1. Evidence that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary
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Sui, Xu-Xia, Luo, Li-Li, Xu, Jin-Jie, and Fu, Yu-Cai
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Oocytes -- Research -- Physiological aspects ,Genes -- Research -- Physiological aspects ,Cell cycle -- Research -- Physiological aspects ,Ovaries -- Research -- Physiological aspects ,Rats -- Physiological aspects -- Research ,Proteins -- Research -- Properties -- Physiological aspects ,Rattus -- Physiological aspects -- Research ,Apoptosis -- Research -- Physiological aspects ,Transcription factors -- Research -- Properties -- Physiological aspects ,Biological sciences - Abstract
Previous studies have proposed that the forkhead transcription factor FOXO3a is involved in cell cycle arrest and apoptosis and that it may also repress follicular development by inducing cell cycle arrest in ovaries. We have recently demonstrated that FOXO3a induces oocyte apoptosis of neonatal rat ovaries under in vitro conditions. In the present study, we evaluated the role of FOXO3a in oocyte apoptosis under in vivo conditions. Ovaries from rats were obtained from newborns on postnatal day (PD) 1, 2, 3, and 4. TUNEL assay results showed that oocyte apoptosis occurred mainly on PD 1 and 2. Immunohistochemical staining of FOXO3a, Bim, Fas ligand (FasL), p27KIP1, caspase-8, and caspase-3 showed that they were all expressed mainly in naked oocytes on PD 1 and 2. The percentage of positive FOXO3a staining of oocytes reached peak levels in the ovaries of 2-day-old rats, which was consistent with the rate of the apoptotic profiles determined by TUNEL. The percentage between TUNEL-positive and FOXO3a-positive oocytes in the nucleus showed no statistical differences within the 4-day-old rat ovaries. Furthermore, the positive oocyte percentage of the target factors of FOXO3a (Bim, p27KIP1, and FasL) and pro-apoptotic proteins (caspase-3 and caspase-8) also reached peak levels in the ovaries of 2-day-old rats, which was similar to the rate of FOXO3a-positive oocytes. These results suggest that FOXO3a in the oocyte nucleus is involved in oocyte apoptosis; that is, FOXO3a-positive oocytes may be the apoptotic cells. To verify this, rat oocytes were subjected to TUNEL and immunofluorescent double-labeling assays. We found that TUNEL-positive cells were also FOXO3a-, Bim-, or FasL-positive. To identify the downstream target of FOXO3a, double immunofluorescent staining with antibodies to Bim and FasL was performed. We found that FOXO3a-positive cells were also Bim- and FasL-positive. We conclude that the overexpression of FOXO3a in the oocyte nucleus of neonatal rat ovaries may play an important role in the apoptosis of naked oocytes, and that Bim, FasL, and p27KIP1 are the key downstream factors of FOXO3a. Key words: FOXO3a, oocyte apoptosis, neonatal rat ovary. Des etudes precedentes ont propose; que le facteur de transcription membre de la famille << forkhead >> FOXO3a soit implique dans l'arret du cycle cellulaire et l'apoptose, et qu'il puisse aussi reprimer le developpement folliculaire en induisant l'arret du cycle cellulaire dans l'ovaire. Nous avons recemment demontre que FOXO3a induit l'apoptose des ovocytes de rates neonatales in vitro. Dans cette presente etude, nous avons evalue le role de FOXO3a dans l'apoptose des ovocytes in vivo. Les ovaires de rates ont ete obtenus de nouveau-nes aux jours postnataux (PN) 1, 2, 3 et 4. Les resultats de l'essai TUNEL ont montre que l'apoptose des ovocytes survenait principalement aux jours PN1 et 2. Une coloration immunohistochimique de FOXO3a, de Bim, du ligand de Fas (FasL), de p27KIP1, de la casapase-8 et de la caspase-3 a montre qu'ils etaient tous exprimes principalement dans les ovocytes nus aux jours PN 1 et 2. Le pourcentage d'ovocytes colores positivement pour FOXO3a atteignait un pic dans les ovaires des rates de 2 jours, ce qui concordait avec le taux d'apoptose determine par TUNEL. Les pourcentages de cellules positives en TUNEL et a FOXO3a nucleaire n'etaient pas significativement differents dans les ovaires des rates de 4 jours. De plus, le pourcentage d'ovocytes positifs aux facteurs cibles de FOXO3a (Bim, p27KIP1 et FasL) et aux proteines pro-apoptose (caspase-2 et caspase-8) atteignait aussi un pic dans les ovaires de rates de 2 jours, similairement aux taux d'ovocytes positifs a FOXO3a. Ces resultats suggerent que FOXO3a present dans le noyau des ovocytes est implique dans l'apoptose des ovocytes, et ce faisant, que les ovocytes positifs a FOXO3a peuvent etre en apoptose. Afin de verifier ceci, les ovocytes de rates ont ete soumis aux essais TUNEL et a un double marquage en immunofluorescence. Nous avons trouve que les cellules positives en TUNEL sont aussi positives a FOXO3a, Bim, et FasL. Afin d'identifier les cibles de FOXO3a, une double coloration en immunofluorescence a l'aide d'anticorps diriges contre Bim et FasL a ete realisee. Nous avons trouve que les cellules positives a FOXO3a etaient aussi positives a Bim et a FasL. Nous concluons donc que la surexpression de FOXO3a dans le noyau des ovocytes des ovaires des rates neonatales peut jouer un role important dans l'apoptose des ovocytes nus, et que Bim, FasL et p27KIP1 sont des facteurs cles en aval de FOXO3a. Mots-cles: FOXO3a, apoptose des ovocytes, ovaire de rate neonatale. [Traduit par la Redaction], Introduction It is widely accepted that the wave of oocyte degeneration, which leads to the formation of a fixed reserve pool of primordial follicles, occurs during the first 3 days [...]
- Published
- 2010
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