Essential role of Bone morphogenetic protein 15 in porcine ovarian and follicular development and ovulation

Abstract

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%...

Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte–cumulus cell complexes <i>in vitro</i>

“…BMP15-null female mice exhibit subfertility [6], ewes carrying BMP15 mutations are infertile, and their follicular development is arrested at the primordial stage [7]. BMP15-knockdown gilts show abnormal ovarian follicular development and ovulation [8].…”

MiR-214-3p Promotes Proliferation and Inhibits Estradiol Synthesis In Porcine Granulosa Cells

“…Ovarian GCs, as the main somatic cells in the follicle, play a significant role in the growth and development of follicles, atresia, oocyte maturation, and ovulation (30). GCs functions such as proliferation and estradiol synthesis are affected by many regulatory factors (31)(32)(33). miRNA plays an important part in these processes (34,35).…”