Regulation of the hypothalamic--pituitary--ovarian axis in women.

To account for the regulation of cyclic gonadotrophin release, the separate and interactive effects of the hormonal variable at the levels of CNS-hypothalamus, the pituitary and the ovary have been reviewed. The pituitary gonadotrophs, as target cells exhibited a remarkable cyclic change in their capacity which was correlated with the oestradiol levels. The ultimate release is determined by the relative size of the two pools' releasable gonadotrophins which are themselves regulated by the relative inputs of LH-RH and oestradiol, respectively. LH-RH appears to serve as a primary influence on the gonadotroph, stimulating gonadotrophin synthesis, storage and release. Oestradiol, for the most part, amplifies the action of LH-RH and induces the development of a self-priming effect of LH-RH, except that it impedes LH-RH-mediated gonadotrophin release. The pituitary capacity increases several-fold from the early to late follicular phase, and this is considered to be the prerequisite for the development of a mid-cycle surge. CNS-hypothalamic dopamine, norepinephrine, prostaglandins as well as LH-RH systems are involved in the negative and positive feedback effect of oestradiol. The possible steps and interactive elements in the triggering of LH-RH release for the initiation of the mid-cycle LH/FSH surge are considered.     

Gonadotroph-lactotroph associations and expression of prolactin receptors in the equine pituitary gland throughout the seasonal reproductive cycle

An interaction between gonadotroph and lactotroph cells of the pituitary gland has long been recognized in several species. The current study was conducted to investigate whether an association between gonadotrophs and lactotrophs occurs in mares and whether prolactin receptors are expressed within the pituitary gland of this species. The effects of both reproductive state and season on these variables were examined in pituitary glands obtained from sexually active mares in July (breeding season), sexually active mares in November (non-breeding season) and anoestrous mares in November. Pituitaries were dissected out immediately after death and immunofluorescent staining was carried out on 6 micrometer sections using specific antibodies to the LHbeta subunit, FSHbeta subunit, prolactin and prolactin receptor. Gonadotrophs were observed in both the pars distalis and pars tuberalis; although they appeared mostly as isolated cells, small groups of gonadotrophs were also identified in the pars distalis. In contrast, lactotrophs were observed only as clusters of cells exclusively in the pars distalis of sexually active and anoestrous mares in November and in most of the sexually active mares in July. A specific gonadotroph-lactotroph association was identified only between large isolated gonadotrophs and lactotroph clusters. Double immunofluorescent staining for FSHbeta and prolactin revealed a similar gonadotroph-lactotroph association to the one detected for LH gonadotrophs. No statistical difference in the gonadotroph:lactotroph ratio was observed as a result of changes in reproductive status or season. However, a tendency for a simultaneous decrease in the number of gonadotrophs and an increase in the number of lactotrophs was detected in anoestrous animals. Prolactin receptor immunoreactivity was found in the pars distalis, but not in the pars tuberalis, of sexually active (July and November) and anoestrous animals for both long and short forms of the receptor. No prolactin receptor co-localization for either form of the receptor was observed in LH or FSH gonadotrophs in either of the reproductive states examined during both summer and winter seasons. Furthermore, no significant difference was apparent in the proportion of cells expressing prolactin receptors between mares of different reproductive state or season. The specific anatomical association between gonadotroph and lactotroph cells and the expression of prolactin receptors in the equine pituitary gland indicate a potential role of prolactin in the regulation of gonadotrophin secretion. However, the absence of evidence for co-localization of prolactin receptors in LH or FSH cells does not support the hypothesis of a direct effect of prolactin on the gonadotroph as reported in a short day breeder. The results raise the possibility that, in horses, an intermediate regulatory cell may mediate the action of prolactin on gonadotroph function.     

Characterization and distribution of gonadotrophs in the pars distalis and pars tuberalis of the equine pituitary gland during the estrous cycle and seasonal anestrus

Little is known about the neuroendocrine control of fertility in the horse. In this species, unusual features characterize the normal estrous cycle such as a prolonged preovulatory LH surge during the follicular phase and a distinctive FSH surge during the midluteal phase. This study investigated the distribution and hormonal identity of gonadotrophs in the pars distalis (PD) and pars tuberalis (PT) of the equine pituitary gland as possible morphological bases for the referred unusual endocrine characteristics. In addition, the proportion of gonadotrophs in relation to other pituitary cell types during both the estrous cycle and anestrus were investigated. Pituitary glands were collected from sexually active (n = 5) and seasonally anestrous (n = 5) mares in November, and single or double immunofluorescent staining was carried out on 6-microm sections using monoclonal antibodies to the LHbeta or FSHbeta subunits and a polyclonal antibody to ovine LHbeta. Gonadotrophs were densely distributed around the pars intermedia in the PD and in the caudal ventral region of the PT. In addition to isolated cells, clusters of gonadotrophs were found surrounding the capillaries. No significant differences were detected in the number of gonadotrophs between sexually active and anestrous mares in either the PD or PT. In the PD, gonadotrophs represented 22.7 +/- 5. 8% and 19.1 +/- 2.1% of the total cell density in sexually active and anestrous animals, respectively (P: > 0.05). However, in the PT, gonadotrophs accounted for a higher proportion of the total cell population in sexually active (6 +/- 0.1%) than in anestrous (1.2 +/- 0.05%) mares (P: < 0.02). Double immunofluorescence revealed that the majority of gonadotrophs were bihormonal (i.e., positive for LH and FSH); however, in the sexually active mare, a larger proportion of gonadotrophs (22.5 +/- 3.6%) were monohormonal for either LH or FSH, when compared to anestrous animals (9.7 +/- 1.2%; P: < 0.02). Based on these findings we conclude that: 1) although the relative distribution of gonadotrophs is similar to those reported for other species, a significantly larger proportion of gonadotroph cells is present in the equine pituitary gland; 2) gonadotroph density does not appear to differ between sexually active and anestrous mares in the PD; 3) a larger proportion of gonadotrophs is apparent in the PT of sexually active animals; and 4) although a large incidence of bihormonal gonadotrophs is present in the horse, specific LH or FSH cells differentiate predominantly during the sexually active phase.     

Receptor-mediated endocytosis of GnRH analogs: differential processing of gold-labeled agonist and antagonist derivatives

The hypothalamic decapeptide, GnRH, stimulates LH and FSH release from pituitary gonadotrophs. Many synthetic peptide analogs, both agonist (GnRH-A) and antagonist (GnRH-AT), have been developed which bind specifically to the GnRH receptor. We have utilized highly potent GnRH-A and GnRH-AT analogs labeled with 18 nm colloidal gold to analyze ultrastructurally the events of binding and interiorization of these specific ligands by gonadotrophs in vitro. To examine internalization of GnRH-A-gold, gonadotrophs were cooled to 4 degrees C and equilibrated with the ligand for 1 h. Next, the cells were either fixed immediately or warmed to 37 degrees C for various times (5, 15 and 30 min) and prepared for electron microscopy. For GnRH-AT-gold, which binds slowly at 4 degrees C, the ligand was incubated with gonadotrophs at 37 degrees C for 15, 30 and 60 min, and the cells were processed for electron microscopy at each time point. In both cases, control gonadotrophs were also incubated in an excess of GnRH-A and GnRH-AT, respectively, in the presence of the gold-conjugated ligands. The results indicated that GnRH-A-gold was bound and rapidly internalized via a receptor-mediated endocytic pathway. GnRH-AT-gold was also bound but showed only limited entry into gonadotrophs; the percentage of intracellular GnRH-AT-gold in gonadotrophs was the same as in other pituitary cells contaminating the gonadotroph fraction and did not increase with time. In the gonadotroph, binding of the specific antagonist ligand to GnRH receptors does not stimulate its interiorization, in contrast to the rapid endocytosis and processing of the agonist ligand. These data suggest that specific ligand internalization requires prior receptor activation, and that GnRH-AT which does not activate the receptor remains bound at the cell surface for a prolonged period.     

Phosphatidic acid and the calcium-dependent actions of gonadotropin-releasing hormone in pituitary gonadotrophs

The stimulation of luteinizing hormone (LH) release and cyclic GMP (cGMP) production in rat anterior pituitary cells by gonadotropin-releasing hormone (GnRH) are receptor mediated and calcium dependent, and have been shown to be accompanied by increased phospholipid turnover and arachidonic acid release. The incorporation of 32Pi into the total phospholipid fraction of pituitary gonadotrophs was significantly elevated by 10(-8) M GnRH, with specific increases in the labeling of phosphatidylinositol and phosphatidic acid (PA). Since PA acts as a calcium ionophore in several cell types, its effects upon calcium-mediated gonadotroph responses were compared with those elicited by GnRH. In rat pituitary gonadotrophs prepared by centrifugal elutriation, PA stimulated LH release and cGMP production by 9-fold and 5-fold, respectively. The stimulation of LH release by 30 microM PA was biphasic in its dependence on extracellular calcium concentration, rising from zero in the absence of calcium to a maximum of 10-fold at 0.5 mM Ca2+ and declining at higher calcium concentrations. In dose-response experiments, PA was 3-fold more potent at 0.5 mM Ca2+ than at 1.2 mM Ca2+. The cGMP response to PA in cultured gonadotrophs was also calcium dependent, and was progressively enhanced by increasing Ca2+ concentrations up to 1.5 mM. The ability of PA to stimulate both LH release and cGMP formation in a calcium-dependent manner suggests that endogenous PA formed in response to GnRH receptor activation could function as a Ca2+ ionophore in pituitary gonadotrophs, and may participate in the stimulation of gonadotroph responses by GnRH and its agonist analogs.     

Morphological changes of pituitary gonadotrophs and thyrotrophs following treatment with LH-RH or TRH in vitro

Summary To investigate the relationship between LH-RH and TRH and the formation of castration cells and thyroidectomy cells, pituitary glands of 14-day old female rats were cultured with LH-RH or TRH for 10 days. Observed in these glands were external and internal zones, the former containing active, healthy appearing cells, and the latter consisting of many degenerative and necrotic cells. Gonadotrophs and thyrotrophs were readily demonstrated in the external zone of the organs by immunocytochemistry. Hypertrophie gonadotrophs (castration cells) and thyrotrophs (thyroidectomy cells) were observed only in the external zone by electron microscopy. Neither the typical signet-ring gonadotrophs nor intracisternal granules of thyroidectomy cells were seen in the present study. However, the hypertrophic gonadotrophs or thyrotrophs were similar to castration cells or thyroidectomy cells observed in the pituitary gland following castration or thyroidectomy. The results indicate that LH-RH and TRH directly stimulate the secretion of hormones and alter the morphological features of pituitary target cells.Supported by USPHS Grant HD 11826. The author wishes to express his thanks to NIAMDD for providing pituitary hormones     

Cytological changes in hypophysis of immatur rats after administration of anti-LH antibodies (author's transl)]

In the present work, cytological changes produced in rats' pars distalis were studied by administration of anti-LH antibodies. The anti-LH sera was obtained by active immunization of adult rabbits using bovine LH hormone with Freund's complete adjuvant. Newborn albino Wistar male and female rats were daily and subcutaneously inoculated with 0.1 ml. to 0.8 ml of anti-LH sera from the first 24 hr. on during 5 weeks. For controls, a similar schedule of inoculations with normal rabbit serum was used. At weekly intervals during the treatment, 5 lots of rats were sacrificed and the rest after a recovery period of 95 days. At the end of the 2nd week of immunization, both male and female animals showed degranulated gonadotroph cells in the central part of the pars distalis; at the 3rd week these cells were hypertrophied and presented developed Golgi complex. At the 4th week, the first large vacuoles of "castration" were seen in the gonadotroph cells of male pars distalis, besides in the females the degranulation of gonadotrophs continued. During the last week of treatment, the gonadotrophs of the male animals presented a highly dilated Golgi complex, more "castration-like cells" and numerous mitosis figures. The gonadotrophs of females pars distalis presented degranulation, but not vacuoles. After the recovery period the gonadotrophs of male rats were similar to those of the control hypophisis and did not show castration vacuoles in their cytoplasm. In female rats, the gonadotrophic cells showed "castration-like vacuoles" and a raised number of mitosis cells compared to control pituitaries. The significance of these findings will be discussed.     

Hypersensitivity of the human gonadotrophs to the repeated administration of small doses of LH-RH.

The functional capacity of the gonadotrophs was assessed by repeated stimulation with small doses of LH-RH (5 microgram intravenously at 2-hour intervals for 3 injections) in normal women during the early and late follicular phases of the menstrual cycle. The results were compared to those obtained when a single dose (100 microgram) of the neurohormone was administered. During the early follicular phase, the release of LH and FSH remained about equal after the 3 successive injections of the small and after the large dose of LH-RH. During the late follicular phase, the release of LH and fsh increased progressively after the repeated administration of the 5 microgram of the neurohormone while the large dose induced a more pronounced and a more sustained pituitary response. This hypersensitivity of the gonadotrophs is observed when the E2 concentrations are higher than in the early follicular phase of the menstrual cycle.     

Effect of an antagonistic analog of LH-RH on haloperidol-induced hyperprolactinemia in female rats

The effects of prolonged treatment with the antagonistic analog of LH-RH (N-Ac-D-p-Cl-Phe1,2, D-Trp3,D-Arg6,D-Ala10) LH-RH (ORG 30276) on the hyperprolactinemia induced by haloperidol were investigated in intact or ovariectomized female rats. Treatment with ORG 30276 for 20 days significantly reduced prolactin levels elevated by daily injections of haloperidol in intact as well as in ovariectomized rats. Administration of ORG 30276 also significantly decreased serum LH levels in both types of rats. It is concluded that the LH-RH antagonist ORG 30276 is able to counteract the hyperprolactinemic effect of haloperidol. This effect might be due to a blockade of the action of endogenous LH-RH on the gonadotrophs, which results in a suppressing of the paracrine action of these cells on the lactotroph.     

Ultrastructure of rat pituitary LH gonadotrophs in relation to serum and pituitary LH levels following repeated LH-RH stimulation

The effects of single and repeated LH-RH injections at 120 min intervals on female rat LH gonadotrophs and on pituitary and serum LH levels were investigated using electronmicroscopy and radioimmunoassay. A temporary stimulation of granule release, of protein and new granule synthesis and of the accumulation of lysosomal structures was found in LH cells after the first LH-RH injection. The temporary stimulations were massively enhanced after the second injection. These consecutive yet in their time-sequence overlapping processes account for the initial depletion of secretory granule content (3--15 min after LH-RH injection), for the subsequent regranulation and accumulation of granules above control levels (60--120 min after injection) and also for the reduction in the number of granules to control levels (150 min after LH-RH injection and thereafter). Increased polymorphic lysosomal structures are believed to be responsible for this reduction of excess granules. The amount of assayable pituitary and serum LH generally corresponds with the morphological changes observed in LH-gonadotrophs, thus further substantiating the above observations. A schema which summarizes the observed morphological and hormonal changes in their time-sequence in response to LH-RH stimulation depicts the short-term regulation of secretory processes in female gonadotrophs.     

Receptors for luteinizing hormone-releasing hormone.

Antisera to luteinizing hormone-releasing hormone (LH-RH) confer on Araldite sections of occasional rat pituitaries moderate immunocytochemical staining to the large secretory granules of gonadotrophs. Treatment of the sections with LH-RH before anti-LH-RH yields strong staining in all animals, irrespective of presence or absence of staining without pretreatment. This enhancement of staining is specific for LH-RH and is a high affinity, saturable reaction. Staining with or without LH-RH pretreatment is absent when anti-LH-RH absorbed with insolubilized LH-RH is used. Staining is inhibited by carboxyterminally-deficient LH-RH, unaffected by aminoterminally deficient LH-RH.     

Maintenance of gonadotrophs in pituitary autografts under the kidney capsules of female rats given sex hormones or LRH

Female Sprague-Dawley rats were hypophysectomized and the anterior pituitary gland was immediately placed under the kidney capsule. For 1 week after surgery, groups of pituitary autograft-bearing animals were treated with twice-daily injections of estradiol 17 beta (E), progesterone (P), estradiol 17 beta and progesterone (EP), or luteinizing hormone-releasing hormone (LRH). Within 2--4 hours following the last injection, the pituitary grafts were removed and placed into organ culture. They were maintained in culture with or without added LRH (10(-7) M) for 1 hour at 37 degrees C. The culture media were then frozen for later radioimmunoassay of FSH and LH. The tissues were kept in culture for an additional 24 hours, at which time they were fixed and prepared for immunocytochemistry or electron microscopy. Results showed that treatment of the animals with E, EP, or LRH enhanced the release of FSH and LH into the culture media, and that the release of these hormones was increased further by acute incubation with LRH. The ultrastructure of the gonadotrophs was well maintained by treating the animals with E or the combination of E and P or with LRH. Graft tissue from animals treated with LRH, which was incubated subsequently for 24 hours with LRH, showed the best maintenance of gonadotroph morphology. This experimental procedure should be useful for obtaining gonadotrophs for use in establishing gonadotroph cell lines.     

Quantitative immunocytochemistry of pituitary receptors for luteinizing hormone-releasing hormone

In Araldite sections of male rat pituitaries, stained after embedding by the unlabeled antibody enzyme method with antisera to native luteinizing hormone-releasing hormone (LH-RH) or LH-RH azo-conjugated to bovine serum albumin, localization is confined mainly to the interior of the large, and to a lesser extent to that of the small, secretion granules of the gonadotrophic cells. Plasma membranes are not demonstrated. Except for weak staining in the granules of corticotrophs, no other pituitary cell is stained. Pretreatment of sections with LH-RH (to dilutions of 4 pg/mul) increases staining intensity in the gonadotrophic granules. Other cells are unaffected. The lesser the gonadotroph staining intensity without pretreatment, the greater the increase (more than 23-fold reactivity). Augmented staining is measurable (P less than 0.001) to antiserum dilutions of 1:240000. Pretreatment with des-Glu-1-LH-RH, porcine corticotropin or rat prolactin has no effect. LH-RH-Gly-10(des-amide) inhibits. Rat glycoprotein hormones enhance staining with anti-azo-conjugated LH-RH. With antinative LH-RH these hormones enhance weak staining, but inhibit strong staining. Thick vibrotome sections of male rat or rabbit pituitaries stained before embedding reveal specific localization on plasma membrane and gonadotrophic secretion granules provided the sections have been pretreated with LH-RH (250 pg/mul). The data show that LH-RH after reaction with receptor is not sterically hindered from binding specific antibodies. Receptor may be found in secretion granules, both in the free state or combined with LH-RH. Plasma membrane receptor, on the other hand, was free under the conditions of the experiments. Immunization with LH-RH elicits not only heteroimmune antibodies specific for LH-RH, but also a group of still ill defined autoimmune antibodies, some of which may conceivably be reactive with glycoprotein hormone alpha-chains.     

Morphology of the mammotrophs and gonadotrophs in the anterior pituitary gland of rats with protein-calorie malnutrition

The structure of the anterior pituitary gland of rats with protein-calorie malnutrition was studied at the light and electron microscopic levels. Male Sprague-Dawley rats were fed a low-protein diet from 20 to 50 days of age. The hypophyses from these animals, along with those from age-matched controls, were then removed and fixed for light microscopic immunocytochemical analyses, using anti-rat PRL or anti-ovine LH beta serum, or for routine ultrastructural study. The overall number of mammotrophs and gonadotrophs appeared to be unaffected by the deficiency in dietary protein. However, the nuclei, in both cell types, were significantly smaller in the malnourished rats. Cytoplasmic volume was also apparently less than in control rats, leading to the "crowding" of adjacent cell types. Additionally, in the malnourished rats, the mammotrophs assumed a less polygonal profile, while the gonadotrophs, which were more irregularly shaped in the central zone, rarely displayed the negative image of a Golgi complex. Ultrastructurally, the mammotrophs in the experimental rats had a less extensively developed granular endoplasmic reticulum and Golgi complex. The number of secretary granules was not different; however, their size was markedly less and their shape was round to ovoid, rather than pleomorphic. Two types of gonadotrophs were distinguished, based on the classification of Kurosumi et al. ('76). The type-I cell (former "FSH" cell) had a smaller Golgi complex, less vesicular endoplasmic reticulum and smaller secretory granules. The major alteration in the type-II gonadotroph (former "LH" cell) was the smaller size of the secretory granules. The morphological changes found in this study support our previous physiological observations (Herbert, '80), which demonstrate that prolactin and gonadotrophin secretion are severely impaired in rats suffering from protein-calorie malnutrition.     

Subcellular localization of gonadotrophic hormones LH and FSH in frog adenohypophysis using double-staining immunocytochemistry

We applied double post-embedding immunocytochemical methods using specific antibodies against bullfrog (Rana catesbeiana) luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with immunogold staining (5- and 20-nm particles) to determine the subcellular localization of both gonadotropins and to observe their immunostaining patterns in anterior pituitary of the frog Rana pipiens. Results showed that individual gonadotrophs may store either one or both gonadotropins in a given secretory granule and in large globules (lysosomes?). Most gonadotrophs (50-88%) contain both hormones; 12-50% contain only FSH, and only a few (0-7%) contain LH alone. Individual secretory granules, even in cells that contain both hormones, may contain only one or both gonadotropin molecules. Evaluation of the percentage of monohormonal and multihormonal secretory granules revealed that multihormonal secretory granules were the most numerous and that LH monohormonal secretory granules were the least numerous. These results indicate that cellular storage of gonadotropin in amphibian pituitary is similar to that described for mammals, where a single cell type containing both gonadotropins predominates. Variability in hormone content both of cells and of granules in all individuals is consistent with the hypothesis that frog pituitary possesses a single multipotential gonadotroph.     

丘脑下部促黄体素释放激素类似物(LRH-A)的作用机制 Ⅲ.对罗非鱼脑垂体促性腺激素分泌细胞超微结构的影响

前文(方永强等,1981)探讨了罗非鱼脑垂体促性腺细胞的生理活动与雌鱼性腺发育不同时期的相互关系,对比注射LRH-A后2、4和10小时对促性腺细胞的影响。根据促性腺细胞核的形态和细胞质中分泌颗粒数量的变化,以及DNA、RNA、酸碱性蛋白和酸碱性磷酸酶等一系列反应,初步阐明了LRH-A的作用机制与细胞核和细胞质的代谢变化有关。在此基础上,本文较详细观察了LRH-A对垂体促性腺细胞超微结构的影响,目的在于从亚细胞的水平上,进一步了解其作用机制。     

Ultrastructural observations of granule extrusion from rat anterior pituitary cells after injection of LH-releasing hormone

Summary In order to observe the ultrastructural aspects of granule extrusion from gonadotrophs, the authors injected pure, natural porcine LH-releasing hormone (LH-RH) into persistent-estrous (PE) rats. Clear-cut extrusions of secretory granules appeared from LH-gonadotrophs 2.5, 5 and 15 minutes after the injection. Massive extrusion was observed at 15 minutes. Serum LH and FSH were also estimated by radioimmunoassay on blood samples taken at the same times after the injections. LH was increased in the blood sera after the injection of LH-RH, but serum FSH was not significantly different among the various treated rats. The rise in serum LH after LH-RH injection was well correlated with the ultrastructural phenomenon of granule extrusion from LH-gonadotrophs.The study was supported by USPHS Grants AM 12583, AM 09094 and AM 07467.The authors acknowledge the able assistance of Mrs. Martha Castilleja in San Antonio and of Meredith M. Nickel in New Orleans.