Stress rapidly suppresses in vivo LH pulses and increases activation of RFRP-3 neurons in male mice

Restraint stress is a psychosocial stressor that suppresses reproductive status, including LH pulsatile secretion, but the neuroendocrine mechanisms underlying this inhibition remains unclear. Reproductive neural populations upstream of gonadotropin-releasing hormone (GnRH) neurons, such as kisspeptin, neurokinin B and RFRP-3 (GnIH) neurons, are possible targets for psychosocial stress to inhibit LH pulses, but this has not been well examined, especially in mice in which prior technical limitations prevented assessment of in vivo LH pulse secretion dynamics. Here, we examined whether one-time acute restraint stress alters in vivo LH pulsatility and reproductive neural populations in male mice, and what the time-course is for such alterations. We found that endogenous LH pulses in castrated male mice are robustly and rapidly suppressed by one-time, acute restraint stress, with suppression observed as quickly as 12­18 min. This rapid LH suppression parallels with increased in vivo corticosterone levels within 15 min of restraint stress. Although Kiss1, Tac2 and Rfrp gene expression in the hypothalamus did not significantly change after 90 or 180 min restraint stress, arcuate Kiss1 neural activation was significantly decreased after 180 min. Interestingly, hypothalamic Rfrp neuronal activation was strongly increased at early times after restraint stress initiation, but was attenuated to levels lower than controls by 180 min of restraint stress. Thus, the male neuroendocrine reproductive axis is quite sensitive to short-term stress exposure, with significantly decreased pulsatile LH secretion and increased hypothalamic Rfrp neuronal activation occurring rapidly, within minutes, and decreased Kiss1 neuronal activation also occurring after longer stress durations.

Estradiol-Dependent and -Independent Stimulation of Kiss1 Expression in the Amygdala, BNST, and Lateral Septum of Mice.

Kisspeptin, encoded by Kiss1, activates reproduction by stimulating GnRH neurons. Although most Kiss1 neurons are located in the hypothalamus, smaller Kiss1 populations also reside in the medial amygdala (MeA), bed nucleus of the stria terminalis (BnST), and lateral septum (LS). However, very little is known about the regulation and function of these extra-hypothalamic Kiss1 neurons. This study focused on the roles and interactions of two signaling factors, estradiol (E2) and GABA, known to stimulate and inhibit, respectively, extra-hypothalamic Kiss1 expression. First, using estrogen receptor (ER)α knockout (KO) and ßERKO mice, we demonstrated that Kiss1 in both the BnST and LS is stimulated by E2, as occurs in the MeA, and that this E2 upregulation occurs via ERα, but not ERß. Second, using GABABR KO and wild-type mice, we determined that whereas E2 normally increases extra-hypothalamic Kiss1 levels, such upregulation by E2 is further enhanced by the concurrent absence of GABABR signaling in the MeA and LS, but not the BnST. Third, we demonstrated that when GABABR signaling is absent, the additional removal of gonadal sex steroids does not abolish Kiss1 expression in the MeA and BnST, and in some cases the LS. Thus, Kiss1 expression in these extra-hypothalamic regions is not solely dependent on E2 stimulation. Finally, we demonstrated a significant positive correlation between Kiss1 levels in the MeA, BnST, and LS, but not between these regions and the hypothalamus (anteroventral periventricular nucleus/periventricular nucleus). Collectively, our findings indicate that both E2 and GABA independently regulate all three extra-hypothalamic Kiss1 populations, but their regulatory interactions may vary by brain region and additional yet-to-be-identified factors are likely involved.

Acute Psychosocial Stress Inhibits LH Pulsatility and Kiss1 Neuronal Activation in Female Mice.

Psychosocial stress, such as isolation and restraint, disrupts reproductive neuroendocrine activity. Here we investigate the impact of psychosocial stress on luteinizing hormone (LH) pulses and gene expression and neuronal activation within Rfrp and Kiss1 cells in female mice. Mice were ovariectomized (OVX) and handled daily to habituate to the tail-tip blood collection procedure. Blood was collected every 5 minutes for 180 minutes for measurement of LH. After 90 minutes, stress animals were placed into restraint devices and isolated to new cages. No-stress control animals remained in their home cages. LH pulses occurred at regular intervals during the entire 180-minute sampling period in controls. In contrast, stress induced a rapid and robust suppression of pulsatile LH secretion. Stress reduced the frequency of pulses by 60% and diminished basal LH levels by 40%; pulse amplitude was unaffected. In a separate cohort of OVX females, brains were collected after 45, 90, or 180 minutes of stress or in no-stress controls. At all time points, stress induced a potent decrease in arcuate Kiss1 neuronal activation, using cfos induction as a marker, with a 50% to 60% suppression vs control levels, whereas Rfrp and cfos coexpression in the dorsal-medial nucleus was elevated after 45 minutes of stress. Although arcuate Kiss1 gene expression remained stable, Rfrp expression was elevated 20% after 180 minutes of stress. These findings demonstrate rapid suppression of LH pulsatile secretion by psychosocial stress, associated with reduced cfos induction in Kiss1 neurons and time-dependent increases in Rfrp neuronal activation and messenger RNA.

Effects of Selective Deletion of Tyrosine Hydroxylase from Kisspeptin Cells on Puberty and Reproduction in Male and Female Mice.

The neuropeptide kisspeptin, encoded by Kiss1, regulates reproduction by stimulating GnRH secretion. Kiss1-syntheizing neurons reside primarily in the hypothalamic anteroventral periventricular (AVPV/PeN) and arcuate (ARC) nuclei. AVPV/PeN Kiss1 neurons are sexually dimorphic, with females expressing more Kiss1 than males, and participate in estradiol (E2)-induced positive feedback control of GnRH secretion. In mice, most AVPV/PeN Kiss1 cells coexpress tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis (in this case, dopamine). Dopamine treatment can inhibit GnRH neurons, but the function of dopamine signaling arising specifically from AVPV/PeN Kiss1 cells is unknown. We generated a novel TH flox mouse and used Cre-Lox technology to selectively ablate TH specifically from Kiss1 cells. We then examined the effects of selective TH knock-out on puberty and reproduction in both sexes. In control mice, 90% of AVPV/PeN Kiss1 neurons coexpressed TH, whereas in mice lacking TH exclusively in Kiss1 cells (termed Kiss THKOs), TH was successfully absent from virtually all Kiss1 cells. Despite this absence of TH, both female and male Kiss THKOs displayed normal body weights, puberty onset, and basal gonadotropin levels in adulthood, although testosterone (T) was significantly elevated in adult male Kiss THKOs. The E2-induced LH surge was unaffected in Kiss THKO females, and neuronal activation status of kisspeptin and GnRH cells was also normal. Supporting this, fertility and fecundity were normal in Kiss THKOs of both sexes. Thus, despite high colocalization of TH and Kiss1 in the AVPV/PeN, dopamine produced in these cells is not required for puberty or reproduction, and its function remains unknown.

Estrogen Stimulation of Kiss1 Expression in the Medial Amygdala Involves Estrogen Receptor-α But Not Estrogen Receptor-ß.

The neuropeptide kisspeptin, encoded by Kiss1, regulates reproduction by stimulating GnRH secretion. Neurons synthesizing kisspeptin are predominantly located in the hypothalamic anteroventral periventricular (AVPV) and arcuate nuclei, but smaller kisspeptin neuronal populations also reside in extrahypothalamic brain regions, such as the medial amygdala (MeA). In adult rodents, estradiol (E2) increases Kiss1 expression in the MeA, as in the AVPV. However, unlike AVPV and arcuate nuclei kisspeptin neurons, little else is currently known about the development, regulation, and function of MeA Kiss1 neurons. We first assessed the developmental onset of MeA Kiss1 expression in males and found that MeA Kiss1 expression is absent at juvenile ages but significantly increases during the late pubertal period, around postnatal day 35, coincident with increases in circulating sex steroids. We next tested whether developmental MeA Kiss1 expression could be induced early by E2 exposure prior to puberty. We found that juvenile mice given short-term E2 had greatly increased MeA Kiss1 expression at postnatal day 18. Although MeA Kiss1 neurons are known to be E2 up-regulated, the specific estrogen receptor (ER) pathway(s) mediating this stimulation are unknown. Using adult ERα knockout and ERß knockout mice, we next determined that ERα, but not ERß, is required for maximal E2-induced MeA Kiss1 expression in both sexes. These results delineate both the developmental time course of MeA Kiss1 expression and the specific ER signaling pathway required for E2-induced up-regulation of Kiss1 in this extrahypothalamic brain region. These findings will help drive future studies ascertaining the potential functions of this understudied kisspeptin population.