P. Matton

CHANGES IN MORPHOLOGICAL APPEARANCE AND FUNCTIONAL CAPACITY OF RECRUITED FOLLICLES IN COWS TREATED WITH FSH IN THE PRESENCE OR ABSENCE OF A DOMINANT FOLLICLE

This study was designed to determine the effect of the presence of a dominant follicle at the beginning of FSH stimulation on the morphological appearance and functional capacity of recruited follicles during FSH stimulation in cattle. Synchronized nonlactating dairy cows were assigned to 1 of 2 groups and treated with FSH in the presence (n = 5) or absence (n = 6) of a dominant follicle between Days 7 and 12 of the estrous cycle (Day 0 = estrus) to stimulate follicular growth. Dominant follicles were identified by daily ultrasonographic observations, beginning on Day 3 of the estrous cycle. Dominant follicle had an ultrasonographic diameter > or = 10 mm and were in a growing phase, or maintaining a constant diameter (> or = 10 mm) for less than 4 d. Ovaries were collected at slaughter on the morning of the third day following initiation of the FSH stimulation. All follicles > 2 mm were dissected, classified according to diameter (Class 1: 2 to 4.4 mm; Class 2: 4.5 to 7.9 mm; Class 3: > 8 mm), and incubated individually for 90 min in medium M-199 (37 degrees C, 5% CO2). Following incubation, integrity of each follicle was evaluated histologically to assess the level of atresia and biochemically to determine the in vitro release of estradiol (E2) and androstenedione in culture media. On Day 3 of the FSH treatment, mean number of follicles in each class was similar (P > 0.1) between the 2 groups. The percentage of atretic follicles in Classes 1 and 3 on Day 3 of the FSH stimulation did not differ (P > 0.1) between the 2 groups. However, the percentage of atretic follicles in Class 2 was higher (P < 0.005) in cows treated with FSH in presence than in absence of a dominant follicle (60.8 vs 38.2%). The release of E2 in culture media by small Class 1 atretic or healthy follicles, by Class 2 atretic and by Class 3 healthy follicles was not affected (P > 0.1) by the ovarian status. However (P < 0.001), the release of E2 in culture media of Class 2 healthy and Class 3 atretic follicles was less for follicles harvested from cows bearing than from those not bearing a dominant follicle. Within each follicular class, concentrations of androstenedione in the culture media did not differ between the 2 groups (P > 0.1). These results suggest that the presence of a dominant follicle at the beginning of FSH stimulation alters the population of follicles recruited FSH stimulation. This may be associated with the reported decrease of the superovulatory response in cows superovulated in presence of a dominant follicle.

Steroid production, cell proliferation, and apoptosis in cultured bovine antral and mural granulosa cells: Development of an in vitro model to study estradiol production

This study was undertaken to characterize the relationship between changes in steroid production, cell cycle activity (ie, cell proliferation) and apoptosis in antral and mural bovine granulosa cells cultured in vitro. This was done to select conditions promoting optimal estradiol production by bovine granulosa cells cultured in completely defined conditions. In the first experiment, antral granulosa cells were cultured over the entire 4 days of the culture period in the presence of either 0, 2, or 10 ng/ml of FSH (chronic conditions) or were maintained under minimal FSH support (0.5 ng/ml FSH) for the first 3 days of culture and then were challenged over the fourth day of culture with either 0, 2, or 10 ng/ml FSH (challenged conditions). Compared with cells exposed to constant FSH levels (chronic conditions), the FSH‐induced production of estradiol was higher (P < 0.006) and that of progesterone was lower (P < 0.02) over the last 24 h of culture, when antral granulosa cells were maintained under minimal FSH support during the first 3 days of culture (challenged conditions). In the second experiment, dynamics of estradiol and progesterone productions, conversion of [14C]androstenedione into subsequent steroid metabolites, DNA content, cell cycle activity, and apoptosis (as assessed by flow cytometry) of antral and mural granulosa cells over the first 3 days of culture under minimal FSH support and in response to a challenge with FSH during the last 24 h of culture were evaluated. Estradiol production as well as the conversion of androstenedione into testosterone and estradiol were greater (P< 0.01) in antral than in mural granulosa cells cultured under challenged conditions. A higher proportion of mural than antral granulosa cells were in the proliferative state at the end of culture (P < 0.03). This may be related to the decreased ability of mural cells to produce estradiol. FSH suppressed (P < 0.05) the spontaneous onset of apoptosis in both cell types. These results suggest that functional differences between these two cell compartments need to be considered in studying bovine granulosa cells in vitro. Because of their large (400 to 600%) FSH‐induced estradiol production, antral granulosa cells cultured under challenged conditions provide a model that can be used to examine substances for their ability to alter estradiol production and apoptosis in bovine granulosa cells. Mol. Reprod. Dev. 50:170–177, 1998.

Plasma LH and androgen levels in the red-winged blackbird (Agelaius phoeniceus) treated with a potent GnRH analogue

1. Low doses of GnRH-A (0.01-0.10-1.0 micrograms) given during the annual testes growth period did not clearly affect plasma LH and androgen levels 10 min following the injection. 2. The first injection of high doses of GnRH-A (2.0-10.0-20.0 micrograms) markedly increased plasma LH and androgen levels measured 10 min following the injection. The increase in plasma LH level was dose-dependent and the maximal LH level was obtained with 10.0 micrograms of GnRH-A. 3. Impairment of the LH response to GnRH-A was assessed by comparing the first and the fourteenth injection of high doses of GnRH-A. Evidences of pituitary gland desensitization are reported since plasma LH levels were reduced following the fourteenth injection in all groups. 4. Plasma androgen levels following high doses of GnRH-A were not clearly affected in red-winged blackbirds.

Pituitary LH content and plasma LH levels following daily GnRH analogue treatment in male red-winged blackbirds (Agelaius phoeniceus)

1. During the 14-day treatment period, plasma LH levels following GnRH analogue (GnRH-A) injections (10.0 micrograms) were significantly reduced after the 6th and the 14th injection. 2. One day post-treatment, the LH pituitary content was significantly reduced in GnRH-A-treated redwings compared to saline-injected controls. 3. Pituitary LH content was significantly higher in GnRH-A treated birds compared to control birds 14 and 28 days post-treatment and plasma LH levels were similar in both groups. 4. Hypersecretion of LH following GnRH-A injections appears to reduce pituitary LH content, acting as a stimulus for its synthesis. 5. These results suggest a higher LH synthesis and storage in the pituitary gland of the GnRH-A-treated birds compared to the control birds during the post-treatment period.