FSH Stores Research Articles

SAT-406 Deletion of Musashi in Gonadotropes Leads to Increased GnRHR Protein Levels and Gonadotrope Dysfunction

Leptin is a critical mediator of metabolic regulation of the hypothalamic-pituitary gonadal (HPG) axis. We have previously shown that leptin is responsible for the optimal expression of GnRHR, a rate-limiting component of the reproductive process. Female mice lacking leptin receptors (Lepr-null) specifically on gonadotropes are sub-fertile. Their reduced GnRHR proteins and normal Gnrhr mRNA levels suggest leptin’s actions on gonadotropes are post-transcriptional. A clue about a candidate translational regulator is seen in our studies showing that Lepr-null gonadotropes have increased expression of Musashi (MSI), which has binding sites within the Gnrhr 3’ UTR. Furthermore, leptin reduced MSI expression specifically in gonadotropes and increased GnRHR expression. We hypothesized that MSI may repress Gnrhr mRNA translation and that leptin alleviates this repression. To determine the effects of MSI on the HPG axis, we developed a gonadotrope-Msi1/2-null mouse line and compared mutant females (MUT) to their littermate controls (CTL) on the morning of diestrus, when the pituitary GnRHR protein levels should reach a peak. The levels of GnRHR proteins (measured by EIA) are significantly increased (1.6X) in the pituitary of the mutant females (CTL: 1.355 ± 0.11 ng/ml vs MUT: 2.202 ± 0.16 ng/ml, p=.0006), with no change in mRNA. The gonadotrope-Msi1/2-null females are subfertile, with litter sizes of 3 ± 0.4 pups, with the first litter at around day 40 and an average of 41 day delay between litters. To understand the downstream effect of the MSI knockout on gonadotropin levels, we measured pituitary and serum LH and FSH protein levels (Luminex EIA) and mRNA (qPCR). Serum FSH levels are decreased by >50% in mutant females (CTL: 0.947 ± 0.14 ng/mL vs MUT: 0.406 ± 0.05 ng/ml, p=.0049), but FSH stores and Fsh mRNAs are unchanged. Additionally, we observed a 2.2X increase in the pituitary LH protein content in mutant females (CTL: 0.328 ± 0.11 ng/ml vs MUT: 0.729 ± 0.09 ng/ml, p=.0174), but no changes in serum LH or Lh mRNA levels. These studies thus show that, as a repressor of GnRHR translation, Musashi can also regulate expression of gonadotropins. In the gonadotrope Lepr-null model, we hypothesized that leptin signals were needed to de-repress Musashi actions and allow GnRHR translation. In contrast, the gonadotrope-MSI-null mice over-express GnRHR, which confirms this role for MSI as a GnRHR regulator. We propose that the increased expression of GnRHR in the gonadotrope MSI-null animals causes the increased LH content, and disrupted FSH secretion, resulting in a higher serum LH:FSH ratio and subfertility. Collectively these data suggest that MSI regulation is necessary for optimal fertility in the adult female mouse. Future studies will determine the impact of loss of MSI on gonadotrope function throughout the estrous cycle.

The activin receptor-like kinase 6 Booroola mutation enhances suppressive effects of bone morphogenetic protein 2 (BMP2), BMP4, BMP6 and growth and differentiation factor-9 on FSH release from ovine primary pituitary cell cultures

Bone morphogenetic proteins (BMPs) have been shown to influence the regulation of FSH synthesis and secretion at the level of the pituitary. Primary pituitary cells were harvested and cultured from Booroola ewes homozygous for a mutation in activin receptor-like kinase 6 (ALK6) also known as BMP receptor IB (BMPRIB), and from wild-type (WT) ewes to determine if the mutation caused alterations in FSH secretion in vitro. The cells were collected 24 h following induction of luteolysis and cultured for 72 h prior to being challenged for 24 h with BMP2, BMP4, BMP6, growth and differentiation factor-9 (GDF9), transforming growth factor-beta 1, activin-A and GnRH. The levels of FSH and LH were measured by RIA and then compared with the untreated controls. Primary pituitary cell cultures from Booroola ewes secreted less FSH than WT cells in the presence of BMP2, BMP4 and BMP6. These BMPs did not affect the FSH stores within the cells, or the levels of LH released. GDF9 appeared to act in a BMP-like manner by suppressing FSH secretion. The ALK6 receptor however, was not found to co-localise with gonadotroph cells in either Booroola or WT pituitary tissues. These findings imply that the increased sensitivity of Booroola cells to BMP2, BMP4, BMP6 and GDF9 cannot be due to the direct action of the ALK6 mutant Booroola receptor in the cells that synthesise FSH.

Characterisation of the pituitary gonadotrophs of FSH‐receptor knockout mice

To investigate further brain‐pituitary‐gonadal interrelationships we have generated mice in which the gene encoding the FSH receptor has been disrupted (FSHRKO). FSHRKO females but not males are infertile. Compared with control animals, serum concentrations of LH and FSH and pituitary content of FSH are significantly increased in female FSHRKO (Abel et al. Endocrinology141, 2000). The aim of the present study was to correlate these physiological differences in gonadotroph function of 8‐week‐old‐female normal and FSHRKO mice with a structural analysis of their gonadotrophs. Gonadotrophs were identified by LH‐immunogold labeling for electron microscopy and gonadotroph frequency estimated from 1 (m sections immunolabelled for LH. In FSHRKO the frequency, cell and nuclear profile areas of gonadotrophs did not differ significantly from that in normal mice. Surprisingly the endoplasmic reticulum appeared normal and undilated despite the lack of oestradiol feedback in the KO animals. LH‐immunoreactive cells in female FSHRKO had significantly (P < 0.05) larger secretory granules 176(7 nm) diameter, mean(SEM) compared to normal mice 139(7 nm) but there was no significant difference in the granule area/cytoplasmic area. The subpopulation of granules in which an electron‐dense core was present in a more electron‐lucent ‘halo’ was absent in FSHRKO gonadotrophs. The increased pituitary FSH stores in FSHRKO mice therefore appear to be represented by the formation of larger secretory granules in which the packaging of hormone is less dense. Interestingly, despite being infertile and lacking biologically active estrogen, the alterations in FSHRKO pituitary morphology are relatively subtle compared to normal mice.

Differential effects of inhibin on gonadotropin stores and gonadotropin-releasing hormone binding to pituitary cells from cycling female rats.

Numerous studies of rat pituitaries have reported that inhibin suppresses the synthesis and release of FSH and decreases the release of LH. The latter effect seems to be related to the down-regulation of receptors for GnRH. The studies reported here identified cellular changes behind the inhibitory effects of inhibin on gonadotropes to learn whether its effects are mediated by changes in subtypes of gonadotropes. Cell populations from diestrous day 2 and proestrous (morning) rats were collected, dispersed to single cell populations, and plated in medium containing either recombinant 32-kDa inhibin or porcine follicular fluid for 24 h. GnRH binding was detected by exposing the cells to a biotinylated analog (Bio-GnRH) for 10 min before fixation, followed by avidin-peroxidase labeling protocols to detect the biotin on the analog. In parallel fields, the cells were further identified by immunolabeling for LH or FSH beta-subunits or for GH with a different colored reaction product. The most striking changes were seen in cells from proestrous rats. Inhibin reduced the percentages of Bio-GnRH target cells in the population by 60% and the area and density of Bio-GnRH label on the remaining cells. Inhibin reduced the percentages of FSH cells by 30% and caused nearly a 60% reduction in the binding of Bio-GnRH by this cell type (from 83% of FSH cells to 32% of FSH cells). Inhibin also reduced the area of FSH cells and the density of FSH stores. Inhibin's effects on LH cells were limited to a reduction in the area of the cells and the density of LH stores, but not the number of LH cells. In addition, it reduced the percentages of LH cells with Bio-GnRH receptors from 84% to 40%. When cells with GH were analyzed, inhibin had no effect on their percentages, areas, or GH stores. In populations from proestrous rats, inhibin reduced the percentages of GH cells with Bio-GnRH binding from 38% to 21%. These data suggest that inhibin's target cell is the abundant multihormonal gonadotrope that contains LH, FSH, and GH and predominates during proestrus. Inhibin's effects are most severe on FSH cells, which suggests that it may either selectively affect FSH synthesis and stores in bihormonal gonadotropes and/or affect monohormonal FSH cells. Thus, mechanisms behind its inhibitory effects include 1) a reduction in the percentage of Bio-GnRH target cells, 2) a reduction in the area of Bio-GnRH-binding sites on individual cells, and 3) a reduction in the stores of FSH and the percentages of FSH cells. These last effects are consistent with known reductions in FSH synthesis. The effects of inhibin on LH secretion may be secondary to the effects on Bio-GnRH receptors in bihormonal gonadotropes.

Comparison of the pretranslational regulation of FSH synthesis by gonadal steroids in rats and mice

There are significant differences between rats and mice in the gonadal regulation of several aspects of gonadotroph function. To investigate whether these extend to the pretranslational regulation of FSH synthesis by gonadal steroids, we have measured FSH-beta mRNA levels following gonadectomy and sex-steroid replacement and have related these to serum and pituitary FSH as a reflection of overall hormone synthesis. In ovariectomized rats, FSH-beta mRNA levels increased by 8 h, decreased, and then rose progressively over the next 28 days. A similar pattern of response was observed in orchidectomized rats. In mice, there were progressive increases in FSH-beta mRNA levels in both males and females following gonadectomy, without evidence of the early peaks observed in rats. In both species, the change in FSH-beta mRNA levels after gonadectomy was greater in females than in males. These changes in FSH-beta mRNA following gonadectomy were paralleled by changes in the serum FSH concentration. In ovariectomized female rats and mice, pituitary FSH stores increased by 8 h and 3 days respectively, whereas in male rats, pituitary FSH content did not rise until 10 days after orchidectomy. The most striking species difference was the marked and prolonged reduction of pituitary FSH after orchidectomy of mice. Treatment of rats and mice from the time of ovariectomy, with a dose of oestradiol that prevents increases in serum LH, only partially attenuated the rises in FSH-beta mRNA and serum FSH and did not prevent the increase in pituitary FSH content. Treatment of intact or orchidectomized rats with testosterone suppressed FSH-beta mRNA levels to 50% below intact control values without affecting pituitary FSH content.(ABSTRACT TRUNCATED AT 250 WORDS)

Minor interference of low-dose pulses of GnRH with the positive effect of oestradiol on the pituitary gland.

In the ovariectomized rat we investigated the effects of prolonged treatment with oestradiol and 'physiological' pulses of exogenous GnRH on the GnRH-responsiveness of the pituitary gland. Rats were for 20 h pre-treated with either GnRH (3-ng pulses of 3 min duration every 20 min) or saline and for 19 h with either oestradiolbenzoate (OB; 3 micrograms/sc injection) or oil. Then LH and FSH responses were evoked by continuous test infusions of GnRH at the rates of either 60, 100, 150 or 1000 ng/h, lasting 10 h. It appeared that, irrespective of pre-treatment with either GnRH or saline, OB caused an increase of the maximal LH and FSH responses (that is the responses to the maximal stimulus of 1000 ng GnRH/h), and an increase of the GnRH-sensitivity of the pituitary gland, as far as the FSH secretion is concerned. GnRH pulses caused, irrespective of pre-treatment with OB or oil, a decrease of the GnRH-sensitivity of the pituitary gland, as well as a decrease of the maximal FSH response and of the pituitary FSH content. It was concluded that low-dose pulses of exogenous GnRH desensitize the pituitary gland and deplete the pituitary FSH stores, but do not change the positive effect of oestradiol on the maximal gonadotropin response.

The depressing (negative) effect of oestradiol benzoate on the in vitro secretion of LH and FSH by the pituitary gland of the LRH-pretreated long-term ovariectomized rat changes into the augmentative (positive) effect after discontinuation of the LRH-pretreatment.

The effect of pretreatment in vivo with oestradiol benzoate on in vitro secretion of LH and FSH was studied in long-term ovariectomized (OVX) rats both at the end of a 5-day continuous in vivo pretreatment with LRH and 4-days after cessation of such LRH pretreatment. Rats were on day 0 sc implanted with osmotic minipumps which released LRH at the rate of 250 ng/h. Control rats were implanted with a piece of silicone elastomer with the dimensions of a minipump. On days 2 and 4 the rats were injected with either 3 micrograms EB or with oil. On day 5 part of the rats were decapitated and the in vitro autonomous (i.e. non-LRH-stimulated) and 'supra-maximally' LRH-stimulated release of LH and FSH was studied using a perifusion system. From other rats the minipumps were removed on day 5 and perifusion was performed on day 9. On the 5th day of the in vivo LRH pretreatment the pituitary LH/FSH stores were partially depleted; the pituitaries of the EB-treated rats more so than those of the oil-injected rats. EB alone had no significant effect on the content of the pituitary LH- and FSH stores. On day 9, i.e. 4 days after removal of the minipumps, the pituitary LH and FSH contents had increased in both the oil- and the EB injected rats, but had not yet recovered to control values. In rats not subjected to the 5-days pretreatment with LRH EB had a positive effect on the supra-maximally LRH-stimulated secretion of LH and FSH as well as on the non-stimulated secretion of LH. EB had no effect on the non-stimulated secretion of FSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Desensitization of the pituitary gland induced in vivo by luteinizing hormone-releasing hormone (LRH) or by the LRH-analogue buserelin does not affect the autonomous secretion of luteinizing hormone and follicle stimulating hormone as observed in vitro.

Three-weeks ovariectomized rats were sc implanted with Alzet osmotic minipumps which released either LRH or the LRH-analogue buserelin at the rate of 250 ng/h. Control rats were implanted with a silastic 'shampump'. After explantation, 6 days later, the pituitary glands of part of these rats were exposed to the maximally active LRH concentration of 1 micrograms/ml for a period of 6 h, using a perifusion system. In a second group of rats explantation and perifusion was done not directly, but 5 days after cessation of the LRH pre-treatment. After 6 days in vivo pre-treatment with LRH or with buserelin the pituitary LH and FSH stores were partially depleted, the depletion after buserelin being stronger than after LRH. The pituitary glands of the first group of rats showed rates of both maximally LRH-stimulated and unstimulated (autonomous) LH- and FSH-secretion which were strongly impaired, the impairment after buserelin being stronger than after LRH. In the group with a 5 days interval between in vivo LRH/buserelin pre-treatment and explantation the pituitary LH and FSH stores were restored to the range of pre-treatment levels. Of these pituitaries the autonomous secretion of LH and FSH as well as the maximally LRH-stimulated secretion of FSH was restored to the normal level; the maximally LRH-stimulated secretion of LH, however, remained depressed, indicating that 5 days after cessation of exposure to LRH or to buserelin, and in spite of restored pituitary LH/FSH contents, the sensitivity of the LH releasing system to LRH was still subnormal.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged combined in vivo pre-treatment with luteinizing hormone-releasing hormone (LRH) and oestradiol benzoate causes long-lasting suppression of the autonomous and the LRH-stimulated secretion of luteinizing hormone and follicle stimulating hormone. An in vitro study.

The effect of a combined in vivo pre-treatment with luteinizing hormone-releasing hormone (LRH) and oestradiol benzoate (EB) on the autonomous and the 'supra-maximally' LRH-stimulated in vitro release of LH and FSH by pituitary glands of 2 weeks ovariectomized (OVX) rats was studied using a perifusion system. The concentration of LRH in the perifusion medium was 1 microgram/ml. Pre-treatment with LRH during 6 days was effected by means of sc implanted Alzet osmotic minipumps (MP). Control rats received a piece of silastic with the dimensions of a minipump ('sham-pump'; Sh-P). EB, 3 micrograms/injection or solvent (arachis oil) was sc injected on days-3 and -1 (day of perifusion: day 0). Of the pituitary glands of EB-injected, Sh-P-implanted rats both the autonomous and the LRH-stimulated secretion of LH and the LRH-stimulated secretion of FSH were significantly higher than those of the oil-injected, Sh-P-implanted rats without EB administration. Pretreatment with LRH for 6 days had a suppressing effect on the autonomous and the LRH-induced depletion of the pituitary LH and FSH stores. In combination with EB, the suppressing effect of LRH pre-treatment on the LRH-stimulated secretion of LH and FSH was still greater: the pituitary gland appeared to be fixed in a relatively unresponsive state with very low autonomous LH and FSH secretion. It is discussed that increase of pituitary LRH-responsiveness due to EB demands withdrawal of the pituitary gland from the influence of LRH, an effect which is in vivo achieved by the negative feedback of oestrogen on the hypothalamus.

Pituitary FSH increase in male protein-deficient rats induced by testosterone propionate.

SummaryMale rats were placed on a purified protein-free diet for 10, 21, or 31 days. Ventral prostate weights declined almost immediately, but pituitary FSH concentrations remained at preexisting levels for at least 10 days. After 21 days of deficiency, when FSH stores had become greatly reduced, daily injections of 60 or 400 μg of TP for 10 consecutive days produced partial restoration of ventral prostate weight and pituitary FSH concentrations. The same treatment increased ventral prostate weight, but not pituitary FSH potency, of control rats fed optimal amounts of protein.

Hypothalamic control of pituitary follicle-stimulating hormone synthesis.

The possibility of the existence of a hypothalamic FSH-synthesizing factor (SF) mechanism was explored. Intravenous administration of stalk-median eminence (SME) extracts from intact, mature female rats led to depletion of pituitary FSH in intact, mature male rats and to repletion of pituitary FSH in mature male rats with previously depressed pituitary FSH levels due to ME lesions. Fortyfive min after the injections of extracts of 2 SME, pituitary FSH concentrations of the MElesioned recipients reached maximal levels, and were conspicuously higher than those of MElesioned control animals; a steady fall to depressed control hypophysial FSH stores occurred between 4 and 6 hi1 post injection. Within a dose range of 0.5–2.0 SME, a straight-line log doseresponse relationship exists for depletion of pituitary FSH in the intact rat and for augmentation of pituitary FSH in the ME-lesioned rat. The collective evidence indicates that synthesis and release are intimately coupled. Whether release or synthesis does oc...

Influence of progesterone on the pituitary and plasma levels of LH and FSH in the female rat.

In order to study the effects of progesterone on pituitary gonadotrophin levels, the steroid was administered (4 mg/day, subcutaneously) for a period corresponding to the duration of pseudopregnancy (12–14 days) to intact, unilaterally and bilaterally spayed rats (4–15 rats/group). In the bilaterally spayed rats the progesterone treatment was started on the day of ovariectomy, 43 days after ovariectomy or 43 days after ovariectomy + estrogen priming. In all experiments, pituitary levels and in some experiments involving bilaterally spayed rats, blood levels of FSH and LH were determined by HGG augmentation and OAAD methods, respectively. The administration of progesterone to intact and unilaterally spayed rats resulted in increased pituitary LH stores which appeared comparable to those which have been reported to normally occur at proestrus during the estrous cycle. In contrast, pituitary FSH stores in these experiments were increased by more than three-fold (p < .05). Progesterone treatment from the day of bilateral spaying failed to alter ovariectomy-induced increases in pituitary LH and FSH stores but was effective in decreasing postovariectomy rises in the plasma LH and FSH. In the long-term spayed rats, whether primed with estrogen or not, progesterone failed to alter pituitary stores of LH and FSH. It appears that progesterone, at least under the experimental conditions employed, in addition to decreasing the release of LH, also decreases its synthesis in the pituitary. To some extent, it is postulated, the same may be true with regard to FSH.

Studies on the mode of action of oral contraceptives: effect of chlormadinone on pituitary FSH and LH contents of the female rat.

Summary. Chlormadinone, a very potent synthetic progestagen, was administered subcutaneously for 10 days (400 \g=m\g/day)to intact, unilaterally and bilaterally spayed rats, either adult or immature, and pituitary levels of fsh (hcg augmentation method of Steelman & Pohley, 1953) and lh (oaad method of Parlow, 1958) were determined. The chlormadinone treatment induced a significant atrophy of the ovaries as well as ovarian interstitial tissue and a total blockade of ovarian compensatory hypertrophy in the unilaterally spayed rats. The pituitary concentration \g=m\g/mg) and the total content (\g=m\g/gland) of both lh and fsh were significantly increased by chlormadinone treatment of the intact and unilaterally spayed rats despite a significant atrophy of the pituitary gland due to treatment. In the bilaterally spayed adult and immature rats, chlormadinone did not alter the ovariectomy-induced rise in lh and fsh stores of the pituitary gland. These results suggest that chlormadinone, when administered for 10 days in the dosages employed, induces storage of fsh and lh in the pituitary gland primarily by impairing the release of these hormones, without exerting any detectable inhibitory effects on their synthesis.

EFFECTS OF EXOGENOUS ANDROGEN AND GONADOTROPINS ON THE TESTES AND HYPOPHYSIAL FOLLICLE-STIMULATING HORMONE CONTENT OF THE IMMATURE MALE RAT.

Administration of 50 μg of LH for 7 days to 30-day-old male rats simultaneously treated with 15, 50, 100, 200 or 300 μg of testosterone propionate (TP) maintained normal testicular weight and morphology. FSH at 200 μg daily was much less effective in preventing the testicular weight reduction associated with 50 μg of TP and did not significantly alter the effect of higher doses. Hypophysial FSH content was directly proportional to testicular weight. This relationship was maintained when FSH was added to doses of androgen up to 300 μg. However, 300 μg of TP plus FSH resulted in a 66 % drop in stored FSH. While LH prevented much of the reduction in FSH potency seen in males treated with 50 μg of TP alone, it did not alter effects produced by higher doses. After 7 days of castration pituitary weight was increased, and FSH content was significantly decreased. Administration of 50 μg of TP alone, or in combination with FSH, prevented the weight increase and elevated pituitary FSH stores, but the latter were st...

Gonadotrophic hormone function in persistent estrous rats with hypothalamic lesions.

SummaryElectrolytic lesions in pre-optic and anterior hypothalamic areas of the brain induced persistent (frequent) vaginal estrus and uterine hypertrophy in rats examined 25-300 days post-operatively. Gonad weight eventually decreased. Compensatory hypertrophy of ovary after unilateral castration was inhibited in persistent estrous lesioned rat. FSH stores in adenohypophysis were maintained but titers in serum decreased. The results signify that adenohypophysis of persistent estrous lesioned rat loses its capacity to augment secretion of FSH under conditions of enhanced demand (unilateral oophorectomy).