Sodium butyrate (NaBu), a common feed additive, has been shown to enhance reproductive performance in livestock and poultry. However, whether NaBu exerts this effect by directly regulating follicular development remains unclear. In this study, a three-dimensional (3D) in vitro culture system of mouse preantral follicles was used to investigate the effects of NaBu on follicular growth, hormone secretion, maturation of oocytes, and subsequent embryonic development. Preantral follicles were treated with different doses of NaBu on the fourth day of culture. Subsequently, the mature oocytes (MII stage) were released from the follicles on the ninth day and subjected to parthenogenetic activation for developmental assessment. The results showed that 0.10 mM NaBu treatment could significantly promote follicular growth, antral formation, and oocyte maturation. Furthermore, NaBu also significantly increased estradiol (E₂) secretion, improved follicular structure, and maintained cellular viability. qPCR analysis revealed that NaBu significantly increased the mRNA levels of STAR, CYP11A1, and CYP1B1. In addition, it significantly enhanced the distribution and organization of F-actin, with increases in the mRNA levels of GDF9, BMP15, and CX37. NaBu treatment significantly reduced intracellular ROS levels and increased the mRNA levels of NRF2 and SOD1, while SOD2 and GSR showed increasing trends without significant differences. NaBu significantly improved oocyte cytoskeletal organization and the morphology of the spindle, but it did not lead to a significant increase in the rates of cleavage and blastocyst formation after parthenogenetic activation. Collectively, these findings indicate that NaBu promotes follicular development and improves oocyte quality, at least partly, by enhancing steroidogenesis, alleviating oxidative stress, and maintaining cytoskeletal integrity, providing insight into its potential application for improving reproductive performance in livestock and poultry.

Cellular inhibitor of apoptosis protein 1 (cIAP1) plays a crucial role in the apoptotic process. This study involved gene cloning, protein expression, bioinformatics analysis, and the preparation of a specific polyclonal antibody. First, the coding sequence of the duck cIAP1 was amplified by PCR and sequenced. Bioinformatics prediction indicated that this protein is a stable hydrophilic protein with no signal peptides and transmembrane domains. It contains characteristic BIR (bacterial IAP repeat) domains. The secondary structure consists of 43% random coils, 28% α-helix, and 8% β-strand, and its 3D structure is highly conserved compared with human and mouse cIAP1. Subsequently, the cIAP1 was cloned into the pET-28a vector. The recombinant His-tagged cIAP1 protein was successfully expressed in Escherichia coli BL21(DE3) upon induction and subsequently purified. Immunization of BALB/c mice with the purified protein emulsified in Freund’s adjuvant generated polyclonal antibodies. Indirect ELISA confirmed that the antibody titer exceeded 1:2,048,000. Additionally, Western blotting and immunofluorescence staining demonstrated that the antibody specifically recognized native duck cIAP1 protein without non-specific reactions with negative serum. This study successfully achieved prokaryotic expression of duck cIAP1 and produced a high-titer, highly specific mouse polyclonal antibody. This antibody is suitable for experiments such as Western blotting and immunofluorescence, providing important biological materials and a theoretical basis for further investigation of the anti-apoptotic mechanism of cIAP1 in major diseases of ducks.

Histone deacetylase 2 (HDAC2)-mediated epigenetic modifications play a crucial role in oogenesis, yet the regulatory mechanisms of HDAC2 throughout the entire process of folliculogenesis remain elusive. In the present study, the localization and expression of HDAC2 were detected in mouse ovaries at distinct postnatal ages and after gonadotropin treatment. The effects and molecular mechanisms of Santacruzamate A (SCA), a selective HDAC2 inhibitor, on folliculogenesis were investigated via an in vitro 3D culture model of mouse preantral follicles. The results showed that HDAC2 was localized in the nuclei of both ovarian granulosa cells and oocytes. Its expression was gradually increased with ovarian development in mice aged from postnatal day 7 to 21, and significantly decreased after 12 h hCG treatment. In follicles cultured in vitro, HDAC2 expression peaked on day 4 of culture. SCA exerted stage-specific and dose-dependent regulatory effects on folliculogenesis. SCA significantly suppressed follicular growth, blocked antrum formation, and triggered follicular atresia. Mechanistically, SCA promoted ovarian granulosa cell apoptosis via increases in pro-apoptotic genes BAX and Caspase-3 levels, and a decrease in anti-apoptotic gene BCL-2 level. Furthermore, SCA disrupted the structural integrity of the follicular microfilament cytoskeleton, interfered with the levels of genes related to oocyte–granulosa cell communication including CX37 and GDF9, caused abnormalities in oocyte spindle structure, chromosome alignment, mitochondrial distribution, and decreased mitochondrial membrane potential (ΔΨm). In conclusion, HDAC2 participates in the transcriptional regulation of mouse folliculogenesis through dynamically changing expression at distinct developmental stages. HDAC2 exerts stage-specific regulatory effects on follicular growth and oocyte maturation, and its mechanisms are associated with maintaining granulosa cell survival, ensuring cytoskeletal integrity, and preserving oocyte mitochondrial function. This study advances our understanding of epigenetic regulation of folliculogenesis and provides novel targets and an experimental basis for optimizing in vitro follicular culture systems and regulating female reproductive performance.

Granulosa cells (GCs) critically govern follicular development through the coordinated regulation of proliferation, apoptosis, and steroidogenesis. Neuromedin B (NMB), a local regulatory peptide, has been implicated in female reproductive processes. However, its precise effects on porcine GCs remain to be elucidated. In this study, we characterized the ovarian distribution of NMBR, investigated the effects of NMB on GC function, and further explored the involvement of NMBR in these effects using the NMBR antagonist PD168368. The results showed that higher NMBR expression was observed in follicles of larger diameter. NMB treatment differentially regulated the functional activities of porcine ovarian GCs. Specifically, certain concentrations of NMB significantly increased cell viability and altered the expression levels of the proliferation-related markers Cyclin B1 and PCNA; reduced the cell apoptosis rate and modulated the expression levels of apoptosis-related genes and proteins (Bax, Bcl-2, and Caspase-3); and affected steroidogenic responses, including increased E₂ concentrations at selected concentrations while altering expression of STAR, CYP11A1, HSD3B1, and CYP19A1. Importantly, several of these effects were attenuated by PD168368 treatment. Collectively, these findings suggest that NMB may regulate multiple functions of porcine with the involvement of NMBR. These findings provide a theoretical basis for elucidating the neuroendocrine mechanisms underlying porcine follicular development and for further investigating the potential role of NMB/NMBR signaling in porcine follicular physiology.