Pituitary Gonadotrophic Hormone Synthesis,Secretion, Subunit Gene Expression and Cell Structure in Normal and Follicle‐Stimulating Hormone β Knockout,Follicle‐Stimulating Hormone Receptor Knockout,Luteinising Hormone Receptor Knockout,Hypogonadal and Ovariectomised Female Mice

To investigate the relationship between gonadotroph function and ultrastructure, we have compared, in parallel in female mice, the effects of several different mutations that perturb the hypothalamic‐pituitary‐gonadal axis. Specifically, serum and pituitary gonadotrophin concentrations, gonadotrophin gene expression, gonadotroph structure and number were measured. Follicle‐stimulating hormone β knockout (FSHβKO), follicle‐stimulating hormone receptor knockout (FSHRKO), luteinising hormone receptor knockout (LuRKO), hypogonadal (hpg) and ovariectomised mice were compared with control wild‐type or heterozygote female mice. Serum levels of LH were elevated in FSHβKO and FSHRKO compared to heterozygote females, reflecting the likely decreased oestrogen production in KO females, as demonstrated by the threadlike uteri and acyclicity. As expected, there was no detectable FSH in the serum or pituitary and an absence of expression of the FSHβ subunit gene in FSHβKO mice. However, there was a significant increase in expression of the FSHβ and LHβ subunit genes in FSHRKO female mice. The morphology of FSHβKO and FSHRKO gonadotrophs was not significantly different from the control, except that secretory granules in FSHRKO gonadotrophs were larger in diameter. In LuRKO and ovariectomised mice, stimulation of LHβ and FSHβ mRNA, as well as serum protein concentrations, were reflected in subcellular changes in gonadotroph morphology, including more dilated rough endoplasmic reticula and fewer, larger secretory granules. In the gonadotophin‐releasing hormone deficient hpg mouse, gonadotrophin mRNA and protein levels were significantly lower than in control mice and gonadotrophs were correspondingly smaller with less abundant endoplasmic reticula and reduced numbers of secretory granules. In summary, major differences in pituitary content and serum concentrations of the gonadotrophins LH and FSH were found between control and mutant female mice. These changes were associated with changes in expression of the gonadotrophin subunit genes and were reflected in the cellular structure and secretory granule appearance within the gonadotroph cells.     

Short-Term Regulation of Gonadotropin Subunit mRNA Levels by Estrogen: Studies in the Hypothalamo-Pituitary Intact and Hypothalamo-Pituitary Disconnected Ewe

In this study the levels of mRNA for the pituitary gonadotropin hormone subunits luteinizing hormone β (LHβ), follicle stimulating hormone β (FSHβ) and the common α-subunit were assessed during the acute feedback stages of estradiol benzoate (EB) action in ovariectomized (OVX) ewes with and without hypothalamo-pituitary disconnection (HPD). In OVX/HPD ewes maintained on hourly pulses of 250 μg of gonadotropin-releasing hormone (GnRH) a single i.m. injection of EB in oil caused a biphasic (decrease and then increase) change in plasma LH levels and a monophasic decrease in FSH levels. There was a decrease in pituitary α-subunit and FSHβ mRNA levels during the acute negative (8 h post EB) and through the positive feedback (20 h post EB) stages of the response. No significant change was seen in LHβ mRNA levels following treatment with EB. In hypothalamic-pituitary intact OVX ewes the same EB treatment as above caused a biphasic change in LH secretion with the positive feedback component being much greater than in GnRH-pulsed OVX-HPD ewes. The levels of mRNA for all three gonadotropin subunits were reduced by 8 h after EB injection and remained low throughout the positive feedback period. These data suggest that the LH surge in this experimental model does not require an increase in LHB mRNA levels. Furthermore, the fall in LHβ subunit mRNA seen after estrogen injection to OVX ewes is most likely due to an effect of estrogen to decrease GnRH secretion, since pulsatile GnRH replacement prevents this effect. These data also show that estrogen feedback can effect rapid alterations in pituitary gonadotropin subunit mRNA levels. Short-term changes in FSHβ mRNA are reflected in changes in FSH secretion; the same is not true for LH.     

Changes of expression of genes related to the activity of the gonadotrophin-releasing hormone pulse generator in young versus middle-aged male rats

In females, it is well established that changes in the expression of neurotransmitters and peptides regulating the activity of the gonadotrophin-releasing hormone (GnRH) pulse generator are altered during ageing. By contrast, little is known about whether those age-related changes also occur in males. Therefore, we designed an animal study with orchidectomised young and middle-aged male rats to investigate changes in luteinising hormone (LH) secretion profiles and changes in the mRNA expression of genes regulating the activity of the GnRH pulse generator. Our results demonstrate that middle-aged rats exhibit lower serum LH levels and relatively fewer LH pulses with attenuated amplitude compared to young animals. Furthermore, upon ageing, GnRH mRNA expression is up-regulated in the preoptic area and the septum where GnRH neurones reside. Analysis of mRNA levels of glutamate decarboxylase (GAD) enzymes revealed that GAD(65) and GAD(67) mRNA expression increased in the mediobasal hypothalamus (MBH) and that GAD(67) mRNA levels decreased in the suprachiasmatic nucleus. In addition, we observed an age-related increase of oestrogen receptor (ER)alpha mRNA in the MBH, and both ERalpha and ERbeta mRNA expression was up-regulated in the pituitary of middle-aged rats compared to young animals. Taken together, our data support the existence of a male 'andropause' that is, like the menopause in females, accompanied by changes in neurotransmitter and hormone receptor expression that are involved in regulating the function of the GnRH pulse generator.     

Ontogeny of Growth Hormone and LHβ-, FSHβ- and α-Subunit mRNA Levels in the Porcine Fetal and Neonatal Anterior Pituitary

The mechanism underlying the ontogenetic increase in plasma growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentration during fetal life in mammalian species and the prenatal sex difference in these hormones in some species is not fully understood. To this end anterior pituitaries were collected from German Landrace fetuses and piglets at day (d) 50, 65, 80, 95, 110 pc and d 6 pp and pituitary GH, LHβ, FSHβ and α-subunit mRNA levels were determined by measuring Northern blot hybridization signals. GH mRNA was detected in both sexes as early as d 50 pc. The mRNA level markedly increased with age in both sexes (males>females, P≤0.05) reaching its maximum at d 95/110 pc. LHβ mRNA signals were first detected at d 50 pc in females and at d 65 pc in males increasing thereafter to a maximum at d 6 pp in both sexes (P≤0.05). In males the augmentation in LHβ mRNA was delayed compared to females (P≤0.01). Before d 80 pc no FSHβ mRNA hybridization signals were apparent. Thereafter the mRNA level continuously increased with age (P≤0.01) in both sexes reaching its maximum at d 6 pp. The FSHβ mRNA level in females was always higher than in males (P≤0.01). As early as d 50 pc the α-subunit mRNA level was high in both sexes and further increased without sex difference to d 6 pp (P≤0.05). In conclusion, the mRNA levels of GH, LHβ and FSHβ are age and sex dependent during fetal development. We suggest that the fetal increase in plasma GH concentration can be accounted for by changes in GH mRNA levels, while the dramatic perinatal decrease in plasma GH concentration seems to be primarily controlled at the posttranslational and/or secretion level. The fetal sex difference and the increase in plasma LH and FSH concentrations seems to be primarily dependent on the cellular concentration of the gonadotropin β-subunit mRNAs and/or number of gonadotrophs.     

Kisspeptin expression in guinea pig hypothalamus: effects of 17β-estradiol

Kisspeptin is essential for reproductive functions in humans. As a model for the human we have used the female guinea pig, which has a long ovulatory cycle similar to that of primates. Initially, we cloned a guinea pig kisspeptin cDNA sequence and subsequently explored the distribution and 17β-estradiol (E2) regulation of kisspeptin mRNA (Kiss1) and protein (kisspeptin) by using in situ hybridization, real-time PCR and immunocytochemistry. In ovariectomized females, Kiss1 neurons were scattered throughout the preoptic periventricular areas (PV), but the vast majority of Kiss1 neurons were localized in the arcuate nucleus (Arc). An E2 treatment that first inhibits (negative feedback) and then augments (positive feedback) serum luteinizing hormone (LH) increased Kiss1 mRNA density and number of cells expressing Kiss1 in the PV at both time points. Within the Arc, Kiss1 mRNA density was reduced at both time points. Quantitative real-time PCR confirmed the in situ hybridization results during positive feedback. E2 reduced the number of immunoreactive kisspeptin cells in the PV at both time points, perhaps an indication of increased release. Within the Arc, the kisspeptin immunoreactivity was decreased during negative feedback but increased during positive feedback. Therefore, it appears that in guinea pig both the PV and the Arc kisspeptin neurons act cooperatively to excite gonadotropin-releasing hormone (GnRH) neurons during positive feedback. We conclude that E2 regulation of negative and positive feedback may reflect a complex interaction of the kisspeptin circuitry, and both the PV and the Arc respond to hormone signals to encode excitation of GnRH neurons during the ovulatory cycle.     

5HT1A-receptor binding in the brain of cyclic and ovariectomized female rats

Although it has been reported that hypothalamic 5HT1A-receptor functioning is modulated by oestrogen and that this modulation contributes to the regulation of female sexual behaviour, there have been no reports up to now showing changes in numbers of these receptors during the oestrus cycle and after oestrogen treatment. We therefore analysed 5HT1A-receptors in eight brain areas of female rats at different stages of the oestrus cycle, and in ovariectomized (OVX) females without and with oestrogen replacement. In-vitro receptor autoradiography with the agonist 3H-8-OH-DPAT(3H-8-hydroxy-2-[di-n-propylamino]tetralin) was used to determine numbers and affinities of 5HTA1A-receptors. To evaluate the hormonal state of the animals, serum concentrations of oestradiol, progesterone, luteinizing hormone (LH), and prolactin were also measured. Hormone determinations confirmed the expected endocrine states of the animals. In the ventromedial hypothalamic nucleus, the number of 3H-8-OH-DPAT binding sites (Bmax-value) during oestrus was increased compared to dioestrus yielding significant differences when using ANOVA statistics. In OVX females, the number of binding sites was decreased compared to pro-oestrus and oestrus, and after oestrogen replacement, it was as high as during oestrus. All other brain areas analysed (medial preoptic area, bed nucleus of the stria terminalis, lateral septum, cingulate cortex, amygdala, hippocampal region CA1, and layers V and VI of the occipital cortex) showed no significant changes in 3H-8-OH-DPAT binding site numbers. Also the affinity of 3H-8-OH-DPAT binding sites did not change during the oestrus cycle, but in the medial preoptic area, oestradiol-treated OVX animals showed a tendency for increased affinity compared to untreated OVX females. This was indicated by a change in Kd which appeared to be significant when groups were compared with the t-test. We conclude from our data, that in the ventromedial hypothalamic nucleus, which is involved in the regulation of sexual function, 5HT1A-receptors are up-regulated during oestrus, that ovariectomy reduces the receptor numbers, and that oestradiol replacement counteracts the effect of ovariectomy. Since the ventromedial hypothalamic nucleus contains a high number of oestrogen receptive cells, our data indicate that oestrogen up-regulates 5HT1A-receptor expression in this nucleus.     

Altered functional responses with preserved morphology of gonadotrophic cells in congenitally athymic mice

Neonatal thymectomy or congenital absence of the thymus induces severe reproductive deficiencies in female mice, which are associated with reduced levels of circulating and pituitary gonadotropins. In contrast, the reproductive function is well preserved in nude males. It was therefore of interest to assess gonadotrophic cell morphology and function in congenitally athymic male mice. Circulating gonadotropins were measured under basal and stressful conditions, taking as a reference their haired counterparts. Adult normal (+/+), heterozygous nude (nu/+), and homozygous (nu/nu) CD-1 mice were subjected to 1-h immobilization stress. Serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were assessed by RIA at 0, 30, and 60 min poststress. Athymic animals showed significantly lower basal levels of serum LH and FSH than their heterozygous littermates. Immunohistochemical assessment of LH and FSH cell populations revealed a normal morphology and cell number in the athymic animals compared to their normal littermates. Immobilization stress induced a significant reduction in gonadotrophin levels, particularly LH, in normal mice but had only a weak effect in athymic animals. It is concluded that congenital athymia in the adult male mouse is associated with decreased basal levels of serum LH and FSH, in the presence of a normal gonadotroph number and morphology. The anomalous responses of athymic mice to stress do not appear to be due to primary hypopituitarism but, rather, to an altered modulation of pituitary hormone secretion. .     

Acute Action of Oestrogen on Medial Preoptic Gamma-Aminobutyric Acid Neurons: Correlation with Oestrogen Negative Feedback on Luteinizing Hormone Secretion

The acute effects of oestrogen on the medial preoptic area (MPOA) γ-aminobutyric acid (GABA) system were examined by delivering an intravenous bolus of 17β-oestradiol (5 μg/100 g body wt) to conscious ovariectomized rats implanted with microdialysis probes. Fifteen-min blood samples were taken to determine the time-course of negative feedback effects of oestrogen on luteinizing hormone (LH) secretion. Two h after administration of 17β-oestradiol, GABA release from the MPOA was significantly elevated compared with vehicle-treated controls (P<0.05). The rise in GABA levels continued until the end of the experiment, 4 h after 17β- oestradiol, at which time it was over 50% higher than controls (P<0.01). The pulsatile pattern of LH secretion was significantly depressed 2 and 3 h after administration of 17β-oestradiol compared with controls (P<0.05). To determine the effects of the 17β-oestradiol treatment on pituitary responsiveness to LH-releasing hormone (LHRH), a further group of rats were given exogenous LHRH (50ng/100g body wt, intravenously) before and 3 h after vehicle or 17β-oestradiol treatment and blood samples taken to determine the effect on LH secretion. The maximal LH response to LHRH in 17β-oestradiol-treated rats was approximately 50% that of control-treated values. This study demonstrates the acute and potent action of 17β-oestradiol on GABA release in the MPOA and lends support to a genomic site of action for oestrogen in modulating neural elements regulating GABA release from the MPOA. These results, showing a parallel decrease in LH secretion with increased GABA levels in the MPOA, suggest a role for GABA elements within the MPOA as a site of oestrogen negative feedback on LH secretion.     

Hormonal Regulation of GnRH and LHβ mRNA Expression in Cultured Rat Granulosa Cells

We have recently demonstrated that the rat ovary expresses LHβ, FSHβ, and the common alpha subunit mRNA. In the present report, we studied the regulation of LHβ and of gonadotropin-releasing hormone (GnRH) mRNA expression in granulosa cells that were isolated from immature rats treated with either estrogen or pregnant mare serum gonadotropin (PMSG). In both cell types, GnRH agonist treatment resulted in a decrease in LHβ mRNA expression. However, only in cells derived from PMSG-treated rats, GnRH treatment produced an increase in GnRH mRNA expression. A markedly increased GnRH mRNA expression was also obtained in granulosa cells derived from PMSG-primed rats in response to LH. In addition, FSH reduced the expression of LHβ mRNA in granulosa cells from estrogen-primed rats. These results suggest that the expression of LHβ in the ovary is regulated by locally produced GnRH and by FSH from either the ovary or the pituitary.     

Developmental programming: prenatal androgen exposure alters the gonadotroph population of the ovine pituitary gland

In utero exposure of the female foetus to androgens during development disrupts the reproductive axis and results in hypersecretion of luteinising hormone (LH) (but not follicle-stimulating hormone) in postnatal life. Abnormalities in the neural circuits controlling hypothalamic gonadotrophin-releasing hormone have been documented; however, androgens could also programme abnormalities in the pituitary gland. Ovine foetuses were exposed to either testosterone propionate or the non-aromatisable androgen dihydro-testosterone from days 30-90 of gestation (term 147 days) and the effects on the functional morphology of the pituitary were determined. Exogenous testosterone propionate exposure resulted in pituitary glands in adult male and female sheep that were 40% heavier than controls. Because this effect was not observed in the dihydro-testosterone-exposed animals, these actions are mediated via the oestrogen receptor (ER). No significant differences were apparent in 90- or 140-day foetuses. There was no difference between control and androgen-exposed animals in the density of LHβ or ERα immunoreactive cells in the pituitary although the density of follicle-stimulating hormone-β immunoreactive cells was lower in the testosterone-treated animals. The percentage of cells co-localising LHβ and ERα was lower in the testosterone-treated ewes and this may, in part, explain a reduced ability to respond to steroid feedback. Thus, enlargement of the pituitary gland, coupled with a reduced sensitivity to oestrogen negative-feedback, may contribute to the hyper-secretion of LH observed in animals that have been exposed to excess androgens during foetal life.     

Quantitative analysis of synaptic input to gonadotropin-releasing hormone neurons in normal mice and hpg mice with preoptic area grafts

Mutant hypogonadal (hpg) mice with a truncated gene for the precursor to gonadotropin-releasing hormone (GnRH) show certain aspects of recovery of reproductive function after receiving grafts of normal preoptic area into the third ventricle. We have previously shown that GnRH neurons from within the grafts can innervate the appropriate neural-hemal target in the host. To determine if in turn these exogenously derived neurons receive a synaptic input comparable to the GnRH neurons in the normal animal we have now carried out a quantitative ultrastructural analysis to compare the synaptic input to GnRH neurons in the normal preoptic area and in the grafts. In almost all cases GnRH cells or dendrites in normal brains and within the grafts received a synaptic input. In normal animals, input to GnRH dendritic profiles was significantly greater (P less than 0.001) than to the somatic plasma membrane and this trend was also observed within the grafts though the difference was not statistically significant. In addition, no statistically significant difference was found between the input to GnRH structures within the grafts and in normal preoptic area. However, a substantial variability in input among grafted animals was evident which was not observed in normal animals. The sources of variability within the grafts are discussed and we suggest that the deficiencies and differences that exist in regulation of gonadotropin secretion among grafted hpg animals may be reflected in aberrant synaptic input.     

Neuropeptide Y and Luteinizing Hormone Releasing Hormone Synergize to Stimulate the Development of Cellular Follicle-Stimulating Hormone in the Hamster Adenohypophysis

Luteinizing hormone releasing hormone (LHRH) stimulates the development of cellular FSH immunoreactivity in the perinatal hamster adenohypophysis. Because neuropeptide Y (NPY) can act directly on rat adenohypophysial cells to stimulate FSH and LH release and potentiate the stimulatory effect of LHRH on FSH and LH release, we investigated the effects of NPY alone and in combination with a low, ineffective dose of LHRH on inducing cellular FSH immunoreactivity in the neonatal hamster adenohypophysis. Neonatal female pituitary glands were grafted beneath the right renal capsules of hypophysectomized-ovariectomized adult hamster hosts with a catheter implanted in the external jugular vein. After treatment, hosts were decapitated and graft tissue was stained for FSH and LH immunoreactivity. The mean percentage of adenohypophysial cells that stained for FSH was low (2.8%) in grafts in hosts infused continuously with heparinized saline vehicle for 7 days. In other hosts, peptides were pulsed through the catheter every 12 h for 7 days. The mean percentage of FSH cells also was low after pulsing 6 ng LHRH or 2 μg NPY but increased substantially when the two peptides were pulsed simultaneously. No differences in the mean percentage of LH cells existed between any of the groups. The results demonstrate that NPY and LHRH can synergize to induce cellular FSH immunoreactivity in the neonatal female hamster.     

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.     

Comparison of Afferent and Efferent Competence of Transplanted GnRH Cells in the Brains of Hypogonadal Female Mice

A deletion in the gene encoding gonadotropin-releasing hormone (GnRH) induces hypogonadism in mice caused by the deficiency of GnRH. Activation of the reproductive axis can be achieved in these hypogonadal (hpg) mice by third cerebro-ventricular transplantation of preoptic area (POA) containing GnRH neurons, obtained from normal fetal mice. The present study was carried out in female hpg mice with POA grafts (hpg/POA) to investigate anatomical integration of the GnRH cells required for the functional activation of the reproductive system. Ovarian development was present only in mice in which the graft tissue was located close to the median eminence (ME). The total lack of ovarian development in individuals with grafts containing GnRH cells located elsewhere in the brain suggests that the mere presence of GnRH cells does not guarantee ovarian development, but that the location of the graft may be important. Activation of the grafted GnRH cells following mating, as evidenced by the induction of Fos immunoreactivity, was observed in hpg/POA mice in which there was no ovarian development or detectable GnRH immunoreactive fiber innervation of the ME. Although ovarian development was evident in individuals with grafts located close to the ME, release of luteinizing hormone (LH) in response to mating was apparent in only some of these mice. The occurrence of mating and pregnancy only in hpg/POA mice with ovarian development and the reflex release of LH in response to mating suggests that both the efferent and afferent connections of the GnRH system are important for the full functioning of the system.     

LHRH release depends on Locus Coeruleus noradrenergic inputs to the medial preoptic area and median eminence

We tested the hypothesis that Locus Coeruleus (LC) inputs to the medial preoptic area (MPOA) and median eminence (ME) are essential for gonadotropin release. Proestrus and ovariectomized (OVX) rats were decapitated at 16:00 h. LC electrolytic lesion was performed at 11:00 h during proestrus and 24h before decapitation in OVX rats. Plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured and MPOA and ME were microdissected for LHRH content measurement. In addition, FOS protein in LC and MPOA were studied in proestrus and OVX rats at 12:00, 15:00, and 17:00 h. On proestrus, LC lesion blocked the LH surge and only decreased plasma FSH; in OVX rats the lesion induced only a slight decrease on plasma LH without affecting FSH secretion. An increased content of LHRH in the MPOA and ME of both groups accompanied the decreases of plasma LH. In proestrus, the number of FOS-immunoreactive (FOS-ir) neurons increased from 12:00 to 17:00 h in the LC and MPOA. In OVX rats, there was an increase at 15:00 h in the LC and a decrease at 17:00 h in both areas. The number of FOS-ir neurons was lower in OVX than in proestrus animals. Thus, LC (1) is responsible, at least in part, for gonadotropin release through the activation of LHRH neurons, (2) is more closely related to the positive than the negative feedback, and (3) seems to show an intrinsic cyclic activity which is amplified by ovarian steroids.     

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.     

Lamprey gonadotropin hormone-releasing hormone-III has no selective follicle-stimulating hormone-releasing effect in rats

Lamprey gonadotropin releasing-hormone (LGnRH)-III, a hypothalamic neurohormone recently isolated from sea lamprey, was reported to have a selective stimulatory effect on follicle-stimulating hormone (FSH) release in rats and suggested to be the mammalian FSH-releasing factor. In this study, we determined the relative luteinizing hormone (LH)- and FSH-releasing potency of LGnRH-III compared to mammalian gonadotropin-releasing hormone (LHRH) in normal female rats, ovariectomized (OVX) and oestrogen/progesterone substituted rats and the superfused rat-pituitary cell system. The specificity of LGnRH-III for the mammalian LHRH receptor was investigated by blocking the receptor with an LHRH antagonist, MI-1544. In vitro, LGnRH-III dose-dependently stimulated both LH and FSH secretion from rat pituitary cells at 10(-7) to 10(-5) M concentrations, while LHRH stimulated gonadotropin secretion at a 1000-fold lower doses (10(-10) to 10(-8) M). The difference between its LH- and FSH-releasing potency was similar to that of LHRH. LGnRH-III bound to high affinity binding sites on rat pituitary cells with a Kd of 6.7 nM, B(max)=113 +/- 27 fmol/mg protein. In vivo, LGnRH-III also stimulated both LH and FSH secretion in a dose-dependent manner and, similar to LHRH, induced a greater rise in the serum LH than the FSH level. In normal cycling rats, it showed 180-650-fold weaker potency than LHRH in stimulating LH secretion and 70-80-fold weaker effect in stimulating FSH secretion. In OVX rats, LGnRH-III demonstrated a similarly weak effect on both gonadotropins. It was found to be 40-210-fold less potent than LHRH regarding LH release and 50-160-fold weaker regarding FSH release. LHRH-receptor antagonist MI-1544 prevented both the LH- and the FSH-releasing effect of LGnRH-III both in vitro and in vivo. These results do not support the hypothesis that LGnRH-III might be the mammalian FSH-releasing factor but demonstrate that it is a weak agonist for the pituitary LHRH receptor and stimulates both gonadotropins in a dose-dependent fashion.