Differential hypothalamic expression of Musashi protein 2 in Mongolian sheep with single and double ovulation.

Introduction: Musashi-2 (MSI2) is an RNA-binding protein involved in the regulation of mammalian reproduction. However, its expression characteristics and role in hypothalamic reproductive regulation in Mongolian sheep remain unclear. We investigated the differential expression of MSI2 in key hypothalamic nuclei of Mongolian ewes and analyzed its potential association with gonadotropin-releasing hormone (GnRH)-related neuroendocrine characteristics. Methods: Sixty 12-month-old Mongolian ewes were selected and divided into single corpus luteum (single-CL) and double corpora lutea (double-CL) groups. Immunofluorescence was used to detect the colocalization of MSI2 and GnRH in the supraoptic nucleus (SON), anteroventral periventricular nucleus (AVPV), arcuate nucleus (ARC), and paraventricular nucleus (PVN). Reverse transcription-quantitative PCR (RT-qPCR) and western blotting were performed to determine MSI2 mRNA and protein expression levels, as well as differences in GnRH protein expression. Sanger sequencing was used to preliminarily screen for MSI2 sequence variations, and enzyme-linked immunosorbent assay (ELISA) was performed to detect serum estradiol (E2), GnRH, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and progesterone (P4) levels. Results: MSI2 was expressed in the SON, AVPV, ARC, and PVN of ewes in both groups. MSI2 mRNA and protein levels were significantly higher in the single-CL group. By contrast, GnRH protein expression was higher in the double-CL group, whereas MSI2 protein levels were significantly negatively correlated with GnRH protein levels in all examined hypothalamic nuclei. Immunofluorescence showed colocalization of MSI2 and GnRH, suggesting that MSI2 may be involved in GnRH-related neuroendocrine regulation. The double-CL group showed significantly higher serum E2, GnRH, LH, and P4 levels, but lower FSH levels. In the single-CL group, an A/G overlapping peak signal was observed at the c.66 site of exon 6 of MSI2, which was identified as a synonymous variant based on sequence alignment. In contrast, the corresponding site in the double-CL group showed a single A peak signal. Conclusion: Differential hypothalamic MSI2 expression may be associated with GnRH-related neuroendocrine differences between Mongolian ewes with single and double corpora lutea. This study provides a theoretical basis for further exploration of the potential role of MSI2 in ovulation-related traits in Mongolian sheep.