Alan H. Kaynard

Pineal Melatonin Mediates Photoperiodic Control of Pulsatile Luteinizing Hormone Secretion in the Ewe

Seasonal breeding in the ewe is regulated by photoperiod through a pineal-dependent mechanism. Changes in the ability of estradiol to inhibit tonic LH secretion are critical. During anestrus, this ovarian steroid gains the ability to slow the frequency of pulsatile LH secretion through an action on the brain. Exposure of ovariectomized, estradiol-implanted ewes to short photoperiods during summer anestrus revealed that daylength can control LH pulse frequency. After removal of estradiol, LH pulse frequency still differed between long- and short-day ewes, suggesting photoperiodic modulation of LH and presumably GnRH secretion independent of gonadal steroids. Significantly, the effects of daylength expressed both in the presence and the absence of estradiol failed to occur in pinealectomized ewes. Long-term infusions of melatonin, given in physiological patterns to pinealectomized ewes, mimicked the effects of photoperiod on pineal-intact ewes. Specifically, a pattern of melatonin characteristic of that in short days (16-hour night-time rise) led to an increase in LH pulse frequency to a breeding season rate. Conversely, melatonin infusions typifying a long-day pattern (8-hour night-time rise) produced an anestrous pulse pattern. Pituitary sensitivity to GnRH was not reduced in sheep which were reproductively suppressed by photoperiod or melatonin treatments. These observations support the conclusion that day-length acts through pineal melatonin secretion to regulate a neural LH pulse generator which, by changing the frequency of GnRH pulses, determines the ewes seasonal reproductive state.

Importance of Pituitary and Neural Actions of Estradiol in Induction of the Luteinizing Hormone Surge in the Ewe

Two experiments were performed to test the importance of both pituitary and neural sites of action of estradiol in inducing the surge of luteinizing hormone (LH) in the ewe. Both experiments were conducted using an animal model in which pulsatile secretion of gonadotropin-releasing hormone (GnRH) and endogenous secretion of ovarian steroids were eliminated by ovariectomy during seasonal anestrus and treatment with Silastic implants which maintained a luteal-phase level of serum progesterone. The hormonal requirements for the surge were then evaluated by systematic application of GnRH and estradiol signals using pulsatile infusion pumps (for GnRH) and Silastic implants (for estradiol). In experiment 1, the circulating level of estradiol and frequency of GnRH pulses were increased either abruptly or progressively (i.e. mimicking the changes in the estrous cycle between luteolysis and just before the LH surge). Abrupt increments led to an LH surge in all ewes; progressive rises to the same absolute levels did not. However, sudden application of a further large increase in GnRH upon the progressive rise elicited an LH surge in every instance. In experiment 2, a GnRH pulse pattern known to be effective in inducing the LH surge was applied under conditions of differing estradiol concentration: no estradiol, basal estradiol, basal rising to peak estradiol. The GnRH signal elicited high-amplitude surges of LH only in the presence of a peak estradiol concentration. Our findings are consistent with the conclusion that two actions are required for a rise in estradiol to elicit a full-amplitude surge of LH in the ewe: an action on the brain to evoke a sudden increase in GnRH release and an action on the pituitary to maximize its response to GnRH.

Does Melatonin Alter Pituitary Responsiveness to Gonadotropin-Releasing Hormone in the Ewe?

The diurnal secretion of melatonin from the pineal gland transduces information about day length to the reproductive axis of many seasonal breeders including the ewe. In the sheep the target for melatonin is thought to be neural, such that the hormone acts through the GnRH pulse generator to produce seasonal alterations in the frequency of pulsatile LH secretion. These effects on the pulse generation mechanism take approximately 50 days to become evident. It is possible that melatonin also exerts direct effects at the level of the pituitary gland to alter responsiveness to GnRH. Such effects have been noted in other species. The site of action of melatonin to regulate pulsatile LH secretion was assessed in the ewe by determining whether the animals endogenous melatonin acutely modifies pituitary responsiveness to sustained pulsatile administration of GnRH. Using an animal model in which endogenous GnRH was blocked, pituitary responsiveness to hourly pulses of exogenous GnRH was assessed under conditions of both high (dark period) and low (light period) melatonin. No evidence for acute effects of melatonin on pituitary response to GnRH was found. In another experiment, the amplitude and frequency of endogenously generated LH pulses in ovariectomized ewes was found not to change during the 24-hour light/dark cycle. These data lead to the conclusion that melatonin does not act at the pituitary gland to produce acute effects on LH secretion. Rather, our findings are consistent with the hypothesis that the action of melatonin, in this short-day breeder is long term, and is directed towards the neural elements of the hypothalamic pulse-generating mechanism.

Feedback Regulation of Pulsatile LH Secretion in the Ewe: Stimulation of Frequency by Estradiol

This study tested the hypothesis that estradiol can enhance LH pulse frequency in the ewe by an action which does not depend on other ovarian hormones. Long-term ovariectomized ewes were treated with a small subcutaneous estradiol implant at a time equivalent to the early breeding season (October), and frequent blood samples (6-min intervals) were obtained during sequential 3-hour periods over the next 84 h. All ewes responded with an increase in frequency of LH pulses, a response evident by 60 h and maintained at 84 h after initiation of the estradiol stimulus. Mean (+/- SE) pretreatment frequency was one pulse every 41 +/- 2 min; that at the height of the response was one pulse every 34 +/- 2 min (p less than 0.01). This increased rate was equivalent to the annual mid-winter maximum observed in ovariectomized ewes not treated with estradiol. These findings are consistent with the hypothesis that estradiol can enhance LH pulse frequency by an action which does not depend on other ovarian steroids. It is suggested that this action contributes to the heightened pace of LH pulses during the follicular phase of the estrous cycle.

FSH and LH Stimulation of Granulosa Cell Prodynorphin Gene Expression Abolished by Overexpression of a Nonfunctional Mutant of the RI Subunit of Protein Kinase A

At least three second-messenger systems are potentially involved in follicle stimulating hormone (FSH) activation of follicular development. They are cAMP/protein kinase A (cAMP-dependent protein kinase; PKA) (1), phosphoinositide turnover (2), and calcium/calmodulin(3); the latter two both act to increase the activity of protein kinase C. FSH has many actions on the ovary, and these three systems may be of different importance in contributing to each action of FSH. One mechanism by which FSH drives follicular development is its ability to stimulate the expression of mRNAs that encode important ovarian anabolic enzymes (4) and secretory products (5), including the endogenous opioid peptide genes prodynorphin (6) and proopiomelanocortin (7). To delineate the relative importance of different second-messenger systems in transducing the stimulatory effects of FSH on granulosa cells (GC) we tested the hypothesis that suppression of the cAMP/PKA system in these cells would alter FSH and luteinizing hormone (LH) stimulation of prodynorphin gene expression.