Effects of species differences on oocyte regulation of granulosa cell function

The aims were to investigate whether oocyte-secreted growth factors from a high (i.e. rat) and low (i.e. sheep) ovulation rate species could stimulate 3H-thymidine incorporation in granulosa cells (GC) from antral follicles from the same or across species. Denuded oocytes (DO) were co-incubated with GC with or without specific antibodies to growth differentiating factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15). Co-incubations of DO-GC from the same or across species significantly increased thymidine incorporation in GC with increasing numbers of DO. GDF9 immuno-neutralisation reduced thymidine incorporation in rat GC co-incubated with either rat or ovine DO and in ovine GC co-incubated with ovine or rat DO. BMP15 immuno-neutralisation only reduced thymidine incorporation when ovine DO were co-incubated with either ovine or rat GC. Western blotting of oocytes co-incubated with GC identified GDF9 and BMP15 proteins for sheep and GDF9 protein for rats in oocyte lysates and incubation media. With respect to rat BMP15, a promature protein was identified in the oocyte lysate but not in media. Expression levels of GDF9 relative to BMP15 mRNA in DO co-incubated with GC were highly correlated (R 2=0.99) within both species. However, the expression ratios were markedly different for the rat and sheep (4.3 vs 1.0 respectively). We conclude that during follicular development, rat oocytes secrete little, if any, BMP15 and that GDF9 without BMP15 can stimulate proliferation of rat and ovine GC. In contrast, ovine oocytes secrete both BMP15 and GDF9, and both were found to stimulate proliferation in ovine and rat GC.

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Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function

Reproduction ◽

10.1530/rep.1.0511 ◽

2005 ◽

Vol 129 (4) ◽

pp. 473-480 ◽

Cited By ~ 113

Author(s):

Kenneth P McNatty ◽

Jennifer L Juengel ◽

Karen L Reader ◽

Stan Lun ◽

Samu Myllymaa ◽

...

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Bone Morphogenetic Protein ◽

Granulosa Cells ◽

Thymidine Incorporation ◽

Progesterone Production ◽

Differentiation Factor ◽

Morphogenetic Protein ◽

Growth Differentiation Factor 9 ◽

Bone Morphogenetic Protein 15

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular growth and function. The effects of murine (m) and ovine (o) GDF9 as well as oBMP15, alone or together, on 3H-thymidine uptake and progesterone and inhibin production by granulosa cells from rats were determined. Murine GDF9 stimulated thymidine incorporation by granulosa cells whereas oGDF9 and oBMP15 alone had no effect. However, oBMP15 given together with mGDF9 or oGDF9 was very potent in stimulating 3H-thymidine incorporation by granulosa cells with a greater than 3-fold stimulation compared with any growth factor alone. The synergistic effect of oBMP15 and oGDF9 was almost completely blocked by antibodies generated against these growth factors when administered either alone or in combination. While neither GDF9 (murine or ovine) nor oBMP15 were able to modulate FSH-stimulated progesterone production on their own, FSH-stimulated progesterone production by granulosa cells was potently inhibited when BMP15 and GDF9 were administered together. Immunoreactive α-inhibin levels increased more than 15-fold from granulosa cells when BMP15 and GDF9 were given together whereas consistent stimulatory effects of either growth factor alone were not observed. The effects of GDF9 and BMP15, when added together, were different than those observed for the growth factors alone. Therefore, we hypothesize that within the ovary, these oocyte-secreted growth factors co-operate to regulate proliferation and gonadotropin-induced differentiation of granulosa cells in mammals.

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Species Differences in the Expression and Activity of Bone Morphogenetic Protein 15

Endocrinology ◽

10.1210/en.2012-2015 ◽

2013 ◽

Vol 154 (2) ◽

pp. 888-899 ◽

Cited By ~ 22

Author(s):

Sara L. Al-Musawi ◽

Kelly L. Walton ◽

Derek Heath ◽

Courtney M. Simpson ◽

Craig A. Harrison

Keyword(s):

Growth Factor ◽

Bone Morphogenetic Protein ◽

Species Differences ◽

Site Directed Mutagenesis ◽

Type I ◽

Binding Interface ◽

Morphogenetic Protein ◽

Bone Morphogenetic Protein 15 ◽

Species Specific ◽

Expression And Activity

Oocyte-derived bone morphogenetic protein 15 (BMP15) regulates ovulation rate and female fertility in a species-specific manner, being important in humans and sheep and largely superfluous in mice. To understand these species differences, we have compared the expression and activity of human, murine, and ovine BMP15. In HEK293F cells, human BMP15 is highly expressed (120 ng/ml), ovine BMP15 is poorly expressed (15 ng/ml), and murine BMP15 is undetectable. Because BMP15 synthesis is dependent upon interactions between the N-terminal prodomain and the C-terminal mature domain, we used site-directed mutagenesis to identify four prodomain residues (Glu46, Glu47, Leu49, and Glu50) that mediate the high expression of human BMP15. Substituting these residues into the prodomains of murine and ovine BMP15 led to significant increases in growth factor expression; however, maximal expression was achieved only when the entire human prodomain was linked to the mature domains of the other species. Using these chimeric constructs, we produced and purified murine and ovine BMP15 and showed that in a COV434 granulosa cell bioassay, these molecules displayed little activity relative to human BMP15 (EC50 0.2nM). Sequence analysis suggested that the disparity in activity could be due to species differences at the type I receptor binding interface. Indeed, murine BMP15 activity was restored when specific residues through this region (Pro329/Tyr330) were replaced with the corresponding residues (Arg329/Asp330) from human BMP15. The identified differences in the expression and activity of BMP15 likely underlie the relative importance of this growth factor between species.

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Effects of active immunization against growth differentiation factor 9 and/or bone morphogenetic protein 15 on ovarian function in cattle

Reproduction ◽

10.1530/rep-09-0009 ◽

2009 ◽

Vol 138 (1) ◽

pp. 107-114 ◽

Cited By ~ 37

Author(s):

Jennifer L Juengel ◽

Norma L Hudson ◽

Martin Berg ◽

Keith Hamel ◽

Peter Smith ◽

...

Keyword(s):

Bone Morphogenetic Protein ◽

Ovarian Function ◽

Follicular Development ◽

Active Immunization ◽

Ovulation Rate ◽

Differentiation Factor ◽

Morphogenetic Protein ◽

Growth Differentiation Factor 9 ◽

Bone Morphogenetic Protein 15 ◽

Ovarian Follicular Development

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are essential for ovarian follicular growth in sheep, whereas only GDF9 is essential in mice suggesting that the roles of these oocyte-derived growth factors differ among species. At present, however, there is only limited information on the action of BMP15 and GDF9 in other species. Thus, the aim of this experiment was to determine the effect of neutralizing GDF9 and/or BMP15in vivoon ovarian follicular development and ovulation rate in cattle through active immunization using the mature regions of the proteins or peptides from the N-terminal area of mature regions. Immunization with the BMP15 peptide, with or without GDF9 peptide, significantly altered (increased or decreased) ovulation rate. In some animals, there were no functional corpora lutea (CL), whereas in others up to four CL were observed. From morphometric examination of the ovaries, immunization with GDF9 and/or BMP15 reduced the level of ovarian follicular development as assessed by a reduced proportion of the ovarian section occupied by antral follicles. In addition, immunization against GDF9 and/or BMP15 peptides reduced follicular size to <25% of that in the controls. In conclusion, immunization against GDF9 and BMP15, alone or together, altered follicular development and ovulation rate in cattle. Thus, as has been observed in sheep, both GDF9 and BMP15 appear to be key regulators of normal follicular development and ovulation rate in cattle.

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Efficient generation of bone morphogenetic protein 15-edited Yorkshire pigs using CRISPR/Cas9†

Biology of Reproduction ◽

10.1093/biolre/ioaa138 ◽

2020 ◽

Vol 103 (5) ◽

pp. 1054-1068

Author(s):

Xuan Shi ◽

Tao Tang ◽

Qiyuan Lin ◽

Hongbo Liu ◽

Yufeng Qin ◽

...

Keyword(s):

Bone Morphogenetic Protein ◽

Transforming Growth Factor ◽

Follicular Development ◽

Female Fertility ◽

Efficient Generation ◽

Morphogenetic Protein ◽

Yorkshire Pigs ◽

Bone Morphogenetic Protein 15 ◽

Ovarian Follicular Development

Abstract Bone morphogenetic protein 15 (BMP15), a member of the transforming growth factor beta superfamily, plays an essential role in ovarian follicular development in mono-ovulatory mammalian species. Studies using a biallelic knockout mouse model revealed that BMP15 potentially has just a minimal impact on female fertility and ovarian follicular development in polyovulatory species. In contrast, our previous study demonstrated that in vivo knockdown of BMP15 significantly affected porcine female fertility, as evidenced by the dysplastic ovaries containing significantly decreased numbers of follicles and an increased number of abnormal follicles. This finding implied that BMP15 plays an important role in the regulation of female fertility and ovarian follicular development in polyovulatory species. To further investigate the regulatory role of BMP15 in porcine ovarian and follicular development, here, we describe the efficient generation of BMP15-edited Yorkshire pigs using CRISPR/Cas9. Using artificial insemination experiments, we found that the biallelically edited gilts were all infertile, regardless of different genotypes. One monoallelically edited gilt #4 (Δ66 bp/WT) was fertile and could deliver offspring with a litter size comparable to that of wild-type gilts. Further analysis established that the infertility of biallelically edited gilts was caused by the arrest of follicular development at preantral stages, with formation of numerous structurally abnormal follicles, resulting in streaky ovaries and the absence of obvious estrous cycles. Our results strongly suggest that the role of BMP15 in nonrodent polyovulatory species may be as important as that in mono-ovulatory species.

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Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants

Reproduction ◽

10.1530/rep.1.00517 ◽

2005 ◽

Vol 129 (4) ◽

pp. 481-487 ◽

Cited By ~ 121

Author(s):

Kenneth P McNatty ◽

Jennifer L Juengel ◽

Karen L Reader ◽

Stan Lun ◽

Samu Myllymaa ◽

...

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Bone Morphogenetic Protein ◽

Granulosa Cells ◽

Progesterone Production ◽

Differentiation Factor ◽

Morphogenetic Protein ◽

Growth Differentiation Factor 9 ◽

Bone Morphogenetic Protein 15

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular development and ovulation rate. In addition, it is known from both in vivo and in vitro studies that these factors co-operate in some manner. To date, most studies examining the in vitro effects of these growth factors have used the rodent model. However, the evidence suggests that these growth factors have somewhat different roles between rodents and ruminants. Therefore, the objectives of these studies were to examine the effects of GDF9 and BMP15, alone and together, on the functions of ovine and bovine granulosa cells under in vitro conditions. Ovine (o)BMP15 given together with murine (m)GDF9 or oGDF9 was more potent in stimulating 3H-thymidine incorporation by ovine granulosa cells compared with each growth factor alone. For bovine granulosa cells, there appeared to be little or no co-operativity between oBMP15 and oGDF9 as oBMP15 alone was as potent as any combination of the two growth factors in stimulating 3H-thymidine uptake. The species of origin of GDF9 affected the progesterone response in ovine granulosa cells with mGDF9 stimulating and oGDF9 inhibiting progesterone production. Ovine BMP15 alone had no effect on progesterone production by ovine granulosa cells and these growth factors did not appear to co-operate. FSH-stimulated progesterone production by bovine granulosa cells was most potently inhibited when oBMP15 and murine or ovine GDF9 were administered together. As was observed for progesterone, the species of origin of GDF9 affected inhibin production by ovine granulosa cells where mGDF9 inhibited while oGDF9 stimulated production. Murine GDF9 also inhibited inhibin production from bovine granulosa cells. For both ovine and bovine granulosa cells, BMP15 alone had no effect on inhibin production and there did not appear to be any co-operation between GDF9 and BMP15. These results indicate that the effects of BMP15 and GDF9 varied with respect to the species of origin of the growth factor. Moreover, the effects of GDF9 and BMP15 together were often co-operative and not always the same as those observed for these growth factors alone.

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Development of an in Vitro Assay to Assess Gap Junction Activity in Cumulus-Oocyte Complexes (COC) in the Rat

10.26686/wgtn.16967377.v1 ◽

2021 ◽

Author(s):

◽

Shruti Patel

Keyword(s):

Growth Factors ◽

Gap Junctions ◽

Gap Junction ◽

Oocyte Maturation ◽

Follicular Development ◽

Cumulus Cells ◽

Dye Transfer ◽

Antral Follicles ◽

Fluorescence Dye

<p>The capacity of an oocyte to mature during ovarian follicular development is a key process in reproductive biology. Bidirectional communication between mammalian oocytes and their associated follicular somatic cells (cumulus-cells) is essential for oocyte maturation. Historically, studies examining the control of ovarian follicular development focused mainly on the endocrine (external) signalling but recently intraovarian (paracrine) regulation has also been shown to be important. In addition, signalling via gap junctions between follicular cells had also been crucial for oocyte maturation and follicular development. In antral follicles, gap junction activity between the oocyte and adjacent cumulus cells first increase during follicular growth and shortly before ovulation they decrease as the oocyte resumes meiosis once more before ovulation. The range of factors that modulate gap junction activity of oocyte-cumulus cell complexes (COC) is largely unknown. The aims of these studies were to develop an assay to assess the rate of transfer of low molecular weight materials from cumulus cells to the oocyte via gap junctions. The first objective was to validate a bioassay by which to test the effects of hormones, second messengers, and growth factors on gap junction activity in rat cumulus-oocyte complexes. In this study, COCs were collected from antral follicles of untreated post-pubertal Sprague Dawley rats. Gap junction activity was measured in the presence or absence of different treatments using the fluorescence dye, Calcein-AM and in the presence of a phosphodiesterase type 3 inhibitor (PDE3) milrinone. Transfer of the calcein dye from cumulus cells into the oocyte was measured at various times using CRAIC fluorescence system. The results showed that removal of the COCs from their follicular environments disrupted the gap junction activity which recovered over time in culture media. COC were sensitive to changes in pH concentration and gap junction activity could be blocked with 8 ocatnol-1 but not carbenoxolone. Treating rat COCs with dibutyryl cAMP or agents that maintained or increased intracellular cAMP levels like milrinone or forskolin were unable to modulate gap junction activity. Further, the combined effect of the oocyte-derived growth factors: growth differentiating factor 9 (GDF9) with bone morphogenetic protein 15 (BMP15) was also unable to modulate the rate of calcein dye transfer from cumulus cells to the oocyte. Ovarian steroids such as oestradiol and testosterone by themselves were unable to modulate the gap junction activity of rat COC but the combined treatment of testosterone plus forskolin or testosterone plus forskolin plus insulin-like growth factor 1 (IGF-1) increased the rate of dye transfer from cumulus cells to the oocyte. In conclusion, a fluorescence dye transfer assay was developed to measure the effects of different treatments on gap junction activity in rat COC. Under in vitro conditions, it was established that the combination of steroid and cAMP stimulators or a steroid, cAMP stimulator with IGF1 but not these reagents individually could enhance the recovery of gap junction function in rat COC. The outcomes of these experiments may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes. Also, the newly developed assay may serve as a useful in vitro model to evaluate the effects of hormones, nutritional supplements and other factors on COC functions.</p>

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Genes controlling ovulation rate in sheep

Reproduction ◽

10.1530/rep.0.1210843 ◽

2001 ◽

pp. 843-852 ◽

Cited By ~ 72

Author(s):

GW Montgomery ◽

SM Galloway ◽

GH Davis ◽

KP McNatty

Keyword(s):

New Zealand ◽

Recent Progress ◽

Follicular Development ◽

Ovulation Rate ◽

Morphogenetic Protein ◽

Speed Up ◽

Follicular Size ◽

Heterozygous Carriers ◽

Bone Morphogenetic Protein 15 ◽

Sites Of Action

Sheep provide a valuable model for studying the genetic control of ovulation rate. Recent progress includes the identification of mutations in BMP15 (bone morphogenetic protein 15) that increase ovulation rate in heterozygous carriers and block follicular development in homozygous carriers. The genes characterized to date appear to act principally within the ovary and result in earlier maturity of granulosa cells and reduced follicular size. There may also be other sites of action, and increased FSH concentrations appear to be important in the expression of the FecB phenotype. A new locus on the X chromosome in New Zealand Coopworth sheep increases ovulation rate by about 0.4 and is maternally imprinted. Results from studies in the Cambridge and Belclare breeds indicate that further genes remain to be characterized. Finding the first mutations leading directly to variation in ovulation rate is likely to speed up the identification and molecular analysis of these other genes. There is still much to learn about follicular development and the control of litter size from genetic models in sheep.

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