| Embryonic and neonatal periods are important window period to the development of ovary in female animal. The alterations of ovarian structure and function in neonatal animals can predispose adult ovary function, further affecting the reproductive function of adult animals. The maternal nutrition during pregnancy and lactation can program ovarian development in offspring. Folliculogenesis and steroidogenesis are two key biological process to determine ovarian development and function. However, so far, the studies that maternal nutrition programs ovarian folliculogenesis and steroidogenesis of offspring mainly focus on rodent animals. It has few reports in pigs. Moreover, the effect of maternal nutrition on the development of ovary in neonatal animals has not been described. In addition, the molecular mechanism of maternal nutrition on the folliculogenesis and steroidogenesis at different developmental stages of ovary in offspring and linking these two biological processes remain unclear.Therefore, this study was designed to study the effect of sows fed Low-Protein (LP) diet during pregnancy and lactation on ovarian folliculogenesis and steroidogenesis in newborn piglets and young gilts, and to explore its underlying molecular mechanism. Moreover, we analysed the regulation mechanism of FasL and CYP19A1 genes in the ovary of young gilts under the maternal protein restriction.1 Effects of maternal LP diet during pregnancy on the ovarian expression of folliculogenesis and steroidogenesis genes in neonatal piglets and its molecular mechanismSixteen multiparous Landrace x Yorkshire crossbred sows in the second parity were artificially inseminated, at the observation of estrus, with a mixture of Duroc semen samples obtained from two littermate boars. One week after artificial insemination, sows were randomly divided into standard-(SP) and low-protein (LP) diet groups (n= 8 per group). SP sows were fed diets containing 15% of crude protein throughout gestation and those in the LP group were fed diets containing 7.5% of crude protein. Sows were fed three times daily (05:00,10:00 and 17:00 h), with the ration of 1.2 kg/d,1.8 kg/d and 2.4 kg/d during early, middle and late stage of pregnancy and had free access to water. After parturition of sows, one female piglet of the mean body weight was selected from all female piglets of each litter and exsanguinated. The blood and ovaries were collected.Female piglets born to LP sows showed significantly decreased ovary weight relative to body weight (P< 0.05) at birth, which was accompanied with increased serum 17β-estradiol (E2) level (P< 0.05). LP piglets demonstrated higher ratio of BAX/Bcl-2 mRNA (P< 0.01), which was associated with up-regulated mRNA expression of BMP4 (P < 0.05) and PCNA (P< 0.05). Moreover, the steroidogenic gene, CYP19A1 and CYP17A1 was significantly down-regulated (P(?) 0.05), in LP piglets. The alterations in ovarian gene expression were associated with a significant down-regulation of FSHR and GPR30 mRNA expression (P< 0.05) in LP piglets. Maternal LP diet did not affect the methylation level in the promoter of BMP4, PCNA and. CYP19A1. However, three miRNAs, including miR-423-5p targeting both CYP19A1 and PCNA, miR-378 targeting CYP19A1 and miR-210 targeting BMP4, were significantly down-regulated (P< 0.05) in the ovary of LP piglets. miR-423-3p targeting FSHR tended to be significantly down-regulated (P= 0.08).These results suggest that miRNAs are involved in mediating the effect of maternal LP diet during pregnancy on ovarian function through regulating the expression of folliculogenic and steroidogenic genes in neonatal piglets. Moreover, Higher serum E2 level caused by maternal protein restriction in neonatal piglets may play a role through its receptor, GPR30.2 Effects of maternal LP diet during pregnancy and lactation on the folliculogenesis and steroidogenesis of ovary in young gilts and its molecular mechanismAfter parturition, SP sows were fed diets containing 18% of crude protein during lactation. LP sows were fed an isoenergy and protein-restricted diet containing 9% of crude protein. Sows were fed two times daily (10:00 and 17:00 h), with the ration of 4.2 kg/d and had free access to water. All the piglets were weaned at 28 days of age and were fed the same standard diet until six-month-old. Blood and ovaries were collected immediately. The right ovaries were snap-frozen in liquid nitrogen and stored at-70℃ for subsequent analysis of molecular mechanism. The left ovaries were fixed immediately in 4% paraformaldehyde for histological processing.Maternal LP diet during pregnancy and lactation didn’t affect the ovary weight relative to body weight in the young gilts of offspring. LP offspring had less graafian follicles with more apoptotic granulosa cells (P< 0.05). Among the apoptosis genes, Fas and FasL mRNA expression was markedly up-regulated (P< 0.05) in the ovary of LP offspring. Moreover, the protein content of FasL and the activated Caspase-3 in the ovary were significantly higher (P< 0.05) in LP offspring. However, maternal protein restriction did not affect the expression of autophagy related protein LC3 and ATG12. LP offspring had higher serum E2 level (P< 0.01), which was accompanied by lower mRNA (P< 0.05) but higher protein (P< 0.05) expression of CYP19A1 in the ovary. Maternal LP diet during pregnancy and lactation made the ratio of ERa gene expression to ERβ significantly increased (P< 0.05) in the ovary of LP offspring. Moreover, the ovarian ERa protein content was significantly up-regulated (P< 0.05).These results suggest that maternal LP diet in pregnancy and lactation induced granulosa apoptosis in graafian follicles through up-regulating the expression of Fas, then activated the Fas/FasL-Caspase 3 pathway, leading to the decrease of the number in graafian follicles. Maternal protein restriction elevated serum E2 level of young gilts by regulating the expression of CYP19A1. Higher serum E2 level caused by maternal protein restriction in young gilts may play a role through its receptor, ERa.3 Regulated expression of FasL and CYP19A1 genes in the ovary of young gilts under the maternal LP diet during pregnancy and lactationThe ChIP assay revealed that maternal LP diet had no effect on the ER binding to the promoter of CYP19A1 gene. However, ERa binding to the FasL gene promoter was significantly higher (P< 0.05) in the ovary of LP offspring. No alterations were detected for the expression of miRNAs predicted to target the FasL gene. However, among 9 predicted miRNAs targeting CYP19A1,6 miRNAs were significantly down-regulated in LP offspring (P< 0.05).These results suggest that Maternal LP diet during pregnancy and lactation regulated the expression of FasL gene through the transcription factor at the transcriptional level, which activates the ovarian folliculogenesis, and regulated the ovarian expression of CYP19A1 gene in the post-transcriptional level, leading to activation of ovarian steroidogenesis.In conclusion, Maternal LP diet in pregnancy regulated the ovarian expression of folliculogenesis and steroidogenesis genes in the post-transcriptional level through miRNAs in neonatal piglets. Maternal LP diet during gestation and lactation elevated serum E2 level by activating CYP19A1 through miRNA-mediated mechanism. Higher serum E2 level in LP group activated the Caspase cascade through enhanced ERa binding to FasL promoter, leading to the apoptosis of granulosa cells in graafian follicles and decrease of the number of graafian follicles in young gilts. The higher E2 caused by maternal protein restriction may play a role through GPR30 in the ovary of neonatal piglets, while through ERa in the ovary of young gilts. |
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