Project description:Background: Follicular growth and maturation in semi-synchronously spawning fish involve numerous cell signaling cascades and different molecular cascades are activated or inhibited during specific stages of oocyte development. The objectives of the current study were to identify molecular pathways and temporal gene expression patterns throughout a complete breeding cycle in female wild LMB to characterize the molecular sequence of events underlying follicle and ovary development. Methods: Microarray analysis was performed on eight morphologically distinct stages, from primary stages of oocyte growth to ovulation and atresia. Ovarian tissue histology, plasma vitellogenin, and sex steroids (E2 and T) were also measured to correlate molecular signaling cascades to higher levels of biological organization. Results: Global expression patterns revealed dramatic differences between early and late stages of ovarian follicle progression, with over 200 and 500 genes being differentially expressed during both ovulation and atresia respectively (p < 0.01). Time course analysis for all stages leading to ovulation and atresia identified increased expression of GABAA receptor subunits and peroxisome proliferator-activated receptor gamma during ovulation. Gene set enrichment analysis (GSEA) revealed that early stages of oocyte growth involved increases in pathways of natural killer cell and mast cell activation, as well as gap junction regulation. GSEA revealed that arachidonic acid metabolism was significantly up-regulated while CD2, fibronectin, and neuropeptides Y receptor signaling cascades were down-regulated at ovulation. Expression targets for LH signaling were decreased during vitellogenesis but increased at ovulation. GSEA revealed decreases in actin cytoskeleton regulation and receptor mediated signaling pathways involving TGFβ and ephrin receptor regulation at atresia. Conclusions: This study offers new insight into the molecular pathways involved in vitellogenesis, ovulation and atresia in LMB and provides new hypotheses about the cellular pathways involved in oocyte growth and maturation.

Project description:Background: In mammals, female fertility is determined by the outcome of follicular development (ovulation or atresia). Follicular atresia is a complex physiological process that results in the degeneration of oocytes from the ovary. However, the molecular mechanisms of oocyte degeneration and key protein markers of follicular atresia remain unclear. In this study, we explored the complex transcriptional regulatory mechanisms and protein profiles in oocytes and follicular fluid in atretic follicle stages using single-cell RNA sequencing and tandem mass tag proteomics. Results: First, through paired analysis of different follicle development stages, we identified 175 atresia-specific genes and eight candidate oocyte-secreted factors, including PKG1, YTHDF2, and MYC. Meanwhile, we also characterized unique features of the oocyte transcriptional landscape in the atretic follicle stage that displayed cell death-related transcriptional changes and mechanisms, such as autophagy (TBK1 and IRS4), necroptosis (PKR), and apoptosis (MARCKS). Moreover, we identified atresia-specific genes, namely FTH1, TF, and ACSL4, which may participate in regulation of oocyte ferroptosis in atretic follicles through a series of mechanisms including ferritinophagy, ferritin transport, and lipid metabolism. Additionally, we uncovered 333 differentially expressed proteins that may coordinate follicular atresia and revealed key pathways, such as negative regulation of angiogenesis, metabolic pathways, and transcription and mRNA splicing, that lead to oocyte degeneration. Finally, by combining transcriptome and proteomics analyses, we identified two oocyte-secreted biomarkers, PGK1 and ANGPT2, that may be associated with follicular atresia. Conclusions: In conclusion, our work provides a comprehensive characterization of oocyte degeneration in ovine atretic follicles, which provides a basis for establishing an oocyte quality evaluation system and understanding the mechanism of follicular atresia in sheep, as well as an important reference for in vitro production of embryos.

Project description:Nylon microarrays displaying 9152 rainbow trout cDNAs were hybridized using RNA samples originating from ovarian tissue collected during late vitellogenesis, post-vitellogenesis and oocyte maturation. Differentially expressed genes were identified using a statistical analysis. A supervised clustering analysis was performed using only differentially expressed genes in order to identify gene clusters exhibiting similar expression profiles. In addition, specific genes were selected and their preovulatory ovarian expression was analyzed using real-time PCR. From the statistical analysis, 310 differentially expressed genes were identified. Among those genes, 90 were up-regulated at the time of oocyte maturation while 220 exhibited an opposite pattern. After clustering analysis, 90 clones belonging to 3 gene clusters exhibiting the most remarkable expression patterns were kept for further analysis. Using real-time PCR analysis, we observed a strong up-regulation of ion and water transport genes such as aquaporin 4 (aqp4) and pendrin (slc26). In addition, a dramatic up-regulation of vasotocin (avt) gene was observed. Furthermore, angiotensin-converting-enzyme 2 (ace2), coagulation factor V (cf5), adam 22, and the chemokine cxcl14 genes exhibited a sharp up-regulation at the time of oocyte maturation. Finally, ovarian aromatase (cyp19a1) exhibited a dramatic down-regulation over the post-vitellogenic period while a down-regulation of Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) was observed at the time of oocyte maturation. We showed the over or under expression of more that 300 genes, most of them being previously unstudied or unknown in the fish preovulatory ovary. Our data confirmed the down-regulation of estrogen synthesis genes during the preovulatory period. In addition, the strong up-regulation of aqp4 and slc26 genes prior to ovulation suggests their participation in the oocyte hydration process occurring at that time. Furthermore, among the most up-regulated clones, several genes such as cxcl14, ace2, adam22, cf5 have pro-inflammatory, vasodilatory, proteolytics and coagulatory functions. The identity and expression patterns of those genes support the theory comparing ovulation to an inflammatory-like reaction. Late vitellogenesis: 3 samples Post-vitellogenesis: 4 samples Oocyte maturation (germinal vesicle breakdown): 6 samples

Project description:Nylon microarrays displaying 9152 rainbow trout cDNAs were hybridized using RNA samples originating from ovarian tissue collected during late vitellogenesis, post-vitellogenesis and oocyte maturation. Differentially expressed genes were identified using a statistical analysis. A supervised clustering analysis was performed using only differentially expressed genes in order to identify gene clusters exhibiting similar expression profiles. In addition, specific genes were selected and their preovulatory ovarian expression was analyzed using real-time PCR. From the statistical analysis, 310 differentially expressed genes were identified. Among those genes, 90 were up-regulated at the time of oocyte maturation while 220 exhibited an opposite pattern. After clustering analysis, 90 clones belonging to 3 gene clusters exhibiting the most remarkable expression patterns were kept for further analysis. Using real-time PCR analysis, we observed a strong up-regulation of ion and water transport genes such as aquaporin 4 (aqp4) and pendrin (slc26). In addition, a dramatic up-regulation of vasotocin (avt) gene was observed. Furthermore, angiotensin-converting-enzyme 2 (ace2), coagulation factor V (cf5), adam 22, and the chemokine cxcl14 genes exhibited a sharp up-regulation at the time of oocyte maturation. Finally, ovarian aromatase (cyp19a1) exhibited a dramatic down-regulation over the post-vitellogenic period while a down-regulation of Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) was observed at the time of oocyte maturation. We showed the over or under expression of more that 300 genes, most of them being previously unstudied or unknown in the fish preovulatory ovary. Our data confirmed the down-regulation of estrogen synthesis genes during the preovulatory period. In addition, the strong up-regulation of aqp4 and slc26 genes prior to ovulation suggests their participation in the oocyte hydration process occurring at that time. Furthermore, among the most up-regulated clones, several genes such as cxcl14, ace2, adam22, cf5 have pro-inflammatory, vasodilatory, proteolytics and coagulatory functions. The identity and expression patterns of those genes support the theory comparing ovulation to an inflammatory-like reaction. Keywords: ovarian stages comparisons

Project description:Lake Apopka (Fl, USA) experienced heavy uses of organochlorine pesticides (OCPs) in the 1950s-1970 during an intense agricultural period. As a result, the region remains heavily contaminated and was declared a Superfund site by the US Environmental Protection Agency. Aquatic organisms inhabit the freshwater systems in and around Lake Apopka, however the impacts of sub chronic exposure to OCPs in this natural environment are not known. The objectives of this study were to determine the effects of the contamination on the reproductive axis of largemouth bass (LMB) (Micropterus salmoides). In October (2007), healthy LMB were stocked into natural mesocosms and remained in mesocosms before being sampled in January 2008 (at early oogenesis). Additional LMB were placed into mesocosms for 2 months in February (2008) and sampled in April (2008) (oocyte maturation). LMB placed in these mesocosms for four months had a 2-20X higher contaminant load for OCPs (e.g. DDE, dieldrin, methoxychlor) than LMB collected from reference sites. Gonadosomatic index for LMB collected in April from the mesocosms were not different that LMB from reference sites. Vitellogenin levels in LMB collected from the mesocosms in January and April were not significantly different than fish collected from Welaka in late vitellogenin or early maturation respectively. Steroids were depressed in mesocosm fish. Microarray analysis revealed that the expression profiles of genes in the LMB ovary were unique compared to LMB collected form reference sites. Transcripts that showed altered abundance in LMB from the mesocosms were insulin-like growth factor I and steroidogenic acute regulatory protein. Interestingly, differentially expressed transcripts showed a significant and positive correlation for LMB sampled in January and April despite the 3 month period in between samplings. Sub-network enrichment analysis for cellular processes showed that retinoic acid metabolism and germ-cell development were decreased in mesocosm-exposed fish but processes such as vitellogenesis, amino acid catabolism, granulosa cell function, vitamin D metabolism, and hormone biosynthesis were increased in mesocosm-exposed fish. These data suggest that (1) LMB from the mesocosms are exhibiting unique gene profiles that may impair normal reproduction and that (2) microarray analysis in the field can provide site specific information by discriminating LMB from reference and polluted sites.

Dataset Information

Ovarian transcriptomics over a complete breeding cycle in wild Largemouth bass (Micropterus salmoides) characterizes pathways underlying oocyte maturation and atresia

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