Chronic estradiol exposure suppresses luteinizing hormone surge without affecting kisspeptin neurons and estrogen receptor alpha in anteroventral periventricular nucleus†.

Mammalian ovulation is induced by a luteinizing hormone surge, which is triggered by elevated plasma estrogen levels; however, chronic exposure to high levels of estradiol is known to inhibit luteinizing hormone secretion. In the present study, we hypothesized that the inhibition of the luteinizing hormone surge by chronic estradiol exposure is due to the downregulation of the estrogen receptor alpha in kisspeptin neurons at hypothalamic anteroventral periventricular nucleus, which is known as the gonadotropin-releasing hormone/luteinizing hormone surge generator. Animals exposed to estradiol for 2 days showed an luteinizing hormone surge, whereas those exposed for 14 days showed a significant suppression of luteinizing hormone. Chronic estradiol exposure did not affect the number of kisspeptin neurons and the percentage of kisspeptin neurons with estrogen receptor alpha or c-Fos in anteroventral periventricular nucleus, but it did affect the number of kisspeptin neurons in arcuate nucleus. Furthermore, chronic estradiol exposure did not affect gonadotropin-releasing hormone neurons. In the pituitary, 14-day estradiol exposure significantly reduced the expression of Lhb mRNA and LHß-immunoreactive areas. Gonadotropin-releasing hormone-induced luteinizing hormone release was also reduced significantly by 14-day estradiol exposure. We revealed that the suppression of an luteinizing hormone surge by chronic estradiol exposure was induced in association with the significant reduction in kisspeptin neurons in arcuate nucleus, luteinizing hormone expression in the pituitary, and pituitary responsiveness to gonadotropin-releasing hormone, and this was not caused by changes in the estrogen receptor alpha-expressing kisspeptin neurons in anteroventral periventricular nucleus and gonadotropin-releasing hormone neurons, which are responsible for estradiol positive feedback.

Hypothalamic Kisspeptin Expression in Hyperandrogenic Female Rats and Aging Rats.

Hypothalamic kisspeptin neurons stimulate gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) release. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) of rats induce an LH surge for ovulation, and those in the arcuate nucleus (ARC) regulate pulsatile LH secretion for follicle development and spermatogenesis. Dysfunction of kisspeptin neurons thus reduces the reproductive function. This review focuses on the effect of androgen or aging on kisspeptin expression in rats. Although androgen directly suppresses ARC kisspeptin neurons in female rats, the AVPV kisspeptin neurons are hardly affected. In rats, plasma LH concentrations decrease in both sexes with aging, and ARC kisspeptin expression also decreases in old rats compared with young rats. In addition, kisspeptin neurons may be associated with hyperprolactinemia in old female rats because they are known to release prolactin through hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons. Hypothalamic kisspeptin neurons are thus the main regulator to secrete LH, and inhibition of kisspeptin expression leads to various kinds of reproductive dysfunction.

Age-related alterations in hypothalamic kisspeptin, neurokinin B, and dynorphin neurons and in pulsatile LH release in female and male rats.

Pulsatile secretion of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) decreases during aging. Kisspeptin (encoded by Kiss1) neurons in the arcuate nucleus coexpress neurokinin B (Tac3) and dynorphin (Pdyn) and are critical for regulating the GnRH/LH pulse. We therefore examined kisspeptin neurons by histochemistry and pulsatile LH release in rats aged 2-3 (Young), 12-13 (Young-Middle), 19-22 (Late-Middle), and 24-26 (Old) months. Total LH concentrations, sampled for 3 hours, decreased in both sexes with aging. In females, numbers of Tac3 and Pdyn neurons were significantly reduced in all aging rats, and numbers of Kiss1 neurons were significantly reduced in Late-Middle and Old rats. In males, numbers of all 3 neuron-types were significantly decreased in all aging rats. GnRH agonist induced LH release in all animals; however, the increased LH concentration in all aging rats was less than that in Young rats. These results suggest that expression of each gene in kisspeptin neurons may be controlled individually during aging, and that reduction of their expression or change in pituitary responsiveness may cause attenuated pulsatile LH secretion.

Effect of androgen on Kiss1 expression and luteinizing hormone release in female rats.

Hyperandrogenic women have various grades of ovulatory dysfunction, which lead to infertility. The purpose of this study was to determine whether chronic exposure to androgen affects the expression of kisspeptin (ovulation and follicle development regulator) or release of luteinizing hormone (LH) in female rats. Weaned females were subcutaneously implanted with 90-day continuous-release pellets of 5α-dihydrotestosterone (DHT) and studied after 10 weeks of age. Number of Kiss1-expressing cells in both the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) was significantly decreased in ovary-intact DHT rats. Further, an estradiol-induced LH surge was not detected in DHT rats, even though significant differences were not observed between DHT and non-DHT rats with regard to number of AVPV Kiss1-expressing cells or gonadotrophin-releasing hormone (GnRH)-immunoreactive (ir) cells in the presence of high estradiol. Kiss1-expressing and neurokinin B-ir cells were significantly decreased in the ARC of ovariectomized (OVX) DHT rats compared with OVX non-DHT rats; pulsatile LH secretion was also suppressed in these animals. Central injection of kisspeptin-10 or intravenous injection of a GnRH agonist did not affect the LH release in DHT rats. Notably, ARC Kiss1-expressing cells expressed androgen receptors (ARs) in female rats, whereas only a few Kiss1-expressing cells expressed ARs in the AVPV. Collectively, our results suggest excessive androgen suppresses LH surge and pulsatile LH secretion by inhibiting kisspeptin expression in the ARC and disruption at the pituitary level, whereas AVPV kisspeptin neurons appear to be directly unaffected by androgen. Hence, hyperandrogenemia may adversely affect ARC kisspeptin neurons, resulting in anovulation and menstrual irregularities.

Interactions between Kisspeptin Neurons and Hypothalamic Tuberoinfundibular Dopaminergic Neurons in Aged Female Rats.

Kisspeptin neurons in the arcuate nucleus (ARC) regulate prolactin secretion, and are in physical contact with tuberoinfundibular dopaminergic (TIDA) neurons, which inhibit prolactin secretion. Prolactin levels in the blood are increased with advancing age in rats; therefore, we investigated the interactions with TIDA neurons and kisspeptin neurons in aged female rats (24 months of age), relative to those of young adult female rats (9-10 weeks of age). Plasma prolactin levels in the aged rats were significantly higher than those of young adult rats. Tyrosine hydroxylase (TH)-immunoreactive (ir) cell bodies and kisspeptin-ir nerve fibers were found in the dorsomedial ARC of both groups. The number of TH-ir cell bodies in the dorsomedial ARC did not differ significantly between groups. Additionally, no significant differences in the number of TH-ir cells in contact with kisspeptin-ir fibers was observed between groups. However, the number of kisspeptin-ir or Kiss1 mRNA-expressing cells in the ARC was significantly reduced in the aged rats compared with that of the young rats. These results suggest that the contacts between TIDA neurons and kisspeptin neurons are maintained after reproductive senescence, while production of kisspeptin in the ARC decreases significantly during aging.

Effect of sex steroid hormones on the number of serotonergic neurons in rat dorsal raphe nucleus.

Disorders caused by the malfunction of the serotonergic system in the central nervous system show sex-specific prevalence. Many studies have reported a relationship between sex steroid hormones and the brain serotonergic system; however, the interaction between sex steroid hormones and the number of brain neurons expressing serotonin has not yet been elucidated. In the present study, we determined whether sex steroid hormones altered the number of serotonergic neurons in the dorsal raphe nucleus (DR) of adult rat brains. Animals were divided into five groups: ovariectomized (OVX), OVX+low estradiol (E2), OVX+high E2, castrated males, and intact males. Antibodies against 5-hydroxytryptamine (5-HT, serotonin) and tryptophan hydroxylase (Tph), an enzyme for 5-HT synthesis, were used as markers of 5-HT neurons, and the number of 5-HT-immunoreactive (ir) or Tph-ir cells was counted. We detected no significant differences in the number of 5-HT-ir or Tph-ir cells in the DR among the five groups. By contrast, the intensity of 5-HT-ir showed significant sex differences in specific subregions of the DR independent of sex steroid levels, suggesting that the manipulation of sex steroid hormones after maturation does not affect the number and intensive immunostaining of serotonergic neurons in rat brain. Our results suggest that, the sexual dimorphism observed in the serotonergic system is due to factors such as 5-HT synthesis, transportation, and degradation but not to the number of serotonergic neurons.