Ruochen Yang

Prolactin (PRL) plays an important role in animal follicle development and ovulation. However, its regulatory effects on the different stages of the estrus cycle in ewes are unclear. In this study, bromocriptine (BCR, PRL inhibitor) was used to study the effect of PRL on the secretion of reproductive hormones and gene expressions in order to explore its regulatory effects on the sexual cycle of ewes. Eighty healthy ewes with the same parity and similar weights were randomly assigned to the control group (C, n = 40) and the treatment group (T, n = 40, fed bromocriptine). After estrus synchronization, thirty-one ewes with overt signs of estrus were selected from each group. Six blood samples were randomly obtained by jugular venipuncture to measure the concentration of PRL, estrogen (E₂), progesterone (P₄), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and gonadotropin-releasing hormone (GnRH) in the proestrus, estrus, metestrus, and diestrus. At the same time, we collected the ovaries of the six ewes in vivo after anesthesia in order to detect follicle and corpus luteum (CL) counts and measure the expression of hormone-receptor and apoptosis-related genes. The results show that PRL inhibition had no significant effects on the length of the estrus cycle (p > 0.05). In proestrus, the number of large and small follicles, the levels of E₂, FSH, and GnRH, and the expressions of ER, FSHR, and the apoptotic gene Caspase-3 were increased (p < 0.05); and the number of middle follicles and the expression of anti-apoptotic gene Bcl-2 were decreased (p < 0.05) in the T group. In estrus, the number of large follicles, the levels of E₂ and GnRH, and the expressions of the StAR, CYP19A1, and Bcl-2 genes were increased (p < 0.05), and the number of middle follicles was decreased (p < 0.05) in the T group. In metestrus, the number of small follicles and the expression of LHR (p < 0.05) and the pro-apoptotic gene Bax were increased (p < 0.05); the number of middle follicles was decreased (p < 0.05) in the T group. In diestrus, the number of large follicles, middle follicles, and CL, the level of P₄, and the expressions of PR, 3β-HSD, StAR, Caspase-3, and Bax were increased (p < 0.05); the number of small follicles and the expression of Bcl-2 were decreased (p < 0.05) in the T group. In summary, PRL inhibition can affect the secretion of reproductive hormones, the follicle count, and the gene expression during the estrus cycle. These results provide a basis for understanding the mechanisms underlying the regulation of the ewe estrus cycle by PRL.

High prolactin (PRL) concentration has been shown to induce the apoptosis of ovine ovarian granulosa cells (GCs), but the underlying mechanisms are unclear. This study aimed to investigate the mechanism of apoptosis induced by high PRL concentration in GCs. Trial 1: The optimal concentration of glutathion was determined according to the detected cell proliferation. The results showed that the optimal glutathione concentration was 5 μmol/mL. Trial 2: 500 ng/mL PRL was chosen as the high PRL concentration. The GCs were treated with 0 ng/mL PRL (C group), 500 ng/mL PRL (P group) or 500 ng/mL PRL, and 5 μmol/mL glutathione (P-GSH group). The results indicated that the mitochondrial respiratory chain complex (MRCC) I–V, ATP production, total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and thioredoxin peroxidase (TPx) in the C group were higher than those in the P group (p < 0.05), while they were lower than those in the P-GSH group (p < 0.05). Compared to the C group, the P group exhibited elevated levels of reactive oxygen species (ROS) and apoptosis (p < 0.05) and increased expression of ATG7 and ATG5 (p < 0.05). However, MRCC I–V, ATP, SOD, A-TOC, TPx, ROS, and apoptosis were decreased after the addition of glutathione (p < 0.05). The knockdown of either L-PRLR or S-PRLR in P group GCs resulted in a significant reduction (p < 0.05) in MRCC I–V, ATP, T-AOC, SOD and TPx, while the overexpression of either receptor showed an opposite trend (p < 0.05). Our findings suggest that high PRL concentrations induce apoptotic cell death in ovine ovarian GCs by downregulating L-PRLR and S-PRLR, activating oxidative stress and autophagic pathways.

Prolactin (PRL) has been reported to influence reproductive performance and cell apoptosis. However, its mechanism remains unclear. Hence, in the present study, ovine ovarian granulosa cells (GCs) were used as a cell model to investigate the relationship between PRL concentration and GC apoptosis, as well as its possible mechanisms. We examined the relationship between serum PRL concentration and follicle counts in sexually mature ewes. GCs were isolated from adult ewes and treated with different concentrations of PRL, while 500 ng/mL PRL was selected as the high concentration of prolactin (HPC). Then, we applied the transcriptome sequencing (RNA-Seq) combined with a gene editing approach to explore the HPC contributing to cell apoptosis and steroid hormones. The apoptosis of GCs gradually increased at PRL concentrations above 20 ng/mL, while 500 ng/mL PRL significantly decreased the secretion of steroid hormones and the expression of L-PRLR and S-PRLR. The results indicated that PRL regulates GC development and steroid hormones mainly through the target gene MAPK12. The expression of MAPK12 was increased after knocked-down L-PRLR and S-PRLR, while it decreased after overexpressed L-PRLR and S-PRLR. Cell apoptosis was inhibited and the secretion of steroid hormones increased after interfering with MAPK12, while the overexpression of MAPK12 showed the opposite trend. Overall, the number of follicles gradually decreased with increasing PRL concentration. HPCs promoted apoptosis and inhibited steroid hormone secretion in GCs by upregulating MAPK12 through reducing L-PRLR and S-PRLR.

Dioscorea opposite waste (DOW) has been shown to improve the gastrointestinal microbiome, antioxidation capacity, and immune activity, indicating it is a potential feed resource to improve the physiological health and rumen function of weaned lambs. In the present study, the responses of rumen microbiome to DOW supplementation in diet were profiled using metagenome sequencing. In addition, the potential of DOW to regulate plasma parameters in weaned lambs and its possible mechanisms were investigated. Sixty healthy male small tail Han lambs (22.68 ± 2.56 kg) were selected and equally assigned to four dietary treatments: (1) DOW-free diet (CON), (2) addition of 10% DOW diet (DOW1), (3) addition of 15% DOW diet (DOW2), and (4) addition of 20% DOW diet (DOW3). Experimental lambs were fed a corresponding diet for 62 days. Rumen microbiome and plasma parameters were determined at the end of the experiment. The results showed that dietary supplementation with DOW linearly increased the concentration of aspartate aminotransferase, alkaline phosphatase, Immunoglobulin A, Immunoglobulin M, Immunoglobulin G, Glutathione peroxidase, Superoxide dismutase, and total antioxidant capacity in the plasma of weaned lambs, but an opposite trend was observed in Interleukin-1β, Interleukin-6, tumor necrosis factor-α, and Malondialdehyde between the DOW-supplemented group and the CON group. Sequencing of rumen metagenome revealed that dietary supplementation with 20% DOW significantly affected the microbial composition and function and increased the richness and diversity of rumen microbiota and relative abundance of phylum Verrucomicrobia, Planctomycetes, Fibrobacteres, Chloroflexi, Actinobacteria, and Acidobacteria and species Ruminococcaceae_bacterium, Clostridiales_bacterium_NK3B98, Clostridiales_bacterium, and Clostridia_bacterium. It was concluded that supplementing the weaned lamb’s ration with DOW increased the immune response and antioxidant capacity in a dose-dependent manner. Meanwhile, dietary supplementation with 20% DOW modulated the composition of rumen microbiome function by increasing Ruminococcaceae_bacterium and Clostridiales_bacterium with improving the polysaccharide hydrolase activity in the rumen.