Prolactin (PRL) is known to suppress LH secretion. Kisspeptin neurons regulate LH secretion and express PRL receptors. We investigated whether PRL acts on kisspeptin neurons to suppress LH secretion in lactating (Lac) and virgin rats. Lac rats displayed high PRL secretion and reduced plasma LH and kisspeptin immunoreactivity in the arcuate nucleus (ARC). Bromocriptine-induced PRL blockade significantly increased ARC kisspeptin and plasma LH levels in Lac rats but did not restore them to the levels of non-Lac rats. Bromocriptine effects were prevented by the coadministration of ovine PRL (oPRL). Virgin ovariectomized (OVX) rats treated with either systemic or intracerebroventricular oPRL displayed reduction of kisspeptin expression in the ARC and plasma LH levels, and these effects were comparable with those of estradiol treatment in OVX rats. Conversely, estradiol-treated OVX rats displayed increased kisspeptin immunoreactivity in the anteroventral periventricular nucleus, whereas oPRL had no effect in this brain area. The expression of phosphorylated signal transducer and activator of transcription 5 was used to determine whether kisspeptin neurons in the ARC were responsive to PRL. Accordingly, intracerebroventricular oPRL induced expression of phosphorylated signal transducer and activator of transcription 5 in the great majority of ARC kisspeptin neurons in virgin and Lac rats. We provide here evidence that PRL acts on ARC neurons to inhibit kisspeptin expression in female rats. During lactation, PRL contributes to the inhibition of ARC kisspeptin. In OVX rats, high PRL levels suppress kisspeptin expression and reduce LH release. These findings suggest a pathway through which hyperprolactinemia may inhibit LH secretion and thereby cause infertility.

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.

Kisspeptin (Kp) regulates prolactin (PRL) in an estradiol-dependent manner. We investigated the interaction between ovarian steroid receptors and Kp in the control of PRL secretion. Intracerebroventricular injections of Kp-10 or Kp-234 were performed in ovariectomized (OVX) rats under different hormonal treatments. Kp-10 increased PRL release and decreased 3,4-dihydroxyphenylacetic acid levels in the median eminence (ME) of OVX rats treated with estradiol (OVX+E), which was prevented by tamoxifen. Whereas these effects of Kp-10 were absent in OVX rats, they were replicated in OVX rats treated with selective agonist of estrogen receptor (ER)α, propylpyrazole triol, but not of ERβ, diarylpropionitrile. Furthermore, the Kp-10-induced increase in PRL was two times higher in OVX+E rats also treated with progesterone (OVX+EP), which was associated with a reduced expression of both tyrosine hydroxylase (TH) and Ser40-phosphorylated TH in the ME. Kp-10 also reduced dopamine levels in the ME of OVX+EP rats, an effect blocked by the progesterone receptor (PR) antagonist RU486. We also determined the effect of Kp antagonism with Kp-234 on the estradiol-induced surges of PRL and luteinizing hormone (LH), using tail-tip blood sampling combined with ultrasensitive enzyme-linked immunosorbent assay. Kp-234 impaired the early phase of the PRL surge and prevented the LH surge in OVX+E rats. Thus, we provide evidence that Kp stimulation of PRL release requires ERα and is potentiated by progesterone via PR activation. Moreover, alongside its essential role in the LH surge, Kp seems to play a role in the peak phase of the estradiol-induced PRL surge.

Hyperprolactinemia causes infertility by suppressing gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion. Because effects of prolactin (PRL) on the hypothalamus usually require estradiol (E2), we investigated the role of E2 in PRL-induced suppression of LH pulses. Ovariectomized (OVX) rats treated with oil or E2 (OVX + E2) received a subcutaneous injection of ovine PRL (oPRL) 30 minutes before serial measurement of LH in the tail blood by enzyme-linked immunosorbent assay. E2 reduced pulsatile LH secretion. oPRL at 1.5 mg/kg further reduced LH pulse frequency in OVX + E2 but had no effect in OVX rats. The higher dose of 6-mg/kg oPRL decreased LH pulse frequency in both OVX and OVX + E2 rats, whereas pulse amplitude and mean LH levels were lowered only in OVX + E2 rats. Kisspeptin immunoreactivity and Kiss1 messenger ribonucleic acid (mRNA) levels were decreased in the arcuate nucleus (ARC) of OVX + E2 rats. oPRL decreased both kisspeptin peptide and gene expression in the ARC of OVX rats but did not alter the already low levels in OVX + E2 rats. In the anteroventral periventricular nucleus, oPRL did not change kisspeptin immunoreactivity and, paradoxically, increased Kiss1 mRNA only in OVX + E2 rats. Moreover, oPRL effectively reduced Gnrh expression regardless of E2 treatment. In this study we used tail-tip blood sampling to determine the acute effect of PRL on LH pulsatility in female rats. Our findings characterize the role of E2 in the PRL modulation of hypothalamic components of the gonadal axis and LH release, demonstrating that E2 potentiates but is not essential for the suppression of pulsatile LH secretion caused by hyperprolactinemia.

The locus coeruleus (LC) has been suggested as a CO2 chemoreceptor site in mammals. Most of the studies involving the role of the LC in hypercapnic ventilatory responses have been performed in males. Since ovarian steroids modulate the activity of LC neurons and females have a different respiratory response to CO2 than males, we evaluated the activity of LC noradrenergic neurons during normocapnia and hypercapnia in female and male rats with distinct sex hormone levels. Ovariectomized (OVX), estradiol (E2)-treated ovariectomized (OVX+E2) and female rats on the diestrous day of the estrous cycle were evaluated. Concurrently, males were investigated as gonad-intact, orchidectomized (ORX), testosterone (T)-treated ORX (ORX+T), and E2-treated ORX (ORX+E2). Activation of LC neurons was determined by double-label immunohistochemistry to c-Fos and tyrosine hydroxylase (TH). Hypercapnia induced by 7% CO2 increased the number of c-Fos/TH-immunoreactive (ir) neurons in the LC of all groups when compared to air exposure. Hypercapnia-induced c-Fos expression did not differ between diestrous females and intact male rats. In the OVX+E2 group, there was attenuation in the c-Fos expression during normocapnia compared with OVX rats, but CO2 responsiveness was not altered. Moreover, in ORX rats, neither T nor E2 treatments changed c-Fos expression in LC noradrenergic neurons. Thus, in female rats, E2 reduces activation of LC noradrenergic neurons, whereas in males, sex hormones do not influence the LC activity.