Reproduction in hens: is testosterone necessary for the ovulatory process?

Avian reproduction entails complex endocrine interactions at the hypothalamic and ovarian levels. The initiation of the reproductive season is due to the reduction in melatonin and GnIH production as day length increases. The decline in GnIH permits GnRH and gonadotropin secretion starting follicle growth. Follicular steroids stimulate sexual activity and have important roles for the induction of ovulation. Progesterone (P4) is an inductor of the preovulatory surge of LH, while estradiol (E2) acts as a hypothalamic primer to allow P4 receptor development, as well as a stimulator of yolk production. Conversely, the role of testosterone (T) has been more controversial; however, there is now enough evidence, which demonstrates an essential action of T in the ovulatory process. For instance, blockage of endogenous T, by passive or active immunization or by the use of a specific antagonist of T, inhibits ovulation and the preovulatory surges of P4 and LH. This information is supported by the fact that there is a positive correlation between the occurrences of the T preovulatory surge and those of P4 and LH, in which the absence of T caused a lack of P4 and LH increase in almost 90% of the cases. Additionally, it has been observed that T has a paracrine action within the ovary, to promote P4 secretion by granulosa cells from the larger follicles. This has been related with an increased mRNA expression of StAR and P450scc enzymes, which are essential for P4 production, as well as with LH-R mRNA expression in granulosa cells of preovulatory follicles, an effect that should enhance the positive feedback between P4 and LH necessary for ovulation. Lastly, endocrine activity of hierarchical follicles occurs as a result of a complex interaction between the larger follicles (F1-F3) and the smaller follicles (F4-F6), which is necessary to achieve an adequate preovulatory milieu.

Testosterone stimulates progesterone production and STAR, P450 cholesterol side-chain cleavage and LH receptor mRNAs expression in hen (Gallus domesticus) granulosa cells.

The chicken ovary is organized into a hierarchy of yellow yolky follicles that ovulate on successive days. Active or passive immunization of laying hens against testosterone blocks ovulation without affecting follicle development. Testosterone may play a role in pre-ovulatory follicle maturation by stimulating granulosa progesterone production. We assessed whether this stimulus is dose-related and depends on the maturity of the donor follicle, and if it does so by stimulating granulosa cell STAR, P450 cholesterol side-chain cleavage (P450scc), and LH receptor (LHCGR) mRNAs expression. Progesterone production by granulosa cells from F1, F3, and F4 follicles, cultured for 3 h without testosterone was greater in cells collected 11-14 h than 1-4 h after ovulation. These differences in progesterone production were less pronounced after granulosa cells had been cultured for 24 h. Culture of granulosa cells for 3 or 24 h with testosterone (1-100 ng/ml) stimulated progesterone production in cells collected from F4, F3, or F1 follicles 1-4, or 11-14 h after ovulation. Testosterone (0-4000 ng/ml) alone or in combination with LH (0-100 ng/ml) increased progesterone production by F1 granulosa cells, collected 1-4 and 11-14 h after ovulation and cultured for 3 h. Finally, testosterone (10 or 100 ng/ml) increased STAR, P450scc, and LHCGR mRNAs, when added to 3 h cultures of F1 granulosa cells. In conclusion, testosterone stimulates granulosa cell progesterone production in hen pre-ovulatory hierarchical follicles irrespective of maturational state, acting alone or additively with LH. We propose that testosterone promotes granulosa cell maturation to facilitate the pre-ovulatory release of LH.

Testosterone directly induces progesterone production and interacts with physiological concentrations of LH to increase granulosa cell progesterone production in laying hens (Gallus domesticus).

Blocking testosterone action with immunization or with a specific antagonist blocks the preovulatory surge of progesterone and ovulation in laying hens. Thus, testosterone may stimulate progesterone production in a paracrine fashion within the ovary. To test this hypothesis, we evaluated the effects of testosterone and its interaction with LH on the production of progesterone by granulosa cells in culture. Hen granulosa cells obtained from preovulatory follicles were cultured in 96 well plates. The effects of testosterone (0-100ng/ml) and/or LH (0-100ng/ml) were evaluated. LH-stimulated progesterone production in a dose response manner up to 10ng/ml (p<0.01). Testosterone, up to 10ng/ml, increased progesterone production in a dose response manner in the absence of LH and at all doses of LH up to 1ng/ml (p<0.001). However, at supraphysiological concentrations of LH (10 and 100ng/ml) there was no further increase in progesterone production caused by testosterone (p>0.05). Finally, the addition of 2-hydroxyflutamide (0-1000mug/ml) to hen granulosa cells cultured with 10ng/ml of testosterone reduced progesterone production in a dose response manner (p<0.001). In conclusion, testosterone stimulates progesterone production in preovulatory follicle granulosa cells and interacts with physiological concentrations of LH to increase progesterone production. In addition, testosterone stimulation on granulosa cells is specific since the testosterone antagonist decreased testosterone stimulatory action.

Testosterone immunization blocks the ovulatory process in laying hens without affecting ovarian follicular development.

The role of testosterone in the ovulatory process in hens has been largely neglected. The aim of the present study was to evaluate if testosterone plays an important role on the ovulatory process in laying hens. The effect of active and passive immunization against testosterone on ovarian follicular development and oviposition was studied. Egg laying percentage was evaluated in hens actively immunized against testosterone-BSA (T-AI; n = 6) or BSA (BSA-AI; n = 6). Oviposition was reduced as antibody titer increased in T-AI hens (r = -0.67; P < 0.01). Ovarian structures were assessed in three animals from each group. Follicles reached preovulatory size in both groups, nonetheless, in T-AI hens follicles at different stages of regression indicated that ovulation was blocked by treatment. In the remaining animals, preovulatory concentrations of progesterone and testosterone were determined. A preovulatory surge release of progesterone, preceded by a testosterone peak, was observed in the BSA-AI group (P < 0.05). In contrast, progesterone in T-AI animals remained at basal concentrations. Whereas, testosterone concentrations were significantly greater in T-AI as compared with BSA-AI animals (P < 0.05). Finally, to study the effect of passive immunization on oviposition, hens were passively immunized (PI) on four occasions, on alternate days with anti-T serum (T-PI; n = 10) or anti-BSA serum (BSA-PI; n = 8). During the 13-day period that preceded treatment, oviposition averaged 94.1%. Forty-eight hours after the first immunization, no egg was laid by 8 out of the 10 T-PI hens. During the 10 days following the first passive immunization, there was a reduction in the laying percentage that was significantly greater in T-PI hens (reduction of 52% in T-PI versus 29% in P-BSA, P < 0.01). In summary, these studies show that testosterone immunization hampers egg-laying without affecting ovarian follicular development, suggesting that testosterone has an important role in the ovulatory process in laying hens.

Subdominant hierarchical ovarian follicles are needed for steroidogenesis and ovulation in laying hens (Gallus domesticus).

Ovarian follicle development in avian species is characterized by a strict hierarchical arrangement. The hierarchical follicles secrete progesterone, which induces the LH surge, but the capacity to produce other steroids decreases with development. Our aim was to evaluate the complementary action of subdominant follicles (F4-F6) on ovulation and steroidogenesis of the preovulatory follicles (F1-F3) in domestic laying hens. The first study included four groups: control (C); sham-operated (SO); large hierarchical follicles (LHF) from which F4-F6 follicles were extracted; and subdominant hierarchical follicles (SHF) from which F1-F3 follicles were extracted. Blood samples were collected every 2h from 12h before estimated ovoposition until 2h after ovoposition. Egg laying continued at the same rates in C and SO hens, with normal preovulatory surges of oestradiol, testosterone, progesterone and LH. In contrast, in LHF and SHF groups, ovoposition was blocked; oestradiol concentrations were not affected; but no preovulatory surges of testosterone, progesterone or LH were seen. Further, the testosterone surge was required for the occurrence of progesterone and LH surges. In the second study StAR and steroidogenic enzyme mRNA expression was evaluated within F1-F3 follicles from a LHF group and C-14 and C-8 controls groups, in which follicles were collected 14h and 8h before expected ovoposition, respectively. Extraction of F4-F6 follicles caused a significant reduction in StAR and 3ß-HSD expressions within theca, but not in granulosa cells. In conclusion, subdominant hierarchical follicles (F4-F6) are required for the preovulatory release of testosterone, progesterone and LH, which are highly inter-correlated.

Testosterone antagonist (flutamide) blocks ovulation and preovulatory surges of progesterone, luteinizing hormone and oestradiol in laying hens.

The preovulatory release of luteinizing hormone (LH) in the domestic hen occurs after the initiation of a preovulatory surge of testosterone. The objective of this study was to determine whether this testosterone surge has functional significance in the endocrine control of ovulation. Groups of laying hens (n = 10-22) were treated with the androgen receptor antagonist, flutamide, at 8 h intervals for 24 h at doses of 0, 31.25, 62.5, 125 and 250 mg. All doses reduced egg laying (P < 0.001), with the highest dose being the most effective. In a second study, laying hens (n = 9) were treated with 250 mg flutamide at 8 h intervals for 24 h with a control group being given placebo (n = 10). Blood samples were taken for hormone measurements at 2 h intervals for 18 h starting 4 h before the onset of darkness. The percentage of hens laying per day did not differ between groups before treatment (control, 88% vs flutamide, 86%). Ovulation was blocked in all hens treated with flutamide within 2 days while the control hens continued to lay at the pretreatment rate (80%). Preovulatory surges of plasma testosterone, progesterone, oestradiol and LH were observed in control hens but with the exception of testosterone, flutamide treatment blocked the progesterone, oestradiol and LH surges. LH concentrations declined progressively with time in the flutamide-treated hens. It is concluded that inhibition of testosterone action blocks egg laying and the preovulatory surges of progesterone, luteinizing hormone and oestradiol demonstrating a key role for the preovulatory release of testosterone in the endocrine control of ovulation in the domestic hen.