Search

Bone morphogenetic protein 6 (BMP6) has been suggested as an important local factor capable of modulating the stimulatory actions of follicle-stimulating hormone in granulosa cells in vitro. The objective of this experiment was to determine the effect of direct ovarian infusion of BMP6 (2 microg/h) on ovarian function in ewes with an autotransplanted ovary. Treated ewes (n = 6) and vehicle-treated controls (n = 6) were infused during the early follicular phase, between 12 and 24 h after luteal regression, and ovarian response was determined by collection of samples of ovarian venous blood and transdermal ultrasound. In the absence of any change in circulating gonadotropins or in the antral follicle population, BMP6 infusion resulted in acute but transient increases in ovarian inhibin A, androstenedione, and estradiol secretion (P < 0.05) during the second half of the infusion period. Thereafter, treated animals had an advance in the time of the LH surge by around 10 h (43.3 +/- 2.8 h in treated vs. 53.3 +/- 2.7 h in controls; P < 0.05) and smaller preovulatory follicles (4.1 +/- 0.2 mm in treated vs. 5.3 +/- 0.1 mm in controls; P < 0.01), which gave rise to smaller corpora lutea (9.5 +/- 0.8 mm in treated vs. 11.7 +/- 0.6 mm in controls; P < 0.05). There was, however, no effect of infusion on ovulation rate. Despite the changes in the size of the ovulatory follicles, when the hormonal data were aligned to the time of the luteinizing hormone surge, there were no differences in preovulatory estradiol, androstenedione, or inhibin A between groups. This study therefore provides strong in vivo evidence to support the hypothesis that BMP6 is an important local regulator of ovarian function and that alterations in BMP6 cellular signaling may explain some of the effects of the FecB mutation in inducing precocious maturation of ovulatory follicles.

The aim of this study was to examine the role of LH on the growth of the large preovulatory follicle and its secretion of hormones in sheep. Ewes with ovarian autotransplants were treated with GnRH-antagonist at the time of luteal regression and different LH regimes applied for 60-66 h before administration of an ovulatory stimulus (hCG). In Experiment 1 (N = 24; n = 8), ewes received either no LH or constant or pulsatile infusion of LH at the same dose (1.25 microg/h). In Experiment 2 (N = 12, n = 6), LH was constantly infused at a rate of 1.25 microg or 2.5 microg oLH/h. In Experiment 1, animals receiving either pulsatile or constant LH exhibited increases in estradiol and inhibin A secretion (P < 0.001) and a depression in FSH (P < 0.001) that resembled the normal follicular phase. Similarly in Experiment 2, doubling the dose of LH resulted in a two-fold increase in ovarian estradiol secretion (P < 0.05) but no other changes. All animals receiving LH, regardless of the pattern of stimulation, ovulated and established a normal luteal phase. In contrast, no LH treatment resulted in constant immuno-active LH without pulses, unchanged FSH and inhibin A concentrations (P < 0.05), and basal estradiol secretion (P < 0.001). Morphologically normal large antral follicles were observed in this group and although corpora lutea formed in response to hCG, progesterone profiles were abnormal. In conclusion, these results suggest that LH is an essential requirement for normal ovulatory follicle development and subsequent luteal function and show that a pulsatile mode of LH stimulation is not required by ovulatory follicles.

The objectives of this study were to develop a serum-free bovine granulosa cell culture system in which FSH-responsive estradiol production could be induced and maintained, and to use this system to evaluate the effects of FSH, insulin, and IGF-I on steroidogenesis and proliferation of bovine granulosa cells from different follicle size categories (< 4-, 4-8, and > 8-mm diameter). In the presence of FSH, granulosa cells from small follicles differentiated in vitro, and estradiol secretion increased with time (p < 0.01) so that by the end of the culture period it was similar to that of cells from large follicles. Granulosa cells from medium and large follicles secreted estradiol throughout the culture period. Cells cultured in plasma-coated culture wells had an increased proliferative response but had lower estradiol production compared to cells cultured under serum-free conditions (p < 0.01). Insulin promoted proliferation and estradiol production by granulosa cells from the three follicle-size categories (p < 0.01). Physiological concentrations of FSH induced proliferation and estradiol secretion (p < 0.01) by granulosa cells in a dose-responsive manner. The inclusion of IGF-I in the culture system enhanced proliferation and estradiol production (p < 0.01), even in the absence of gonadotropic support, demonstrating the gonadotropic characteristics of this growth factor. These results demonstrate the development of a relevant physiological culture system for bovine granulosa cells. This system will permit the detailed study of the key factors controlling the differentiation and proliferation of bovine granulosa cells.

A novel experimental model was developed in cattle to investigate the requirement for FSH and LH during ovarian follicle growth and development. On Day 5 of the estrous cycle, 7 heifers were each implanted with an osmotic minipump containing a GnRH agonist (GnRHa), Buserelin (release rate, 2.5 micrograms/h). Another 7 heifers served as controls. Each minipump was replaced 28 days later with a second pump, which was left in place for a further 20 days. Blood samples were collected daily throughout the experimental period, and frequent samples were also collected on both days of minipump insertion and at 10 days after insertion of the second pump. The ovaries of all heifers were scanned daily by real-time ultrasonography to monitor follicular dynamics. All controls displayed 2 or 3 waves of FSH and follicular development per estrous cycle during the experiment. Insertion of the first minipump produced a large LH and FSH surge and induced ovulation in all 7 animals. Within 8 days of the start of treatment, serum LH concentrations fell to basal levels; they then remained constant at this level throughout the infusion period, only beginning to recover 4-5 days after the termination of infusion. After the initial increase, FSH returned to basal levels before showing a normal wave that was coincident with the emergence, growth, and regression of a dominant follicle. However, despite the peak levels of FSH, dominant follicles from the next wave failed to grow beyond 7-9 mm; they remained at this size for 3 wk until 3-4 days after insertion of the second minipump, when FSH fell precipitously to reach low levels that were maintained throughout the remainder of the infusion. After this fall in FSH concentrations, these follicles regressed rapidly, and no antral follicles > 4 mm were detected until after the termination of treatment. Thereafter, FSH concentrations increased significantly; the increase was accompanied by the emergence of a follicular wave and development of a dominant follicle, with estrus observed 8-11 days later. In conclusion, this study has demonstrated clearly that in cattle the early stages of follicle development (< or = 4 mm) are not dependent on acute support by gonadotropins. However, FSH is required for further growth of follicles up to 9 mm, while LH pulses are indispensable for follicle development beyond 9 mm in diameter. The model developed in this study should be valuable for studying the control of ovarian follicle development and atresia in vivo.