Gonadotropins and epidermal growth factor (EGF) play crucial roles in promoting oocyte maturation. The regulatory network downstream of these key factors is not well understood. The present study was designed to investigate the role of the calcium-sensing receptor (CASR) in porcine oocyte in vitro maturation. CASR expression was up-regulated in oocytes matured in gonadotropin-containing medium. Cortical distribution of CASR was enhanced with gonadotropins but not EGF. Supplementation of a CASR agonist (NPS R-568) in the gonadotropin (FSH and/or LH)-containing maturation medium significantly enhanced oocyte nuclear maturation. Addition of NPS2390, a CASR antagonist, compromised oocyte nuclear maturation. Furthermore, increased cortical distribution and decreased expression of CASR was observed after the NPS R-568 treatment. Oocytes treated with NPS R-568 had higher concentration of CYCLIN B1, decreased reactive oxygen species, and increased glutathione levels, indicative of advanced cytoplasmic maturation. In contrast, NPS2390 treatment compromised oocyte cytoplasmic maturation. A higher blastocyst formation rate after parthenogenetic activation was observed when oocytes were matured in the presence of the CASR agonist, NPS R-568. MAPK3/1 phosphorylation was increased during in vitro maturation and after NPS R-568 treatment, and decreased following CASR antagonist supplementation. Taken together, our data showed that the CASR is a gonadotropin-regulated factor that promotes porcine oocyte maturation in a MAPK-dependent manner.

Mammalian spermatogenesis is a highly ordered process that is determined by chromatin-associated moderators which still remain poorly understood. Through a multi-control group proteomics strategy, we confirmed that Sugp2 was a chromatin-associated candidate protein, and its signal arose along spermatogenesis. The expression results showed that Sugp2, which is mainly expressed in the testis, had two transcripts, encoding one protein. During spermatogenesis, Sugp2 was enriched in the nucleus of male germ cells. With the depletion of Sugp2 by CRISPER-Cas9 technology, we found that Sugp2 controlled a network of genes on metal ion and ATP binding, suggesting that alternative splicing regulation by Sugp2 is involved in cellular ion and energy metabolism during spermatogenesis, while it had a little effect on meiotic progression and male fertility. Collectively, these data demonstrated that, as a chromatin-associated protein, Sugp2 mediated the alternative splicing regulatory network during spermatogenesis.

Mammalian oocyte maturation relies on the precise assembly of the acentrosomal spindle, and its disruption causes aneuploidy and developmental failure. Symplekin (SYMPK), a 3'-end processing scaffold with emerging functions in regulating chromosome dynamics, remains unexplored in oocytes. Here, we investigate whether SYMPK governs spindle dynamics and chromosome fidelity during meiotic maturation. We find SYMPK dynamically tracks spindle microtubules during oocyte maturation following germinal vesicle breakdown (GVBD). By generating oocyte-specific Sympk knockout mice, loss of SYMPK in oocytes yields complete female infertility and impaired oocyte quality. Sympk-deficient oocytes show a predominant metaphase I (MI) arrest, accompanied by disorganized spindle architecture and destabilized kinetochore-microtubule attachments. Furthermore, chromosome spreads indicate persistent spindle assembly checkpoint (SAC) activation, and pharmacologic SAC inhibition can partially restore meiotic progression but not spindle integrity in SYMPK-deficient oocytes. Mechanistically, immunoprecipitation-mass spectrometry in MI oocytes reveals that SYMPK interacts with the spindle regulators KIF20A and NUMA1, and is required for their proper localization to the spindle. Collectively, these findings establish that SYMPK supports KIF20A and NUMA1 to coordinate acentrosomal spindle organization, thereby safeguarding oocyte meiotic maturation and ensuring faithful female meiotic progression.

MEIOB is a vital protein in meiotic homologous recombination and plays an indispensable role in human gametogenesis. In mammals, MEIOB and its partner SPATA22 form a heterodimer, ensuring their effective localization on single-strand DNA (ssDNA) and proper synapsis processes. Mutations in human MEIOB (hMEIOB) cause human infertility attributed to the failure of its interaction with human SPATA22 (hSPATA22) and ssDNA binding. However, the detailed mechanism is still unclear. In our study, truncated or full-length hMEIOB and hSPATA22 are traced by fused expression with fluorescent proteins (i.e., copGFP or mCherry), and the live cell imaging system is used to observe the expression and localization of the proteins. When transfected alone, hMEIOB accumulates in the cytoplasm. Interestingly, a covered NLS in the OB domain of hMEIOB is identified, which can be exposed by hSPATA22 and is necessary for the nuclear localization of hMEIOB. When hSPATA22 loses its hMEIOB interacting domain or NLS, the nuclear localization of hMEIOB is aborted. Collectively, our results prove that the NLS in the OB domain of hMEIOB and interaction with hSPATA22 are required for hMEIOB nuclear localization.