Hypothalamic Effects of Tamoxifen on Oestrogen Regulation of Luteinising Hormone and Prolactin Secretion in Female Rats

Oestradiol (E₂) acts in the hypothalamus to regulate luteinising hormone (LH) and prolactin (PRL) secretion. Tamoxifen (TX) has been extensively used as a selective oestrogen receptor modulator, although its neuroendocrine effects remain poorly understood. In the present study, we investigated the hypothalamic effects of TX in rats under low or high circulating E₂ levels. Ovariectomised (OVX) rats treated with oil, E₂ or TX, or E₂ plus TX, were evaluated for hormonal secretion and immunohistochemical analyses in hypothalamic areas. Both E₂ and TX reduced LH levels, whereas TX blocked the E₂‐induced surges of LH and PRL. TX prevented the E₂‐induced expression of progesterone receptor (PR) in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC), although it did not alter PR expression in OVX rats. TX blocked the E₂ induction of c‐Fos in AVPV neurones, consistent with the suppression of LH surge. However, TX failed to prevent E₂ inhibition of kisspeptin expression in the ARC. In association with the blockade of PRL surge, TX increased the phosphorylation of tyrosine hydroxylase (TH) in the median eminence of OVX, E₂‐treated rats. TX also precluded the E₂‐induced increase in TH expression in the ARC. In all immunohistochemical analyses, TX treatment in OVX rats caused no measurable effect on the hypothalamus. Thus, TX is able to prevent the positive‐ but not negative‐feedback effect of E₂ on the hypothalamus. TX also blocks the effects of E₂ on tuberoinfundibular dopaminergic neurones and PRL secretion. These findings further characterise the anti‐oestrogenic actions of TX in the hypothalamus and provide new information on the oestrogenic regulation of LH and PRL.     

Oestrogen‐Independent Circadian Clock Gene Expression in the Anteroventral Periventricular Nucleus in Female Rats: Possible Role as an Integrator for Circadian and Ovarian Signals Timing the Luteinising Hormone Surge

Periodic ovulation in rats, mice and hamsters is the result of a surge in luteinising hormone (LH) that depends on circadian gating signals emerging from the master circadian clock within the suprachiasmatic nucleus (SCN) and rising ovarian oestrogen levels. These two signals converge into the anteroventral periventricular nucleus (AVPV) and lead to the release of kisspeptin, which is responsible for surges of gonadotrophin‐releasing hormone and, in turn, of LH release. How the AVPV integrates circadian and reproductive signals remains unclear. In the present study, we show that the female rat AVPV itself shows circadian oscillations in the expression of the clock genes PER1 and BMAL1, which lie at the core circadian clockwork of mammals. In ovariectomised females treated with oestradiol (E₂), these oscillations are in synchrony with the AVPV rhythmic expression of the KISS1 gene and the gene that codes for the arginine‐vasopressin (AVP) receptor AVPr1a. Although clock gene oscillations are independent of oestrogen levels, circadian expression of Kiss1 and Avpr1a (also referred to as V1a) mRNA is blunted and absent, respectively, in ovariectomised animals without E₂ replacement. Because AVP is considered to be a critical SCN transmitter to gate the LH surge, our data suggest that there is a circadian oscillator located in the AVPV, and that such a putative oscillator could, in an oestrogen‐dependent manner, time the sensitivity to circadian signals emerging from the SCN and the release of kisspeptin.     

Oestrogen‐Induced Activation of Preoptic Kisspeptin Neurones May be Involved in the Luteinising Hormone Surge in Male and Female Japanese Monkeys

The oestrogen‐induced luteinising hormone (LH) surge is evident in male primates, including humans, whereas male rodents never show the LH surge, even when treated with a preovulatory level of oestrogen. This suggests that the central mechanism governing reproductive hormones in primates is different from that in rodents. The present study aimed to investigate whether male Japanese monkeys conserve a brain mechanism mediating the oestrogen‐induced LH surge via activation of kisspeptin neurones. Adult male and female Japanese monkeys were gonadectomised and then were treated with oestradiol‐17β for 2 weeks followed by a bolus injection of oestradiol benzoate. Both male and female monkeys showed an oestrogen‐induced LH surge. In gonadectomised monkeys sacrificed just before the anticipated time of the LH surge, oestrogen treatment significantly increased the number of KISS1‐expressing cells in the preoptic area (POA) and enhanced the expression of c‐fos in POA KISS1‐positive cells of males and females. The oestrogen treatment failed to induce c‐fos expression in the arcuate nucleus (ARC) kisspeptin neurones in both sexes just prior to LH surge onset. Thus, kisspeptin neurones in the POA but not in the ARC might be involved in the positive‐feedback action of oestrogen that induces LH surge in male Japanese monkeys, as well as female monkeys. The present results indicate that oestrogen‐induced activation of POA kisspeptin neurones may contribute to the LH surge generation in both sexes. The conservation of the LH surge generating system found in adult male primates, unlike rodents, could be a result of the capability of oestrogen to induce POA kisspeptin expression and activation.     

Possible Involvement of Microglia Containing Cyclooxygenase‐1 in the Accumulation of Gonadotrophin‐Releasing Hormone in the Preoptic Area in Female Rats

Prostaglandins (PGs), especially PGE₂, are involved in the hypothalamic control of gonadotrophin‐releasing hormone (GnRH) release, acting at least in part on the terminal of GnRH axons in the median eminence. The present study aimed: (i) to clarify the role of PG(s) in regulating GnRH cell function at the level of the perikarya in the preoptic area; (ii) to determine the cyclooxygenase (COX) isozyme responsible for producing PG(s) that regulates GnRH perikarya; and (iii) to identify cell types that contain the responsible COX isozyme in female rats. A surge of luteinising hormone (LH) secretion was induced by oestrogen and progesterone in ovariectomised rats. Treatment of the rat before the LH surge with indomethacin, a nonselective COX inhibitor, or NS‐398, a selective COX‐2 inhibitor, did not interfere with the surge. However, treatment with indomethacin or flurbiprofen, a selective COX‐1 inhibitor, significantly reduced the number of GnRH‐immunoreactive cells in the preoptic area at the time of peak LH secretion during the surge. NS‐398 did not affect the GnRH immunoreactivity. Double‐labelled immunofluorescent histochemistry revealed COX‐1 immunoreactivity in the vicinity of, but not within, GnRH containing neurones in the preoptic area. COX‐2 immunoreactivity was not found in the same area. The COX‐1 immunoreactivity was almost entirely localised in microglia in the preoptic area, but not in neurones or astrocytes. These results suggest that microglia in the preoptic area containing COX‐1 are responsible for producing PG(s), which, in turn, facilitates the accumulation of GnRH during the gonadotrophin surge in female rats.     

Enhancement of the luteinising hormone surge by male olfactory signals is associated with anteroventral periventricular Kiss1 cell activation in female rats

Olfactory stimuli play an important role in regulating reproductive functions in mammals. The present study investigated the effect of olfactory signals derived from male rats on kisspeptin neuronal activity and luteinising hormone (LH) secretion in female rats. Wistar‐Imamichi strain female rats were ovariectomised (OVX) and implanted with preovulatory levels of 17β‐oestradiol (E₂). OVX+E₂ rats were killed 1 hour after exposure to either: clean bedding, female‐soiled bedding or male‐soiled bedding. Dual staining for Kiss1 mRNA in situ hybridisation and c‐Fos immunohistochemistry revealed that the numbers of Kiss1‐expressing cells and c‐Fos‐immunopositive Kiss1‐expressing cells in the anteroventral periventricular nucleus (AVPV) were significantly higher in OVX+E₂ rats exposed to male‐soiled bedding than those of the other groups. No significant difference was found with respect to the number of c‐Fos‐immunopositive Kiss1‐expressing cells in the arcuate nucleus and c‐Fos‐immunopositive Gnrh1‐expressing cells between the groups. The number of c‐Fos‐immunopositive cells was also significantly higher in the limbic system consisting of several nuclei, such as the bed nucleus of the stria terminalis, the cortical amygdala and the medial amygdala, in OVX+E₂ rats exposed to male‐soiled bedding than the other groups. OVX+E₂ rats exposed to male‐soiled bedding showed apparent LH surges, and the peak of the LH surge and area under the curve of LH concentrations in the OVX+E₂ group were significantly higher than those of the other two groups. These results suggest that olfactory signals derived from male rats activate AVPV kisspeptin neurones, likely via the limbic system, resulting in enhancement of the peak of the LH surge in female rats. Taken together, the results of the present study suggests that AVPV kisspeptin neurones are a target of olfactory signals to modulate LH release in female rats.     

Fasting reduces KiSS-1 expression in the anteroventral periventricular nucleus (AVPV): effects of fasting on the expression of KiSS-1 and neuropeptide Y in the AVPV or arcuate nucleus of female rats

Changes in metabolic state, such as those induced by fasting, have profound effects on reproduction. In rats, the time-course over which fasting inhibits luteinising hormone (LH) release is reduced to 48 h by the presence of oestradiol-17beta (E(2)). Hypothalamic kisspeptin plays a key role in mediating the actions of E(2) on gonadotrophin-releasing hormone (GnRH) neurones, and thereby promotes LH release. KiSS-1-expressing neurones are found in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC). Extensive evidence implicates the AVPV in GnRH release and the ARC in energy balance. The latter nucleus also contains neurones that express neuropeptide Y (NPY), an orexigenic peptide implicated in GnRH control. To elucidate the involvement of kisspeptin and/or NPY in hypothalamic responses to fasting, their expression was quantified by in situ hybridisation histochemistry in ovariectomised rats, with or without E(2) replacement, before and after 48 h of fasting. In the presence of E(2), but not in its absence, the fasting suppressed plasma LH. In the AVPV, the low level of KiSS-1 expression found in the absence of E(2) was unaffected by fasting. By contrast, the elevated level found in the presence of E(2) was suppressed by fasting. Independent of E(2), fasting had no effect on KiSS-1 expression in the ARC, but increased NPY expression at that site. The present study has identified the AVPV as a site at which KiSS-1 expression can be influenced by fasting. The results suggest that inhibition of KiSS-1 expression in the AVPV may be a significant factor in restraining the gonadotrophic axis in response to negative energy balance in the presence of oestrogen. The extent to which the concurrent rise in NPY expression in the ARC may contribute to the suppression of LH release by influencing AVPV kisspeptin neurones, directly or indirectly, or by actions independent of kisspeptin, remains to be established.     

The Luteinising Hormone Surge‐Generating System is Functional in Male Goats as in Females: Involvement of Kisspeptin Neurones in the Medial Preoptic Area

A luteinising hormone (LH) surge is fundamental to the induction of ovulation in mammalian females. The administration of a preovulatory level of oestrogen evokes an LH surge in ovariectomised females, whereas the response to oestrogen in castrated males differs among species; namely, the LH surge‐generating system is sexually differentiated in some species (e.g. rodents and sheep) but not in others (e.g. primates). In the present study, we aimed to determine whether there is a functional LH surge‐generating system in male goats, and whether hypothalamic kisspeptin neurones in male goats are involved in the regulation of surge‐like LH secretion. By i.v. infusion of oestradiol (E₂; 6 μg/h) for 16 h, a surge‐like LH increase occurred in both castrated male and ovariectomised female goats, although the mean peak LH concentration was lower and the mean peak of the LH surge was later in males compared to females. Dual staining with KISS1 in situ hybridisation and c‐Fos immunohistochemistry revealed that E₂ treatment significantly increased c‐Fos expression in the medial preoptic area (mPOA) KISS1 cells in castrated males, as well as ovariectomised females. By contrast, dual‐labelled cells were scarcely detected in the arcuate nucleus (ARC) after E₂ treatment in both sexes. These data suggest that kisspeptin neurones in the mPOA, but not those in the ARC, are involved in the induction of surge‐like LH secretion in both male and female goats. In summary, our data show that the mechanism that initiates the LH surge in response to oestrogen, the mPOA kisspeptin neurones, is functional in male goats. Thus, sexual differentiation of the LH surge‐generating system would not be applicable to goats.     

Multiple failures in the lutenising hormone surge generating system in GABAB1KO female mice

Female mice lacking GABAB receptors, GABAB1KO, show disrupted oestrous cycles, reduced pregnancies and increased hypothalamic Gnrh1 mRNA expression, whereas anteroventral periventricular/periventricular preoptic nucleus (AVPV/PeN) Kiss1 mRNA was not affected. In the present study, we characterise the important components of the gonadotrophic preovulatory surge, aiming to unravel the origin of this reproductive impairment. In GABAB1KO and wild‐type (WT) females, we determined: (i) hypothalamic oestrogen receptor (ER)α and β and aromatase mRNA and protein expression; (ii) ovulation index and oestrus serum follicle‐stimulating hormone (FSH) and pituitary Gnrh1r expression; (iii) in ovariectomised‐oestradiol valerate‐treated mice, we evaluated ex vivo hypothalamic gonadotrophin‐releasing hormone (GnRH) pulsatility in the presence/absence of kisspeptin (Kiss‐10, constant or pulsatile) and oestradiol (constant); and (iv) in ovariectomised‐oestradiol silastic capsule‐treated mice (proestrous‐like environment), we evaluated morning and evening kisspeptin neurone activation (c‐Fos+) and serum luteinising homrone (LH). In the medial basal hypothalamus of oestrus GABAB1KOs, aromatase and ERα mRNA and protein were increased, whereas ERβ was decreased. In GABAB1KOs, the ovulation index was decreased together with decreased first oestrus serum FSH and increased pituitary Gnrh1r mRNA. Under constant Kiss‐10 stimulation, hypothalamic GnRH pulse frequency did not vary, although GnRH mass/pulse was increased in GABAB1KOs. In WTs, pulsatile Kiss‐10 together with constant oestradiol significantly increased GnRH pulsatility, whereas, in GABAB1KOs, oestradiol alone increased GnRH pulsatility and this was reversed by pulsatile Kiss‐10 addition. In GABAB1KOs AVPV/PeN kisspeptin neurones were similarly activated (c‐Fos+) in the morning and evening, whereas WTs showed the expected, marked evening stimulation. LH correlated with activated kisspeptin cells in WT mice, whereas GABAB1KO mice showed high, similar LH levels both in the morning and evening. Taken together, all of these alterations point to impairment in the trigger of the preovulatory GnRH surge that entails the reproductive alterations described.     

Facilitation or Inhibition of the Estradiol-Induced Gonadotropin Surge in the Immature Rat by Progesterone: Regulation of GnRH and LH Messenger RNAs and Activation of GnRH Neurons

We have developed and extensively characterized immature female rat models to demonstrate inhibition or facilitation of the estradiol (E₂)-induced gonadotropin surge by progesterone (P). We show here that the surge of free α-subunit is regulated similarly by P in these models. To investigate the possibility that P alters the biosynthesis of GnRH and/or LH, we measured levels of LH subunit mRNAs by Northern blot hybridization and GnRH mRNA by a solution hybridization-RNase protection assay. In the P inhibition model, α-subunit mRNA was significantly decreased when P was administered together with E₂ for 32 or 48 h, and LHβ, at 29 h. In the facilitation model, neither α-subunit nor LHβ mRNA increased with premature and enhanced release of LH and free α-subunit. Levels of GnRH mRNA in E₂-treated rats were significantly higher on the afternoon of the LH surge than on that or the following morning. There was no effect of P on GnRH mRNA levels, however, before, during, or after the LH surge in either paradigm. The time course of activation of GnRH neurons in P-facilitated rats was determined by double-label immunocytochemistry for GnRH and cFos. When serum LH concentrations were basal there was no expression of cFos in GnRH neurons. LH secretion in P-facilitated rats was initiated at ≈14.00 h and remained elevated until at least 19.00 h. During this time 63–78% of GnRH neurons were cFos positive. Both serum LH concentrations and the percentage of cFos-activated GnRH neurons were significantly lower in control rats treated with E₂ alone than in those treated also with P. In conclusion: 1) suppression of LH and free α-subunit secretion by P can be accounted for at least partly by suppression of α-subunit mRNA levels; 2) P facilitation is not associated with changes in LH subunit or GnRH mRNA levels; 3) the large proportion of cFos-positive GnRH neurons in P-facilitated rats closely parallels increases in serum LH concentrations but is not accompanied by changes in GnRH mRNA levels. It is likely, therefore, that P acts in the facilitation model to trigger release of pre-existing GnRH stores by altering synthesis or activity of neuro-transmitters/neuropeptides involved in GnRH regulation and/or release of LH stores by altering, for example, pituitary responsiveness to GnRH (including self-priming) and components of the LH secretory apparatus. Similar possibilities may also obtain for the blockade of the gonadotropin surge in the inhibition model.     

Exposure of female mice to perfluorooctanoic acid suppresses hypothalamic kisspeptin‐reproductive endocrine system through enhanced hepatic fibroblast growth factor 21 synthesis,leading to ovulation failure and prolonged dioestrus

Perfluorooctanoic acid (PFOA) is widely used in household applications. High‐dose exposure to PFOA has been associated with increased risks of infertility and premature ovarian insufficiency in woman. PFOA can alter hepatic gene expression by activating peroxisome proliferator‐activated receptor α (PPARα). The present study investigated whether exposure to PFOA via PPARα activation alters the synthesis of hepatic fibroblast growth factor 21 (FGF21) to disturb female neuroendocrine and reproductive function. In the present study, we show that the oral administration of PFOA (2 or 5 mg kg^‐1) in adult female mice (PFOA mice) caused prolonged dioestrous, a reduction in the number of corpora lutea and decreased levels of hypothalamic gonadotrophin‐releasing hormone, serum progesterone and luteinising hormone (LH). Exposure to PFOA decreased the expression of vasopressin in the suprachiasmatic nucleus (SCN) and kisspeptin in the anteroventral periventricular nucleus (AVPV) with deficits in preovulation or oestrogen‐induced LH surge. PFOA via activation of PPARα increased dose‐dependently hepatic FGF21 expression, leading to elevated serum and hypothalamic FGF21 concentrations. Treatment of PFOA mice with the PPARα antagonist GW6471 or the FGF21 inhibitor PD173074 rescued SCN vasopressin and AVPV‐kisspeptin expression. Either administration of GW6471 and PD173074 or treatment with vasopressin and the G protein coupled receptor 54 agonist kisspeptin‐10 in PFOA‐mice was able to recover the regular oestrous cycle, ovulation ability, LH surge production and reproductive hormone levels. The present study provides in vivo evidence that exposure to PFOA (≥2 mg kg^‐1) in mice causes down‐regulation of the kisspeptin‐reproductive endocrine system by enhancing PPARα‐mediated hepatic FGF21 expression. The liver‐brain reproductive endocrine disorder caused by PFOA exposure may lead to prolonged dioestrous and ovulation failure.     

Neonatal Kisspeptin is Steroid‐Independently Required for Defeminisation and Peripubertal Kisspeptin‐Induced Testosterone is Required for Masculinisation of the Brain: A Behavioural Study Using Kiss1 Knockout Rats

Rodents show apparent sex differences in their sexual behaviours. The present study used Kiss1 knockout (KO) rats to evaluate the role of kisspeptin in the defeminisation/masculinisation of the brain mechanism that controls sexual behaviours. Castrated adult Kiss1 KO males treated with testosterone showed no male sexual behaviours but demonstrated the oestrogen‐induced lordosis behaviours found in wild‐type females. The sizes of some of the sexual dimorphic nuclei of Kiss1 KO male rats are similar to those of females. Plasma testosterone levels at embryonic day 18 and postnatal day 0 (PND0) in Kiss1 KO males were high, similar to wild‐type males, indicating that perinatal testosterone is secreted in a kisspeptin‐independent manner. Long‐term exposure to testosterone from peripubertal to adult periods restored mounts and intromissions in KO males, suggesting that kisspeptin‐dependent peripubertal testosterone secretion is required to masculinise the brain mechanism. This long‐term testosterone treatment failed to abolish lordosis behaviours in KO males, whereas kisspeptin replacement at PND0 reduced lordosis quotients in Kiss1 KO males but not in KO females. These results suggest that kisspeptin itself is required to defeminise behaviour in the perinatal period, in cooperation with testosterone. Oestradiol benzoate treatment at PND0 suppressed lordosis quotients in Kiss1 KO rats, indicating that the mechanisms downstream of oestradiol work properly in the absence of kisspeptin. There was no significant difference in aromatase gene expression in the whole hypothalamus between Kiss1 KO and wild‐type male rats at PND0. Taken together, the present study demonstrates that both perinatal kisspeptin and kisspeptin‐independent testosterone are required for defeminisation of the brain, whereas kisspeptin‐dependent testosterone during peripuberty to adulthood is needed for masculinisation of the brain in male rats.     

Time‐of‐day‐dependent sensitivity of the reproductive axis to RFamide‐related peptide‐3 inhibition in female Syrian hamsters

In spontaneously ovulating rodent species, the timing of the luteinising hormone (LH) surge is controlled by the master circadian pacemaker in the suprachiasmatic nucleus (SCN). The SCN initiates the LH surge via the coordinated control of two opposing neuropeptidergic systems that lie upstream of the gonadotrophin‐releasing hormone (GnRH) neuronal system: the stimulatory peptide, kisspeptin, and the inhibitory peptide, RFamide‐related peptide‐3 (RFRP‐3; the mammalian orthologue of avian gonadotrophin‐inhibitory hormone [GnIH]). We have previously shown that the GnRH system exhibits time‐dependent sensitivity to kisspeptin stimulation, further contributing to the precise timing of the LH surge. To examine whether this time‐dependent sensitivity of the GnRH system is unique to kisspeptin or a more common mechanism of regulatory control, we explored daily changes in the response of the GnRH system to RFRP‐3 inhibition. Female Syrian hamsters were ovariectomised to eliminate oestradiol (E₂)‐negative‐feedback and RFRP‐3 or saline was centrally administered in the morning or late afternoon. LH concentrations and Lhβ mRNA expression did not differ between morning RFRP‐3‐and saline‐treated groups, although they were markedly suppressed by RFRP‐3 administration in the afternoon. However, RFRP‐3 inhibition of circulating LH at the time of the surge does not appear to act via the GnRH system because no differences in medial preoptic area Gnrh or RFRP‐3 receptor Gpr147 mRNA expression were observed. Rather, RFRP‐3 suppressed arcuate nucleus Kiss1 mRNA expression and potentially impacted pituitary gonadotrophs directly. Taken together, these findings reveal time‐dependent responsiveness of the reproductive axis to RFRP‐3 inhibition, possibly via variation in the sensitivity of arcuate nucleus kisspeptin neurones to this neuropeptide.     

Regulation of galanin receptor GalR1 mRNA expression by ovarian steroids in oestrogen receptor alpha-immunoreactive neurones: identification of distinct populations of neurones in the preoptic area

This study examined whether gonadal steroids are involved in regulating galanin receptor 1 (GalR1) mRNA expression in neurones that contain oestrogen receptor alpha (ERalpha), in three regions of the preoptic area (POA) known to be involved in the control of gonadotropin secretion. Double-labelling immunohistochemistry using an antibody against the ERalpha and in situ hybridization experiments using a 35S-labelled riboprobe specific for GalR1 mRNAs revealed that ERalpha is expressed in a large proportion of GalR1 mRNA-expressing neurones of the POA in the ovariectomized (OVX) female rat. Oestradiol (E2) and oestradiol plus progesterone (E2 + P) treatments of OVX rats significantly decreased the proportion of GalR1 mRNA/ERalpha immunoreactive (ERalpha-IR) neurones in the anteroventral periventricular nucleus (AVPV), medial preoptic nucleus (MPN) and medial preoptic area (MPO). The expression of GalR1 mRNA in ERalpha-IR neurones varied according the hormonal status of the female animals. In the AVPV, during the oestrous cycle, the hybridization signal significantly increased at oestrus. E2 and E2 + P treatments of OVX rats did not induced significant variation of levels of GalR1 mRNAs in ERalpha-IR neurones. In the MPN, E2 treatment of OVX rats resulted in significant increase in GalR1 mRNA expression in ERalpha-IR neurones. Similarly, levels of the GalR1 hybridization signal increased during afternoon of proestrus and oestrus. In the MPO, treatment of OVX rats with E2 + P significantly decreased GalR1 mRNA expression in ERalpha-IR neurones. The expression of GalR1 mRNA did not change during the oestrous cycle in this area. These findings suggest that the hypothalamic action of galanin on gonadotopin-releasing hormone (GnRH) secretion may pass through the specific population of GalR1/ERalpha-IR neurones of the MPN in mediating the oestrogen action on the GnRH system at the moment of the luteinizing hormone surge.     

Regulation of glutamic acid decarboxylase 65 and 67 gene expression by ovarian steroids: identification of two functionally distinct populations of GABA neurones in the preoptic area

GABA neurones in the preoptic area (POA) are critical for oestradiol (E2)-dependent surge release of luteinizing hormone (LH); however, it is not clear which population(s) of POA GABA neurones is involved. The goals of the present studies were: (i) to determine whether E2 regulates GABA neurones similarly in two subdivisions of the POA that play a role in LH surge release, the rostral POA region that contains the organum vasculosum of the lamina terminalis (rPOA/OVLT), and the region containing the anteroventral periventricular nucleus (AVPV) and medial preoptic nucleus (MPN) and (ii) to determine whether GABA neurones in either or both regions exhibit temporal changes consistent with a role in the regulation of LH surge release. To accomplish these goals, we measured glutamic acid decarboxylase (GAD) 65 and 67 mRNA levels at several time points in ovariectomized (OVX), E2-treated OVX rats exhibiting LH surge release, and in E2-treated OVX rats in which LH surge release was blocked by prior administration of progesterone (P4). Our findings demonstrate that, despite their close proximity, GABA neurones in the AVPV/MPN region are regulated differently from those in the rPOA/OVLT. Only neurones in the AVPV/MPN region show temporal changes in GAD 67 mRNA expression that appear to be linked to positive-feedback effects of E2 on luteinizing hormone-releasing hormone (LHRH) and LH release. Our findings also indicate that a morning rise and an afternoon fall in GAD 67 mRNA levels marks two E2-dependent signals required for LHRH and LH surge release. Finally, our results suggest that there are distinct E2-induced signals to the rPOA/OVLT and AVPV/MPN regions and that these signals differentially regulate GAD 65 and 67 gene expression.     

Lack of Pulse and Surge Modes and Glutamatergic Stimulation of Luteinising Hormone Release in Kiss1 Knockout Rats

Kisspeptin, encoded by the Kiss1 gene, has attracted attention as a key candidate neuropeptide in controlling puberty and reproduction via regulation of gonadotrophin‐releasing hormone (GnRH) secretion in mammals. Pioneer studies with Kiss1 or its cognate receptor Gpr54 knockout (KO) mice showed the indispensable role of kisspeptin‐GPR54 signalling in the control of animal reproduction, although detailed analyses of gonadotrophin secretion, especially pulsatile and surge‐mode of luteinising hormone (LH) secretion, were limited. Thus, in the present study, we have generated Kiss1 KO rats aiming to evaluate a key role of kisspeptin in governing reproduction via pulse and surge modes of GnRH/LH secretion. Kiss1 KO male and female rats showed a complete suppression of pulsatile LH secretion, which is responsible for folliculogenesis and spermatogenesis, and an absence of puberty and atrophic gonads. Kiss1 KO female rats showed no spontaneous LH/follicle‐stimulating hormone surge and an oestrogen‐induced LH surge, suggesting that the GnRH surge generation system, which is responsible for ovulation, does not function without kisspeptin. Furthermore, challenge of major stimulatory neurotransmitters, such as monosodium glutamate, NMDA and norepinephrine, failed to stimulate LH secretion in Kiss1 KO rats, albeit they stimulated LH release in wild‐type controls. Taken together, the results of the present study confirm that kisspeptin plays an indispensable role in generating two modes (pulse and surge) of GnRH/gonadotrophin secretion to regulate puberty onset and normal reproductive performance. In addition, the present study suggests that kisspeptin neurones play a critical role as a hub integrating major stimulatory neural inputs to GnRH neurones, using newly established Kiss1 KO rats, which serve as a useful model for detailed analysis of hormonal profiles.     

Surge‐Like Luteinising Hormone Secretion Induced by Retrochiasmatic Area NK3R Activation is Mediated Primarily by Arcuate Kisspeptin Neurones in the Ewe

The neuropeptides neurokinin B (NKB) and kisspeptin are potent stimulators of gonadotrophin‐releasing hormone (GnRH)/luteinsing hormone (LH) secretion and are essential for human fertility. We have recently demonstrated that selective activation of NKB receptors (NK3R) within the retrochiasmatic area (RCh) and the preoptic area (POA) triggers surge‐like LH secretion in ovary‐intact ewes, whereas blockade of RCh NK3R suppresses oestradiol‐induced LH surges in ovariectomised ewes. Although these data suggest that NKB signalling within these regions of the hypothalamus mediates the positive‐feedback effects of oestradiol on LH secretion, the pathway through which it stimulates GnRH/LH secretion remains unclear. We proposed that the action of NKB on RCh neurones drives the LH surge by stimulating kisspeptin‐induced GnRH secretion. To test this hypothesis, we quantified the activation of the preoptic/hypothalamic populations of kisspeptin neurones in response to POA or RCh administration of senktide by dual‐label immunohistochemical detection of kisspeptin and c‐Fos (i.e. marker of neuronal activation). We then administered the NK3R agonist, senktide, into the RCh of ewes in the follicular phase of the oestrous cycle and conducted frequent blood sampling during intracerebroventricular infusion of the kisspeptin receptor antagonist Kp‐271 or saline. Our results show that the surge‐like secretion of LH induced by RCh senktide administration coincided with a dramatic increase in c‐Fos expression within arcuate nucleus (ARC) kisspeptin neurones, and was completely blocked by Kp‐271 infusion. We substantiate these data with evidence of direct projections of RCh neurones to ARC kisspeptin neurones. Thus, NKB‐responsive neurones in the RCh act to stimulate GnRH secretion by inducing kisspeptin release from KNDy neurones.     

Evidence for Changes in Numbers of Synaptic Inputs onto KNDy and GnRH Neurones during the Preovulatory LH Surge in the Ewe

Kisspeptin neurones located in the arcuate nucleus (ARC) and preoptic area (POA) are critical mediators of gonadal steroid feedback onto gonadotrophin‐releasing hormone (GnRH) neurones. ARC kisspeptin cells that co‐localise neurokinin B (NKB) and dynorphin (Dyn), are collectively referred to as KNDy (Kisspeptin/NKB/Dyn) neurones, and have been shown in mice to also co‐express the vesicular glutamate transporter, vGlut2, an established glutamatergic marker. The ARC in rodents has long been known as a site of hormone‐induced neuroplasticity, and changes in synaptic inputs to ARC neurones in rodents occur over the oestrous cycle. Based on this evidence, the the present study aimed to examine possible changes across the ovine oestrous cycle in synaptic inputs onto kisspeptin cells in the ARC (KNDy) and POA, and inputs onto GnRH neurones. Gonadal‐intact breeding season ewes were perfused using 4% paraformaldehyde during either the luteal or follicular phase of the oestrous cycle, with the latter group killed at the time of the luteinising hormone (LH) surge. Hypothalamic sections were processed for triple‐label immunodetection of kisspeptin/vGlut2/synaptophysin or kisspeptin/vGlut2/GnRH. The total numbers of synaptophysin‐ and vGlut2‐positive inputs to ARC KNDy neurones were significantly increased at the time of the LH surge compared to the luteal phase; because these did not contain kisspeptin, they do not arise from KNDy neurones. By contrast to the ARC, the total number of synaptophysin‐positive inputs onto POA kisspeptin neurones did not differ between luteal phase and surge animals. The total number of kisspeptin and vGlut2 inputs onto GnRH neurones in the mediobasal hypothalamus (MBH) was also increased during the LH surge, and could be attributed to an increase in the number of KNDy (double‐labelled kisspeptin + vGlut2) inputs. Taken together, these results provide novel evidence of synaptic plasticity at the level of inputs onto KNDy and GnRH neurones during the ovine oestrous cycle. Such changes may contribute to the generation of the preovulatory GnRH/LH surge.     

Aromatase knockout mice show normal steroid-induced activation of gonadotrophin-releasing hormone neurones and luteinising hormone surges with a reduced population of kisspeptin neurones in the rostral hypothalamus

We recently reported that female aromatase knockout (ArKO) mice show deficits in sexual behaviour and a decreased population of kisspeptin‐immunoreactive neurones in the rostral periventricular area of the third ventricle (RP3V), resurrecting the question of whether oestradiol actively contributes to female‐typical sexual differentiation. To further address this question, we assessed the capacity of ArKO mice to generate a steroid‐induced luteinising hormone (LH) surge. Adult, gonadectomised wild‐type (WT) and ArKO mice were given silastic oestradiol implants s.c. and, 1 week later, received s.c. injections of either oestradiol benzoate (EB) followed by progesterone, EB alone, or no additional steroids to activate gonadotrophin‐releasing hormone (GnRH) neurones and generate an LH surge. Treatment with EB and progesterone induced significant Fos/GnRH double‐labelling and, consequently, an LH surge in female WT and in ArKO mice of both sexes but not in male WT mice. ArKO mice of both sexes had fewer cells expressing Kiss‐1 mRNA in the RP3V compared to female WT mice but had more Kiss‐1 mRNA‐expressing cells compared to WT males, reflecting an incomplete sexual differentiation of this system. To determine the number of cells expressing kisspeptin, the same experimental design was repeated in Experiment 2 with the addition of groups of WT and ArKO mice that were given EB + progesterone and sacrificed 2 h before the expected LH surge. No differences were observed in the number of kisspeptin‐immunoreactive cells 2 h before and at the time of the LH surge. The finding that ArKO mice of both sexes have a competent LH surge system suggests that oestradiol has predominantly defeminising actions on the GnRH/LH surge system in males and that the steroid‐induced LH surge can occur in females even with a greatly reduced population of kisspeptin neurones in the RP3V.     

Kisspeptin Regulates Tuberoinfundibular Dopaminergic Neurones and Prolactin Secretion in an Oestradiol‐Dependent Manner in Male and Female Rats

Prolactin (PRL) secretion is inhibited by hypothalamic dopamine. Kisspeptin controls luteinising hormone (LH) secretion and is also involved in PRL regulation. We further investigated the effect of kisspeptin‐10 (Kp‐10) on the activity of tuberoinfundibular dopaminergic (TIDA) neurones and the role of oestradiol (E₂) in this mechanism. Female and male rats were injected with i.c.v. Kp‐10 and evaluated for PRL release and the activity of dopamine terminals in the median eminence (ME) and neurointermediate lobe of the pituitary (NIL). Kp‐10 at the doses of 0.6 and 3 nmol increased plasma PRL and decreased 4‐dihydroxyphenylacetic acid (DOPAC) levels in the ME and NIL of ovariectomised (OVX), E₂‐treated rats but had no effect in OVX. In gonad‐intact males, 3 nmol Kp‐10 increased PRL secretion and decreased DOPAC levels in the ME but not in the NIL. Castrated males treated with either testosterone or E₂ also displayed increased PRL secretion and reduced ME DOPAC in response to Kp‐10, whereas castrated rats receiving oil or dihydrotestosterone were unresponsive. By contrast, the LH response to Kp‐10 was not E₂‐dependent in either females or males. Additionally, immunohistochemical double‐labelling demonstrated that TIDA neurones of male rats contain oestrogen receptor (ER)‐α, with a higher proportion of neurones expressing ERα than in dioestrous females. The dopaminergic neurones of periventricular hypothalamic nucleus displayed much lower ERα expression. Thus, TIDA neurones express ERα in male and female rats, and kisspeptin increases PRL secretion through inhibition of TIDA neurones in an E₂‐dependent manner in both sexes. These findings provide new evidence about the role of kisspeptin in the regulation of dopamine and PRL.