The dominant white phenotype in pigs is thought to be mainly due to a structural mutation in the KIT gene, a splice mutation (G > A) at the first base in intron 17 which leads to the deletion of exon 17 in the mature KIT mRNA. However, this hypothesis has not yet been validated by functional studies. Here, we created two mouse models, KIT D17/+ to mimic the splice mutation, and KIT Dup/+ to partially mimic the duplication mutation of KIT gene in dominant white pigs using CRISPR/Cas9 technology. We found that the splice mutation homozygote is lethal and the heterozygous mice have a piebald coat. Slightly increased expression of KIT in KIT Dup/+ mice did not confer the patched phenotype and had no obvious impact on coat color. Interestingly, the combination of these two mutations reduced the phosphorylation of PI3K and MAPK pathway associated proteins, which may be related to the impaired migration of melanoblasts observed during embryonic development that eventually leads to the dominant white phenotype.

Bone morphogenetic protein 15 (BMP15) is strongly associated with animal reproduction and woman reproductive disease. As a multifunctional oocyte-specific secret factor, BMP15 controls female fertility and follicular development in both species-specific and dosage-sensitive manners. Previous studies found that BMP15 played a critical role in follicular development and ovulation rate in mono-ovulatory mammalian species, especially in sheep and human, but study on knockout mouse model implied that BMP15 possibly has minimal impact on female fertility of poly-ovulatory species. However, this needs to be validated in other poly-ovulatory species. To investigate the regulatory role of BMP15 on porcine female fertility, we generated a BMP15-knockdown pig model through somatic nuclear transfer technology. The BMP15-knockdown gilts showed markedly reduced fertility accompanied by phenotype of dysplastic ovaries containing significantly declined number of follicles, increased number of abnormal follicles, and abnormally enlarged antral follicles resulting in disordered ovulation, which is remarkably different from the unchanged fertility observed in BMP15 knockout mice. Molecular and transcriptome analysis revealed that the knockdown of BMP15 significantly affected both granulosa cells (GCs) and oocytes development, including suppression of cell proliferation, differentiation, and follicle stimulating hormone receptor (Fshr) expression, leading to premature luteinization and reduced estradiol (E2) production in GCs, and simultaneously decreased quality and meiotic maturation of oocyte. Our results provide in vivo evidence of the essential role of BMP15 in porcine ovarian and follicular development, and new insight into the complicated regulatory function of BMP15 in female fertility of poly-ovulatory species.

Summary The melanocortin 1 receptor (MC1R), encoded by the classical extension (E) coat color locus, is expressed on the surface of melanocytes and plays a critical role in switching melanin synthesis from pheomelanin (red/yellow) to eumelanin (black/brown). Different MC1R alleles associated with various coat color patterns in pigs have been identified over the past decades. However, functional analysis of variant porcine MC1R alleles has not yet been performed. Therefore, in this study, we examined the subcellular localization and cyclic adenosine monophosphate (cAMP) signaling capability of MC1R variants in porcine kidney epithelial cells (PK15) overexpressing different MC1R alleles. Transcriptional slippage may partially restore the reading frame of the EP allele, possibly accounting for the observed spot phenotype. The A243T substitution in the e allele severely disrupted the membrane localization of the MC1R receptor, resulting in a severely impaired cAMP signaling capability. Both the V95M and L102P substitutions in the ED1 allele may contribute to the constitutively active function of MC1R, thus accounting for the dominant black phenotype. The D124N substitution in the ED2 allele severely attenuated the cAMP signaling capability of MC1R; however, whether this mutation contributes to the distinct phenotype of Hampshire pigs requires further investigation. Thus, our results provide new insights into the functional characteristics of MC1R variants and their roles in porcine coat color formation.

13Bone morphogenetic protein 15 (BMP15) is a multifunctional oocyte-specific secreted factor. It controls 14 female fertility and follicular development in both species-specific and dosage-sensitive manners. Previous 15 studies found that BMP15 played a critical role on follicular development and ovulation rate of 16 mono-ovulatory mammalian species, but has minimal impact on poly-ovulatory mice. However, whether this 17 is true in non-rodent poly-ovulatory species need to be validated. To investigate this question, we generated a 18 BMP15 knockdown pig model. We found that BMP15 knockdown gilts showed markedly reduced fertility 19 accompanied with phenotype of dysplastic ovaries containing significantly declined number of follicles, 20 increased number of abnormal follicles, and abnormally enlarged antral follicles resulting in disordered 21 ovulation. Molecular and transcriptome analysis revealed that knockdown of BMP15 significantly suppressed 22 cell proliferation, differentiation, Fshr expression, leading to premature luteinization and reduced estradiol 23 production in GCs, and simultaneously decreased the quality and meiotic maturation of oocyte. Our results 24 provide in vivo evidences for the essential role of BMP15 in porcine ovarian and follicular development, and 25 new insight into the complicated regulatory function of BMP15 in female fertility of poly-ovulatory species. 26 KEY WORDS: BMP15; transgenic pig; follicular development; ovarian development 27 28 96cells (PEFs) derived from a male Yorkshire pig. Transfected PEFs then were subjected to G418 selection to 97 screen the cells with stable expression of EGFP as donor cells for somatic cell nuclear transfer (SCNT). Clone 98 embryos then were transferred into Large White sow recipients to generate F0 TG pigs as described in our 99 previous report (Liu et al., 2019) (Fig. S1A). We obtained two healthy F0 TG males at last. After sexual 100 maturity, one F0 TG boar was mated with wild-type sows to generate F1 TG gilts for subsequent experiments. 101Both F0 and F1 TG pigs showed visible intense GFP fluorescence on toes and muscle while subjected to 102 sunlight (Fig. 1C, Fig. S1B), directly suggesting that the pEGFP-BMP15 shRNA plasmid was successfully 103 integrated into the genome of F0 TG boar, and can be transmitted to the next generation through the germline. 104This was confirmed by PCR analysis of fragment of integrated plasmid in muscle tissue of F1 TG gilts ( Fig. 105 S2A). The copy number of integrated plasmid was estimated to be approximate seven in F1 TG pigs through 106 the combination of both qPCR that using a transferrin receptor gene to normalize the genomic DNA (data 107 not shown), and Southern blot analysis ( Fig. S2B). More importantly, evidently decrease level of BMP15 108 mRNA ( Fig. 1E) in 365 days old TG ovaries and BMP15 protein level in 30 days old TG ovaries ( Fig. 1F, G) 109 strongly demonstrated the successful generation of the BMP15 knockdown model, and implied an in vivo 110 BMP15 knockdown efficiency of about 50%...