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

Abstract

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 BMP15 in vivo on 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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Effects of Active Immunization Against Growth Differentiation Factor 9 (GDF9) and/or
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Bone Morphogenetic Protein 15 (BMP15) on Ovarian Function in Cattle.
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Short title: BMP15 and GDF9 in bovine follicular growth
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Jennifer L. Juengel1,*, Norma L. Hudson1,3, Martin Berg2, Keith Hamel1, Peter Smith1, Stephen 9
B. Lawrence1, Lynda Whiting1, & Kenneth P. McNatty1,3 10
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Wallaceville Animal Research Centre1, AgResearch, Upper Hutt 5140, New Zealand
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Reproductive Technologies2, Ruakura Research Centre, AgResearch, Hamilton 3214, New
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Zealand
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*Correspondence: Jenny Juengel, AgResearch Ltd, Invermay Agricultural Centre,
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Puddle Alley, Private Bag 50034, Mosgiel, New Zealand 9053. Tel +64-3-489 9137
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Fax +64-3-489 3739, email jenny.juengel@agresearch.co.nz
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3Current Address: School of Biological Sciences, Victoria University of Wellington, PO Box 600,
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Wellington, New Zealand
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Reproduction Advance Publication first posted on 13 May 2009 as Manuscript REP-09-0009

Copyright © 2009 by the Society for Reproduction and Fertility.

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ABSTRACT
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Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 25
(BMP15) are essential for ovarian follicular growth in sheep whereas only GDF9 is 26
essential in mice suggesting that the roles of these oocyte-derived growth factors 27
differ among species. At present however there is only limited information on the 28
action of BMP15 and GDF9 in other species. Thus, the aim of this experiment was to 29
determine the effect of neutralizing GDF9 and/or BMP15 in vivo on ovarian follicular 30
development and ovulation rate in cattle through active immunization using the 31
mature regions of the proteins or peptides from the N-terminal area of mature regions. 32
Immunization with the BMP15 peptide, with or without GDF9 peptide, significantly 33
altered (increased or decreased) ovulation rate. In some animals, there were no 34
functional corpora lutea (CL) whereas in others up to 4 CL were observed. From 35
morphometric examination of the ovaries, immunization with GDF9 and/or BMP15 36
reduced the level of ovarian follicular development as assessed by a reduced 37
proportion of the ovarian section occupied by antral follicles. In addition, 38
immunization against GDF9 and/or BMP15 peptides reduced follicular size to <25% 39
of that in the controls. In conclusion, immunization against GDF9 and BMP15, alone 40
or together, altered follicular development and ovulation rate in cattle. Thus, as has 41
been observed in sheep, both GDF9 and BMP15 appear to be key regulators of normal 42
follicular development and ovulation rate in cattle.
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INTRODUCTION 44
The use of sheep with naturally occurring mutations in the genes for the 45
related oocyte-derived growth factors bone morphogenetic protein 15 (BMP15) and 46
growth differentiation factor 9 (GDF9) have demonstrated their essential role in 47
regulating ovarian follicular growth and ovulation rate (Galloway et al. 2000; 48
Hanrahan et al. 2004; McNatty et al. 2005b). In mice, the use of “knock-out” models 49
has shown that GDF9 is essential for normal follicular development with BMP15 50
regulating the fertilization potential of oocytes (Chang et al. 2002; Dong et al. 1996; 51
Yan et al. 2001). However, the role of either of these growth factors in regulating 52
ovulation rate in mice is equivocal. Recently, naturally-occurring mutations in either 53
GDF9 or BMP15 in humans have been linked to infertility, including primary 54
amenorrhea (Di Pasquale et al. 2004; Di Pasquale et al. 2006; Dixit et al. 2005; Dixit
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et al. 2006; Kovanci et al. 2007; Laissue et al. 2006), and for GDF9, to increased 56
dizygotic twinning (Palmer et al. 2006). Thus, one or both of these growth factors 57
likely play a critical role in ovarian follicular development in mammals although the 58
absolute requirement and specific roles of the growth factors vary between species, 59
potentially related to the ovulation rate quota of a particular species (Moore et al. 60
2004). 61
Sheep that are homozygous for inactivating mutation of BMP15 or GDF9 are 62
infertile with primary ovarian failure (McNatty et al. 2005b). However, those 63
heterozygous for inactivating mutations of either BMP15 or GDF9 are fertile, with 64
increased ovulation rates (McNatty et al. 2003). This rather unusual phenotype is 65
related to function of GDF9 and BMP15 in co-operatively regulating granulosa cell 66
proliferation and differentiation (McNatty et al. 2005a). Proliferation of granulosa 67
cells of ewes lacking any active BMP15 or GDF9 is insufficient to support the 68
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development of the follicle to ovulation (Juengel et al. 2004a). In contrast, with one 69
gene inactivated, it is postulated the reduced bioactivity of the growth factors leads to 70
the development of follicles that mature earlier in follicular growth than normal with 71
fewer granulosa cells (Juengel et al. 2004a). As FSH concentrations during the 72
follicular phase are regulated by the products from these granulosa cells, namely 73
estradiol and inhibin, more follicles develop LH receptors on the granulosa cells and 74
thus advance to a stage that they can survive in the low FSH environment that is 75
induced through the actions of the granulosa cells of the maturing follicles. In this 76
manner, a greater number of follicles ovulate at each reproductive cycle (Juengel et al. 77
2004a). 78
This effect on ovulation rate can be mimicked in sheep by artificially 79
manipulating their levels of BMP15 or GDF9 through active immunization (Juengel et
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al. 2002; Juengel et al. 2004b; McNatty et al. 2007). Immunization regimens 81
designed to provide high concentrations of antibodies that will neutralize BMP15 or 82
GDF9 result in a cessation of reproductive cycles with the development of follicles 83
altered from a very early stage of development (Juengel et al. 2002). In contrast, by 84
selecting an immunization regime to produce a lesser antibody response, enhanced 85
ovulation rates can be achieved, with individual animals ovulating as many as 10 86
oocytes (Juengel et al. 2002). Thus, active immunization of animals with GDF9 87
and/or BMP15 is a very useful tool for assessing the potential roles of these growth 88
factors on reproductive function in vivo. 89
While GDF9 and BMP15 have both been shown to regulate granulosa and/or 90
theca function in vitro (McNatty et al. 2005a; Spicer et al. 2006; Spicer et al. 2008), 91
the role of GDF9 and BMP15 in regulating ovulation rate or ovarian follicular 92
development in vivo in cattle is unknown. As with sheep and humans, cattle are a 93
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species with a low ovulation rate phenotype, thus if the ovulation rate quota 94
hypothesis was correct, both GDF9 and BMP15 would be expected to be essential for 95
the regulation of follicular growth and ovulation rate. Therefore, the objectives of this 96
experiment in cattle was to explore the role of GDF9 and BMP15 in regulation of 97
ovarian follicular growth and ovulation rate using active immunization to modulate 98
the bioactivity of the growth factors. 99
MATERIALS AND METHODS
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All experiments were performed in accordance with the 1999 Animal Welfare 101
Act Regulations of New Zealand. Except where indicated, laboratory chemicals were 102
obtained from BDH Chemicals New Zealand Ltd (Palmerston North, New Zealand), 103
or Sigma-Aldrich New Zealand Ltd. (Auckland, New Zealand). 104
Immunizations and tissue collection
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Post-pubertal Holstein cross-bred heifers (weight 394 + 3 kg) were allotted to
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treatment group by weight such that the average weight of each group was similar. 107
All animals had normal ovarian morphology at the start of the experiment as assessed 108
by ultrasound. Heifers (n=10 per group) were immunized 3 times at 4 weekly 109
intervals using a Freund’s complete adjuvant for the primary immunization and a 110
Span-Tween-Marcol adjuvant for the booster immunizations. Treatment groups 111
consisted of Keyhole-Limpet Hemocyanin (KLH; control; 0.4 mg antigen primary, 112
0.2 mg antigen boosters), E. coli expressed ovine GDF9 mature region conjugated to 113
KLH (0.8 mg antigen primary, 0.4 mg antigen boosters), E. coli expressed ovine 114
BMP15 mature region conjugated to KLH (0.8 mg antigen primary, 0.4 mg antigen 115
boosters), GDF9 peptide (amino acids 1-14 of the mature region of the bovine protein; 116
0.4 mg antigen primary, 0.2 mg antigen boosters) conjugated to KLH, BMP15 peptide 117
(amino acids 1- 14 of the mature region of the bovine protein; 0.4 mg antigen primary, 118
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