Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep
Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep
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DOI：
10.1095/biolreprod.102.007146
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发表时间：
2002-12-01
影响因子：
3.6
通讯作者：
McNatty, KP
中科院分区：
文献类型：
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作者：
Juengel, JL;Hudson, NL;McNatty, KP
本研究的目的是验证生长分化因子9(GDF9)和骨形态发生蛋白(BMP15；也称为GDF9B)对于排卵率低的哺乳动物的正常卵泡发育是必不可少的假说。将9-10只绵羊(每组9-10只)分别用KLHGDF9特异性多肽(GDF9多肽)、BMP15专一性BMP15多肽(BMP15多肽)或oBMP15成熟部位(oBMP15成熟蛋白)免疫绵羊7mo，观察这些处理对卵泡发育、排卵率和血浆孕酮浓度等卵巢参数的影响。另外，在本研究中，我们用免疫组织化学方法检测了GDF9和BMP15蛋白在羔羊卵巢中的细胞定位。GDF9和BMP15蛋白都专门定位于卵泡和卵母细胞内，从而确立了绵羊卵母细胞是这些生长因子的唯一来源。在收集卵巢时，GDF9和BMP15多肽免疫分别导致10只母羊中的7只和9只母羊无排卵。大多数(7/10)用oBMP15成熟蛋白免疫的母羊在试验期内至少有一次发情，且该发情期的排卵率高于单独免疫KLH的母羊。在KLH-GDF9多肽和KLH-BMP15多肽处理的母羊中，恢复时(即类似于初次免疫后7个月)的卵巢组织学检查显示，大多数动物在发育的主要阶段(即类型2)之外几乎没有正常卵泡。此外，卵母细胞增大，周围有单层扁平和/或立方的颗粒细胞或无卵母细胞的颗粒细胞结节等异常现象，在无排卵母羊中尤为明显。被动免疫母羊，在诱导黄体退化前4天，每只绵羊给予100毫升GDF9多肽或BMP15多肽免疫的血浆池，也扰乱了卵巢功能。给予抗GDF9多肽的血浆的母羊形成了1-2个黄体，但5只动物中有3只没有表现出正常的黄体孕酮浓度模式。血浆对BMP15多肽的影响更为显著，5只动物中有4只无法排卵，5只母羊中有3只在腹腔镜下缺乏表面可见的有腔卵泡。相比之下，注射抗KLH的血浆并不影响任何动物的排卵率或黄体功能。总之，这些发现支持一种假设，即在排卵率低的哺乳动物中，卵母细胞来源的GDF9和BMP15蛋白对于正常的卵泡发育是必不可少的，包括生长的早期和后期。
The aim of this study was to test the hypothesis that both growth differential factor 9 (GDF9) and bone morphogenetic protein (BMP15; also known as GDF9B) are essential for normal ovarian follicular development in mammals with a low ovulation rate phenotype. Sheep (9-10 per group) were immunized with keyhole limpet hemocyanin (KLH; control), a GDF9-specific peptide conjugated to KLH (GDF9 peptide), a BMP15-specific peptide conjugated to KLH (BMP15 peptide), or the mature region of oBMP15 conjugated to KLH (oBMP15 mature protein) for a period of 7 mo and the effects of these treatments on various ovarian parameters such as ovarian follicular development, ovulation rate, and plasma progesterone concentrations evaluated. Also in the present study, we examined, by immunohistochemistry, the cellular localizations of GDF9 and BMP15 proteins in the ovaries of lambs. Both GDF9 and BMP15 proteins were localized specifically within ovarian follicles to the oocyte, thereby establishing for the sheep that the oocyte is the only intraovarian source of these growth factors. immunization with either GDF9 peptide or BMP15 peptide caused anovulation in 7 of 10 and 9 of 10 ewes, respectively, when assessed at ovarian collection. Most ewes (7 of 10) immunized with oBMP15 mature protein had a least one observable estrus during the experimental period, and ovulation rate at this estrus was higher in these ewes compared with those immunized with KLH alone. In both the KLH-GDF9 peptide- and KLH-BMP15 peptide-treated ewes, histological examination of the ovaries at recovery (i.e., similar to7 mo after the primary immunization) showed that most animals had few, if any, normal follicles beyond the primary (i.e., type 2) stage of development. in addition, abnormalities such as enlarged oocytes surrounded by a single layer of flattened and/or cuboidal granulosa cells or oocyte-free nodules of granulosa cells were often observed, especially in the anovulatory ewes. Passive immunization of ewes, each given 100 ml of a pool of plasma from the GDF9 peptide- or BMP15 peptide-immunized ewes at 4 days before induction of luteal regression also disrupted ovarian function. The ewes given the plasma against the GDF9 peptide formed 1-2 corpora lutea but 3 of 5 animals did not display normal luteal phase patterns of progesterone concentrations. The effect of plasma against the BMP15 peptide was more dramatic, with 4 of 5 animals failing to ovulate and 3 of 5 ewes lacking surface-visible antral follicles at laparoscopy. By contrast, administration of plasma against KLH did not affect ovulation rate or luteal function in any animal. In conclusion, these findings support the hypothesis that, in mammals with a low ovulation rate phenotype, both oocyte-derived GDF9 and BMP15 proteins are essential for normal follicular development, including both the early and later stages of growth.