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The sonic hedgehog (SHH) pathway is an important signaling pathway for mammalian ovarian folliculogenesis and oocyte maturation. A previous study demonstrated that low-quality porcine cumulus-oocyte complexes (COCs) have low developmental competence, with lower SHH signaling protein expression before and after in vitro maturation (IVM) than high-quality COCs. However, there is no reported evidence on the restorative effects of SHH protein supplementation during the IVM of low-quality porcine COCs. Therefore, this study investigated the effects of SHH protein supplementation on the IVM of low-quality porcine COCs, as assessed by brilliant cresyl blue (BCB) staining. To examine this, we designed four groups: (i) BCB- (low-quality), (ii) BCB- + SHH, (iii) BCB+ (high-quality), and (iv) BCB+ + SHH. While the supplementation of SHH protein with high-quality COCs had no effect, supplementation with low-quality COCs significantly improved cumulus cell expansion, metaphase II rate, and subsequent embryo development following parthenogenetic activation. Our results provide the first evidence that the low developmental competence of low-quality porcine COCs can be improved by supplementation with the SHH protein. These results indicate that an active SHH signaling pathway is required for the acquisition of developmental competence in porcine COCs.

To elucidate the functional role of V-set and immunoglobulin domain-containing 1 (VSIG1) in spermatogenesis and fertilization, we knocked out (KO) VSIG1 in a mouse embryo using CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) -mediated genome editing. Reverse transcription PCR was performed using cDNA synthesized from VSIG1 KO testis RNA. Although Western blot analysis using a specific antibody to VSIG1 confirmed VSIG1 protein defects in the KO mice, hematoxylin-eosin staining analysis was similar in the KO and wild-type mice. Additionally, computer-assisted sperm analysis and in vitro fertilization experiments were conducted to confirm the activity and fertilization ability of sperm derived from the KO mouse. Mice lacking VSIG1 were viable and had no serious developmental defects. As they got older, the KO mice showed slightly higher weight loss, male mice lacking VSIG1 had functional testes, including normal sperm number and motility, and both male and female mice lacking VSIG1 were fertile. Our results from VSIG1 KO mice suggest that VSIG1 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. VSIG1 in sperm is dispensable for spermatogenesis and male fertility in mice. As several genes are known to possess slightly different functions depending on the species, the importance and molecular mechanism of VSIG1 in tissues of other species needs further investigation.