Abstract: This study aimed to determine the effects of PGD₂ members in the prostaglandin family, which are rarely reported in the reproductive field, on the endocrine function of luteal cells and its apoptosis-related gene expression in a single environment, and to analyze its mechanism in the luteal regression, providing a new theoretical basis for the comprehensive analysis of the biological role of prostaglandin family members. The ovarian tissues of the middle stage of the corpus luteum of the empty pregnant female goat were collected, isolated and purified by collagenase II digestion and pancreatic enzyme differential centrifugation to obtain goat corpus luteal cells. DMEM/F12 was used for in vitro culture to observe the growth status of luteal cells at different time of in vitro culture, and the luteal cells were identified by immunohistochemistry and cell morphological characteristics. PGD₂ was set to three different gradients to determine the dose-dependent relationship of its effect on the luteal cells. Finally, luteal cells were treated with the best dependent dose of PGD₂, the endocrine function changes of luteal cells were detected by ELISA, the apoptosis rate of luteal cells were measured by flow cytometry, and the expression level of mRNA/protein expression of apoptosis-related genes were detected by qRT-PCR/Western blot. The results showed that the goat primary luteal cells were successfully isolated and obtained after identification of the specific expression protein of synaptophysin (SYP). Real-time observation of cell morphology and ELISA detection showed that luteal cells had a full cytoplasm, compact shape, and clear morphology at 5 days of ex vivo culture, and the endocrine P₄ level of luteal cells was the highest. At this time, the cell growth curve results also confirmed that cell growth peaked at 5 days of ex vivo culture, and the cell growth curve was inverted "S" shaped. After 48 h of logarithmic peak cells seeding and PGD₂ treatment, it was found that the order of P₄ secretion levels of luteal cells in different dose groups was 2 μg group < 3 μg group < 1 μg group. At the same time, in the 2 μg dose treatment group, the concentration of P₄ in the medium decreased significantly compared with the control group (P<0.01). The expression of the steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) genes were significantly downregulated (P<0.05). In addition, the results of flow cytometry detection and Flow Jo software analysis showed that the apoptosis rate of luteal cells increased significantly (P<0.05). qRT-PCR and Western blot results confirmed that the anti-apoptotic factor B lymphoma-2 (BCL-2) mRNA and protein were significantly downregulated (P<0.05), and the pro-apoptotic factor cysteine aspartate-specific protease-3 (Caspase-3) mRNA and protein were significantly upregulated (P<0.05). The above results show that PGD₂ can not only inhibit the endocrine function level of luteal cells by downregulating the expression of StAR/3β-HSD gene, but also accelerate the apoptosis process of luteal cells by downregulating the expression of anti-apoptotic BCL-2 gene and upregulating the expression of pro-apoptosis Caspase-3 gene, and finally participate in the molecular regulation of luteal cells in the form of dual pathways. This lays a solid foundation for further improving the biological functional effects of prostaglandin family members and subsequently comprehensively exploring the molecular regulatory network mechanism of luteal maintenance and degradation in domestic animals.

Key words: corpus luteum, prostaglandin, endocrine, apoptosis gene