Effects of Steroid Hormone in Avian Follicles

Abstract

The aim of the present study was to examine the effects of testosterone (T) and estradiol-17β (E2) on the production of progesterone (P4) by granulosa cells, and of the E2 on the production of P4 and T by theca internal cells. In the first experiment, granulosa cells isolated from the largest (F1) and third largest (F3) preovulatory follicle were incubated for 4 h in short-term culture system, P4 production by granulosa cells of both F1 and F3 was increased in a dose-dependent manner by ovine luteinizing hormone (oLH), but not T or E2. In the second experiment, F1 and F3 granulosa cells cultured for 48 h in the developed monolayer culture system were recultured for an additional 48 h with increasing doses of various physiological active substances existing in the ovary, including T and E2. Basal P4 production for 48 h during 48 to 96 h of the cultured was about nine fold greater by F1 granulosa cells than by F3 granulosa cells. In substances examined oLH, chicken vasoactive intestinal polypeptide (cVIP) and T, but not E2, stimulated in a dose-dependent manner P4 production in both F1 and F3 granulosa cells. In addition, when the time course of P4 production by F1 granulosa cells in response to oLH, cVIP, T and E2 was examined for 48 h during 48 to 96 h of culture, although E2 had no effect on P4 production by granulosa cells of F1 during the period from 48 to 96 h of culture, P4 production with oLH was found to be increased at 4 h of the culture, with a maximal 9.14 fold level at 6 h. By contrast, P4 production with cVIP and T increased significantly (p<0.05) from 8 and 12 h of the culture, respectively, with maximal 6.50 fold response at 12 h and 6, 48 fold responses at 36 h. Furthermore, when F1 granulosa cells were precultured with E2 for various times before 4 h culture with oLH at 96 h of culture, the increase in P4 production in response to oLH with a dose-related manner was only found at a pretreatment time of more than 12 h. In the third experiment, theca internal cells of F1, F2 and the largest third to fifth preovulatory follicles (F3-5) were incubated for 4 h in short-term culture system with increasing doses of E2. The production of P4 and T by theca internal cells were increased with the addition of E2 of 10(-6) M. These increases were greater in smaller follicles. These results indicate that, in granulosa cells of the hen, T may have a direct stimulatory action in the long term on P4 production, and on E2 in long-term action which may enhance the sensitivity to LH for P4 production, and thus, in theca internal cells, E2 in short term action may stimulate the production of P4 and T.

Keywords: Granulosa; Preovulatory Follicles; Steroids Hormone; Theca Internal and External Cells.

Figure 1

Progesterone production by granulosa cells of the largest and third largest preovulatory follicles cultured with ovine LH for 4 h in short-term culture system. LH, luteinizing hormone.

Figure 2

Progesterone production by granulose cells of the largest and third largest preovulatory follicles incubated with testosterone or estradiol-17β for 4 h in short-term culture system.

Figure 3

Morphological appearance (×100) of hen granulosa cells cultured for 96 h in serum-free medium and serum-containing medium.

Figure 4

Cell proliferation and progesterone production of granulosa cells cultured in serum-free medium and serum-containing medium and their progesterone production.

Figure 5

Basal and LH-stimulated progesterone production by granulosa cells cultured in serum-free medium and serum-containing medium. LH, luteinizing hormone.

Figure 6

Progesterone production by granulosa cells of the largest and third largest preovulatory follicles cultured with ovine LH or FSH for 48 h in monolayer culture system. LH, luteinizing hormone; FSH, follicle stimulating hormone.

Figure 7

Progesterone production by granulosa cells of the largest and third largest preovulatory follicles cultured with various physiological substances for 48 h in monolayer system. cVIP, chicken vasoactive intestinal peptide; AVT, arginine vasotocin; MT, mesotocin testosterone; T, testosterone; E2, estradiol-17β; E, epinephrine; NE, norepinephrine; PGE₁, prostaglandin E₁; PGF2α, prostaglandin F₂α.

Figure 8

Time-course of progesterone production by granulosa cells of the largest preovulatory follicle with oLH, cVIP, testosterone and estradiol-17β. oLH, ovine luteinizing hormone; cVIP, chicken vasoactive intestinal peptide.

Figure 9

Effect of pretreatment with estradiol-17β on LH-stimulated progesterone production. LH, luteinizing hormone.

Figure 10

Time dependence for the enhancing effect of estradiol–17β on LH-stimulated progesterone production. LH, luteinizing hormone.

Figure 11

Progesterone production by theca interna cells of the largest, second and third-fifth preovulatory follicles incubated with estradiol-17β for 4 h.

Figure 12

Testosterone production by theca interna cells of the largest, second and third-fifth preovulatory follicles incubated with estradiol-17β for 4 h.

Figure 13

Scheme proposed by the interaction of gonadotropins and steroids hormones in ovarian follicles of domestic fowl.