocyte cyst breakdown and follicle formation in mice.

Mammalian oogonia proliferate without completing cytokinesis, forming cysts. Within these, oocytes differentiate and initiate meiosis, promoting double-strand break (DSBs) formation, which are repaired by homologous recombination (HR) causing the pairing and synapsis of the homologs. Errors in these processes activate checkpoint mechanisms, leading to apoptosis. At the end of prophase I, in contrast with what is observed in spermatocytes, oocytes accumulate unrepaired DSBs. Simultaneously to the cyst breakdown, there is a massive oocyte death, which has been proposed to be necessary to enable the individualization of the oocytes to form follicles. Based upon all the above-mentioned information, we hypothesize that the apparently inefficient HR occurring in the oocytes may be a requirement to first eliminate most of the oocytes and enable cyst breakdown and follicle formation. To test this idea, we compared perinatal ovaries from control and mutant mice for the effector kinase of the DNA Damage Response (DDR), CHK2. We found that CHK2 is required to eliminate ~50% of the fetal oocyte population. Nevertheless, the number of oocytes and follicles found in Chk2-mutant ovaries three days after birth was equivalent to that of the controls. These data revealed the existence of another mechanism capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2-/- mice, implying that CHK1 regulates postnatal oocyte death. Moreover, we found that CHK1 and CHK2 functions are required for the timely breakdown of the cyst and to form follicles. Thus, we uncovered a novel CHK1 function in regulating the oocyte population in mice. Based upon these data, we propose that the CHK1- and CHK2-dependent DDR controls the number of oocytes and is required to properly break down oocyte cysts and form follicles in mammals.

Environmentally-relevant exposure to diethylhexyl phthalate (DEHP) alters regulation of double-strand break formation and crossover designation leading to germline dysfunction in Caenorhabditis elegans.

Exposure to diethylhexyl phthalate (DEHP), the most abundant plasticizer used in the production of polyvinyl-containing plastics, has been associated to adverse reproductive health outcomes in both males and females. While the effects of DEHP on reproductive health have been widely investigated, the molecular mechanisms by which exposure to environmentally-relevant levels of DEHP and its metabolites impact the female germline in the context of a multicellular organism have remained elusive. Using the Caenorhabditis elegans germline as a model for studying reprotoxicity, we show that exposure to environmentally-relevant levels of DEHP and its metabolites results in increased meiotic double-strand breaks (DSBs), altered DSB repair progression, activation of p53/CEP-1-dependent germ cell apoptosis, defects in chromosome remodeling at late prophase I, aberrant chromosome morphology in diakinesis oocytes, increased chromosome non-disjunction and defects during early embryogenesis. Exposure to DEHP results in a subset of nuclei held in a DSB permissive state in mid to late pachytene that exhibit defects in crossover (CO) designation/formation. In addition, these nuclei show reduced Polo-like kinase-1/2 (PLK-1/2)-dependent phosphorylation of SYP-4, a synaptonemal complex (SC) protein. Moreover, DEHP exposure leads to germline-specific change in the expression of prmt-5, which encodes for an arginine methyltransferase, and both increased SC length and altered CO designation levels on the X chromosome. Taken together, our data suggest a model by which impairment of a PLK-1/2-dependent negative feedback loop set in place to shut down meiotic DSBs, together with alterations in chromosome structure, contribute to the formation of an excess number of DSBs and altered CO designation levels, leading to genomic instability.

Specification and spatial arrangement of cells in the germline stem cell niche of the Drosophila ovary depend on the Maf transcription factor Traffic jam.

Germline stem cells in the Drosophila ovary are maintained by a somatic niche. The niche is structurally and functionally complex and contains four cell types, the escort, cap, and terminal filament cells and the newly identified transition cell. We find that the large Maf transcription factor Traffic jam (Tj) is essential for determining niche cell fates and architecture, enabling each niche in the ovary to support a normal complement of 2-3 germline stem cells. In particular, we focused on the question of how cap cells form. Cap cells express Tj and are considered the key component of a mature germline stem cell niche. We conclude that Tj controls the specification of cap cells, as the complete loss of Tj function caused the development of additional terminal filament cells at the expense of cap cells, and terminal filament cells developed cap cell characteristics when induced to express Tj. Further, we propose that Tj controls the morphogenetic behavior of cap cells as they adopted the shape and spatial organization of terminal filament cells but otherwise appeared to retain their fate when Tj expression was only partially reduced. Our data indicate that Tj contributes to the establishment of germline stem cells by promoting the cap cell fate, and controls the stem cell-carrying capacity of the niche by regulating niche architecture. Analysis of the interactions between Tj and the Notch (N) pathway indicates that Tj and N have distinct functions in the cap cell specification program. We propose that formation of cap cells depends on the combined activities of Tj and the N pathway, with Tj promoting the cap cell fate by blocking the terminal filament cell fate, and N supporting cap cells by preventing the escort cell fate and/or controlling the number of cap cell precursors.

Developmental effects of imatinib mesylate on follicle assembly and early activation of primordial follicle pool in postnatal rat ovary.

Imatinib mesylate is an anti-cancer agent that competitively inhibits several receptor tyrosine kinases (RTKs). RTKs play important roles in the regulation of primordial follicle formation, the recruitment of primordial follicles into the pool of growing follicles and maturation of the follicles. In the present study, we investigated the effects of the tyrosine kinase inhibitor imatinib on primordial follicle assembly and early folliculogenesis in postnatal rats. Female Sprague-Dawley rats were treated with either imatinib (150mg/kg) or placebo (water) on postnatal days 2-4. Bilateral ovariectomy was performed on postnatal day 2 and 5. Histology, immunohistochemistry, and mRNA analysis were performed. Imatinib treatment was associated with increased density of the multi-oocyte follicles (P<0.01), oogonia (p<0.01) and germline clusters (P<0.05), decreased activation of primordial follicles, increased expression of c-Kit and AMH, and decreased protein expression of Kit-ligand and GDF9 when compared to age-matched controls. In conclusion, imatinib affects folliculogenesis in postnatal rat ovaries by delaying the cluster breakdown, follicular assembly and early activation of the primordial follicle pool.

c-Fos Repression by Piwi Regulates Drosophila Ovarian Germline Formation and Tissue Morphogenesis.

Drosophila melanogaster Piwi functions within the germline stem cells (GSCs) and the somatic niche to regulate GSC self-renewal and differentiation. How Piwi influences GSCs is largely unknown. We uncovered a genetic interaction between Piwi and c-Fos in the somatic niche that influences GSCs. c-Fos is a proto-oncogene that influences many cell and developmental processes. In wild-type ovarian cells, c-Fos is post-transcriptionally repressed by Piwi, which destabilized the c-Fos mRNA by promoting the processing of its 3' untranslated region (UTR) into Piwi-interacting RNAs (piRNAs). The c-Fos 3' UTR was sufficient to trigger Piwi-dependent destabilization of a GFP reporter. Piwi represses c-Fos in the somatic niche to regulate GSC maintenance and differentiation and in the somatic follicle cells to affect somatic cell disorganization, tissue dysmorphogenesis, oocyte maturation arrest, and infertility.

Scp3 expression in relation to the ovarian differentiation in the protogynous hermaphroditic ricefield eel Monopterus albus.

Synaptonemal complex protein 3 (Scp3), which is encoded by scp3, is a meiotic marker commonly used to trace the timing of gonadal differentiation in vertebrates. In the present study, the ricefield eel scp3 cDNA was cloned, and a fragment encoding amino acids 49 to 244 was overexpressed. The recombinant Scp3 polypeptide was purified and used to generate a rabbit anti-Scp3 polyclonal antiserum. In adult ricefield eels, scp3 mRNA was predominantly detected in the gonads and faintly detected in discrete brain areas. In the gonads, Scp3 immunoreactivities were shown to be localized to the germ cells, including meiotic primary growth oocytes, spermatocytes, and pre-meiotic spermatogonia. During early ovarian differentiation, immunoreactive Scp3 was not detected in the gonads of ricefield eels at 6 days post-hatching (dph) but was found to be abundantly localized in the cytoplasm of some oogonia at 7 dph, coinciding with the appearance of the ovarian cavity and ovarian differentiation. At 14 dph, strong Scp3 immunostaining was detected on one side of the nucleus with a distinct polarity in some germ cells, presumably at the leptotene stage. Consistent with these results, the expression of scp3 mRNA was faintly detected in the gonads of ricefield eels at 6 dph, increased at 8 dph, and then remained relatively high thereafter. Taken together, these results suggest that the appearance of immunoreactive Scp3 in oogonia could be a marker for early ovarian differentiation in ricefield eels. The translocation of the Scp3 protein from the cytoplasm to the nucleus in the oogonium of ricefield eels appears to be a controlled process that warrants further study.

Female germ cell mutagenicity of model chemicals in Drosophila melanogaster: mechanistic information and analysis of repair systems.

In spite of differences between female and male germ cells, and although both of them contribute to the gene pool of future generations, most germ cell mutagenicity studies in higher eukaryotes have been carried out on males. To study the response of female germ cells to mutagen/carcinogen exposure, the mutagenicity of two model chemicals like diethyl sulfate (DES) and hexamethylphosphoramide (HMPA), and the monofunctional methylating chemotherapeutic drug streptozotocin (STZ), has been analysed on repair efficient females of Drosophila melanogaster. Results previously obtained with N-ethyl-N-nitrosourea (ENU), another model chemical, have also been included in the analysis. The activity of bypass tolerance mechanism (BTM; represented by the mus308 locus) and nucleotide excision repair (NER) on the removal of oxygen and nitrogen ethylations was studied by determining DES mutagenicity in NER deficient females, comparing it with existing results for ENU, and by analysing both chemicals on BTM deficient females. Results indicate that (1) all chemicals are mutagenic on repair efficient females; (2) a measure of mutagenic activity ranked from the lowest DES to STZ, HMPA, and ENU as the highest. This order correlates with the repair of the respectively induced DNA damages, and with the mutagenic and carcinogenic potency of these compounds, considering the toxicity of cross-linking agents; (3) NER efficiently repairs nitrogen ethylation damage and seems to contribute to the processing of oxygen damage in female germ cells; and (4) BTM is involved on the processing of oxygen ethylation damage, whereas the results on nitrogen ethylation are not clear. Finally, these results indicate that differences between male and female germ cells affect the response to chemical exposure, and therefore demonstrate the necessity of analysing also female cells in germinal mutagenicity studies. In addition, these studies can provide important mechanistic information about germ cell chemical mutagenesis, and even when the analysis of oogonia is not possible, since all female germ cells are pre-meiotic, studies of oocytes could be a model for pre-meiotic cells.

Developmentally regulated expression of a mouse germ cell nuclear antigen examined from embryonic day 11 to adult in male and female mice.

A rat IgM monoclonal antibody has been developed which recognized a mouse germ cell nuclear antigen (GCNA1). GCNA1 is present in prospermatogonia (gonocytes) in males and in oogonia and oocytes of females within the gonadal ridge from Embryonic Day 11.5 onward, but rarely in primordial germ cells prior to their arrival at the gonadal ridge. Immunolocalization demonstrates that GCNA1 is abundant in nuclei of spermatogonia and early spermatocytes, but decreases during subsequent spermatocyte and round spermatid development, and is not detected beyond step 10 elongating spermatids. The antigen is approximately 80-110 kDa on immunoblots of isolated pachytene spermatocytes and round spermatids. However, GCNA1 appears to be absent from sperm in the epididymis and vas deferens, Sertoli cells, TM3 cells (Leydig-like) and TM4 cells (Sertoli-like), lung, liver, kidney, spleen, heart, skin, brain, epididymis, and ovary. GCNA1 is present in prepuberal male mice (Days 2-14) in all stages of prespermatogonial and spermatogonial development. It is also present in prepuberal male mice (Days 2-14) in all stages of prespermatogonial and spermatogonial development. It is also present in oocytes of neonatal females until Postpartum Day 12. GCNA1 is first lost from oocytes in the medulla of the ovary as they arrest at the dictyate stage and gain a layer of granulosa cells. In addition, antigen is present in moderate amounts in F9 embryonal carcinoma cells and SCC-PSA1 pluripotent terato-carcinoma cells. Thus, GCNA1 serves as a common marker of the germ cell lineage in male and female mice after primordial germ cells arrive in the gonadal ridge until they reach the diplotene/dictyate stage of the first meiotic division.

Extrachromosomal DNA and its activity in RNA synthesis in oogonia and oocytes in the pupal ovary of Creophilus maxillosus (Staphylinidae, Coleoptera-Polyphaga).

The participation of extrachromosomal DNA (extra DNA) in RNA synthesis in the nuclei of terminal oogonial cells and oocytes in the pupal ovary of Creophilus maxillosus (Staphylinidae, Coleoptera) was examined by autoradiography. It was found that extra DNA in the nuclei of terminal oogonial cells, although predominantly in a condensed and heterochromatic state, produces numerous nucleoli and incorporates 3H-uridine during the interphases between successive differential divisions. Moreover, it was shown that extra DNA is active in RNA synthesis at the same stage of pupal development in which it is synthesized and accumulated, i.e. in the nuclei of terminal oogonial cells. As soon as the oocyte forms RNA synthesis ceases in the extrachromosomal DNA body cells showed that nucleolar material does not disappear during division but remains, at least partly, connected with the extra DNA body.