Bone Morphogenetic Protein 15 Knockdown Inhibits Porcine Ovarian Follicular Development and Ovulation.

Zuyong He,Tao Tang,Xiaoliang Yang,Xuan Shi,Wei Li,Xiaofeng Liu,Delin Mo,Xinyu Liang,Min Wang,Yufeng Qin,Peiqing Cong,Guanjie Sun,Yaosheng Chen,Xiaohong Liu,Zhiguo Liu

doi:10.3389/fcell.2019.00286

Zuyong He, Tao Tang + Show 13 more

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https://doi.org/10.3389/fcell.2019.00286

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Abstract

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.

Highlights

We found that shRNA1 was the most effective with a RNA inference efficiency reaching 76% (Figure 1B), and this shRNA was selected for transfection into embryonic fibroblast cells (PEFs) derived from a male Yorkshire pig
Studies on animals immunized with different regions of Bone morphogenetic protein 15 (BMP15) peptide revealed that an increased ovulation rate can be found in females where some of the BMP15 have been neutralized, but an inhibition of follicular growth and ovulation was found in females where most of the active BMP15 has been neutralized (McNatty et al, 2007)
We found that TGF and TGS ovaries could be concurrently present in a single TG gilt (Figure 3A), and this may be caused by epigenetic mechanisms including DNA methylation and histone modifications (Spencer et al, 2015), which could cause dynamic and heterogeneous expression of shRNA from integrated TG construct in different individuals and even in different ovaries within a single individual, explaining the varied in vivo levels of active BMP15 in each ovary of each individual

Summary

Introduction

In the past three decades, increasing studies have revealed the important role of the oocytespecific secreted factor, bone morphogenetic protein 15 (BMP15), in mammalian ovarian and follicular development through its multiple functions including promoting granulosa cells (GCs) proliferation and steroidogenesis (Otsuka et al, 2000, 2001; Moore et al, 2003; Moore and Shimasaki, 2005), preventing cell apoptosis and premature luteinization (Hussein et al, 2005; BMP15 for Porcine Ovarian FolliculogenesisMcNatty et al, 2005; Juengel et al, 2011; Chang et al, 2013; Zhai et al, 2013), regulating glycometabolism and lipid metabolism (Sugiura et al, 2007; Su et al, 2008), and controlling oocyte competence and ovulation (Fabre et al, 2006; Hussein et al, 2006). As a key signaling molecule mediating the dialogue between oocyte and its surrounding somatic cells (Gilchrist et al, 2008), BMP15 is expressed initially in the early follicle stage, and the expression gradually increases in subsequent follicle stages till the period of ovulation and/or luteinization (Paradis et al, 2009; Sun et al, 2010). This expression pattern differs with species, for example, the initial expression of BMP15 protein can be found in primary follicle (PF) stage of sheep, human, and pig, but not until the pre-ovulatory stage in mice (Paulini and Melo, 2011). BMP15/GDF9 heterodimers are more potent than BMP15 homodimers in the regulation of GCs, oocytes, and zygote development (Peng et al, 2013)

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