Anterior Pituitary Gonadotropes Research Articles

The expression, regulation and signal transduction pathways of the mammalian gonadotropin-releasing hormone receptor

Normal mammalian sexual maturation and reproductive functions require the integration and precise coordination of hormones at the hypothalamic, pituitary, and gonadal levels. Hypothalamic gonadotropin-releasing hormone (GnRH) is a key regulator in this system; after binding to its receptor (GnRHR), it stimulates de novo synthesis and release of gonadotropins in anterior pituitary gonadotropes. Since the isolation of the GnRHR cDNA, the expression of GnRHR mRNA has been detected not only in the pituitary, but also in extrapituitary tissues, including the ovary and placenta. It has been shown that change in GnRHR mRNA is one of the mechanisms for regulating the expression of the GnRHR. To help understand the molecular mechanism(s) involved in transcriptional regulation of the GnRHR gene, the 5' flanking region of the GnRHR gene has recently been isolated. Initial characterization studies have identified several DNA regions in the GnRHR 5' flanking region which are responsible for both basal expression and GnRH-mediated homologous regulation of this gene in pituitary cells. The mammalian GnRHR lacks a C-terminus and possesses a relatively short third intracellular loop; both features are important in desensitization of many others G-protein coupled receptors (GPCRs), Homologous desensitization of GnRHR has been shown to be regulated by various serine-threonine protein kinases including protein kinase A (PKA) and protein kinase C (PKC), as well as by G-protein coupled receptor kinases (GRKs). Furthermore, GnRHR was demonstrated to couple with multiple G proteins (Gq/11, Gs, and Gi), and to activate cascades that involved the PKC, PKA, and mitogen-activator protein kinases. These results suggest the diversity of GnRHR-G protein coupling and signal transduction systems. The identification of second form of GnRH (GnRH-II) in mammals adds to the complexity of the GnRH-GnRHR system. This review summaries our recent progress in understanding the regulation of GnRHR gene expression and the GnRHR signal transduction pathways.

Approaches to Define the Role of SF-1 at Different Levels of the Hypothalamic-Pituitary-Steroidogenic Organ Axis

Targeted gene disruption has produced knockout mice globally deficient in the orphan nuclear receptor steroidogenic factor 1 (SF-1). These SF-1 knockout mice lacked adrenal glands and gonads and consequently exhibited adrenocortical insufficiency and sex reversal of their internal and external genitalia. They also had impaired expression of gonadtropins in the anterior pituitary gonadotropes and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at all three levels of the hypothalamic-pituitary-steroidogenic organ axis. Ongoing experiments are directed at using evolving techniques for tissue-specific gene inactivation to define the roles of SF-1 within discrete sites of the hypthalamic-pituitary-steroidogenic organ axis.

Cystatin-related epididymal spermatogenic protein colocalizes with luteinizing hormone-beta protein in mouse anterior pituitary gonadotropes.

The CRES (cystatin-related epididymal spermatogenic) protein, a member of the cystatin superfamily of cysteine protease inhibitors, exhibits highly restricted expression in the mouse testis and epididymis, suggesting roles in reproduction. Considering the well-established relationship that exists between the gonads and the neuroendocrine system, the present studies were undertaken to determine whether the CRES messenger RNA and protein are expressed in the anterior pituitary gland and, if so, whether the expression is regulated by hormones. RT-PCR analysis of whole pituitary gland RNA preparations, and Northern blot analyses of pituitary gland cell lines, demonstrated that the CRES gene is expressed in the male and female anterior pituitary gland gonadotropes. Furthermore, Western blot analysis demonstrated that CRES protein was present in whole mouse pituitary glands and was synthesized and secreted by the LbetaT2 gonadotrope cell line. Interestingly, whereas the predominant CRES proteins present in epididymal lysates, LbetaT2 secretory granules, and whole pituitary gland lysates were 19 and 14 kDa, the predominant CRES proteins present in the cell culture conditioned media were 17 and 12 kDa. Deglycosylation studies revealed that the higher-molecular-mass CRES proteins (19 and 17 kDa) were the result of N-linked glycosylation, caused by the presence of high mannose residues. Double-label immunofluorescence and confocal microscopic analysis of male and female mouse pituitary gland tissue confirmed the RNA studies and showed that CRES protein colocalized with LHbeta protein in the gonadotropes. Finally, gonadectomy and hormone replacement studies suggest that CRES protein in the gonadotropes is hormonally regulated. These studies suggest that CRES protein may perform a role in the gonadotrope-mediated control of reproduction.

ADAM7, a member of the ADAM (a disintegrin and metalloprotease) gene family is specifically expressed in the mouse anterior pituitary and epididymis.

The maturation of spermatozoa in the epididymis is a complex process that requires the active involvement of the epididymal epithelium. The primary focus toward elucidating the role of the epididymis in the maturation process has been the study of epididymal secretory proteins and their interaction with spermatozoa. To date there is a paucity of information regarding epididymal epithelial cell surface proteins, which may also play important roles in epididymal function. Through a subtractive hybridization approach to identify genes specifically expressed in the caput epididymidis, the mouse homologue of a member of the ADAM (a disintegrin and metalloprotease) family of proteins was identified. This rapidly growing gene family encodes cell surface proteins that possess putative adhesion and protease domains. Northern blot analyses demonstrated that the mouse ADAM gene, termed ADAM7, is expressed in the caput region of the epididymis and in the anterior pituitary gonadotropes with no detectable expression in the twenty-six other tissues examined. Furthermore, in situ hybridization experiments revealed that the ADAM7 messenger RNA (mRNA) exhibited an apical localization within the proximal caput epididymal epithelium that may correlate with an unusual sparsely granulated endoplasmic reticulum uniquely present in the proximal region of the epididymidis and to which no known function has been ascribed. Hormonal, surgical, and genetic strategies demonstrated that ADAM7 gene expression requires, in a region-dependent manner, androgens as well as testicular factors for expression. Interestingly, the apical localization of ADAM7 mRNA is dependent upon an intact testis, because in situ hybridization analyses of the proximal caput epididymidis from a testosterone maintained castrate mouse did not show the apical localization of ADAM7 mRNA. Finally, chromosomal mapping demonstrated that the ADAM7 gene maps to the central region of mouse Chromosome 14, approximately 4-5 cM distal from the fertilin beta locus, which encodes another reproductive-specific ADAM protein.

Novel independent and synergistic regulation of gonadotropin- α subunit gene by luteinizing hormone/human choriogonadotropin and gonadotropin releasing hormone in the αT3-1 gonadotrope cells

The αT3-1 cells are immortalized anterior pituitary gonadotropes which express gonadotropin- α subunit gene. These cells contain receptors for gonadotropin releasing hormone (GnRH) as well as for luteinizing hormone (LH) which can also bind human choriogonadotropin (hCG). Like GnRH, LH and hCG can upregulate the expression of gonadotropin- α subunit gene. While 0.1–1.0 ng/ml hCG can upregulate, higher concentrations have no effect. However, these higher hCG concentrations can act in a synergistic manner with GnRH to increase the steady state mRNA and protein levels of gonadotropin- α subunit. The synergism between hCG and GnRH was mimicked by LH but not by other hormones in the glycoprotein hormone family or α or β subunits of hCG, suggesting that the synergism is hormone specific and requires the conformation of native hormone. The hCG induced increase in gonadotropin- α subunit mRNA levels was due to a significant increase in the half-life of gonadotropin- α subunit transcripts from 7.8±1.0 h in the controls to 16.5±3.8 h after treatment with hCG. The GnRH induced increase in gonadotropin- α subunit mRNA levels was due to both a significant increase in the half-life to 26.2±3.0 h as well as a significant increase in the transcription rate of the gene (159.0±7.7% of the control). A greater increase in gonadotropin- α subunit mRNA levels following a combined treatment with GnRH and hCG was due to a further increase in half-life to 37.6±3.1 h as well as a greater increase in the transcription rate of the gene (295.1±24.2% of the control) as compared to the treatment with GnRH alone. In summary, we conclude that LH and hCG can independently and synergistically act with GnRH to increase the expression of gonadotropin- α subunit gene by transcriptional as well as by post-transcriptional mechanisms in αT3-1 cells. These effects may be important for the increase of LH levels during the preovulatory surge.

Exercise training-induced changes in anterior pituitary gonadotrope of the female rat.

Chronic exercise training may alter the hypothalamic-pituitary-ovarian axis by affecting gonadotropin secretion and structural indexes of the anterior pituitary gonadotropes. Young female Harlan Sprague-Dawley rats were randomly assigned to either a 12-wk intensive treadmill-running program (n = 16) or to a sedentary untrained group [untrained-proestrus (UTP), n = 16]. After training for 3 mo, animals were killed on proestrous morning, and individual pituitary glands were enzymatically dispersed into single-cell suspensions. Trained rats maintained either predominantly regular estrous cycles [trained-proestrus (TP), n = 11] or exhibited anestrous vaginal cytology [trained-anestrus (TA), n = 5]. At death, plasma estradiol concentrations for TP (57.4 +/- 6 pg/ml, mean +/- SE) and UTP (54.6 +/- 5 pg/ml) were similar. However, TA had plasma estradiol levels (2.7 +/- 0.2 pg/ml; P < 0.05) reflecting their anestrous status. Basal luteinizing hormone (LH) secretion (20-h static incubation) was significantly reduced in gonadotropes from TA (7.8 +/- 1.1 ng/pooled wells) compared with TP (13.6 +/- 1.6 ng/pooled wells) and UTP (12.6 +/- 1.0 ng/pooled wells). Immunofluorescence LH staining and cytoplasmic granularity were significantly lower within the TA gonadotrope population compared with those of TP and UTP rats. Evidence of impaired basal LH secretion, reduced gonadotrope LH-specific fluorescein content, and reduced gonadotrope cytoplasmic granularity in TA rats suggests that, concomitant with proposed changes in the central neural drive, there are important exercise training-induced alterations of the anterior pituitary gonadotrope that influence rats' estrous cycles and affect their reproductive status.

Inward membrane currents and electrophysiological responses to GnRH in ovine gonadotropes.

We have used conventional whole-cell patch-clamp to investigate the membrane currents of ovine anterior pituitary gonadotropes, and nystatin-perforated whole-cell patch-clamp to record the membrane potential changes elicited by the natural hypothalamic secretagogue, gonadotropin-releasing hormone (GnRH). A large basal inward current found by voltage clamp was blocked by tetrodotoxin (TTX) (ED50 < 10 nM), identifying it as a Na+ current. Slowly inactivating inward current, activated at potentials more positive than -30 mV, remained in Na(+)-free medium or in the presence of 1 microM TTX. This current was abolished by ionic Ca2+ channel blockade. In the presence of nifedipine about 70% of this high voltage-activated Ca2+ current was abolished, leaving a slowly inactivating component. No transient Ca2+ current was found. The nifedipine-insensitive slowly inactivating inward current was eliminated by 1 microM omega-conotoxin GVIA (CGTX), consistent with the presence of N-type channels. Outward K+ currents sensitive to membrane voltage and intracellular Ca2+ concentration ([Ca2+]i) were present. The resting membrane potential lay between -20 and -75 mV (mean = -43 +/- 1.5) with spontaneous TTX-sensitive action potentials occurring in 34% of cells. GnRH had concentration-dependent effects on gonadotrope membrane potential. Application of 100 nM GnRH resulted in a rapid hyperpolarization, followed by a gradual depolarization during which action potentials returned briefly. This was followed by protracted electrical quiescence. Application of 1 or 10 nM GnRH led to hyperpolarization, followed by gradual depolarization, upon which rhythmic hyperpolarizations were superimposed, giving membrane potential oscillations. During the depolarising stage of each oscillation, a burst of action potentials occurred. Action potentials, then oscillations, ceased after 5-15 min. Depolarization was then maintained (at -20 to -35 mV) for up to 1 h. Apamin, the SK-type Ca(2+)-dependent K+ channel blocker, prevented the hyperpolarizing oscillations and produced membrane depolarisation, but Ca2+ channel blockade did not. Microfluorimetric detection of [Ca2+]i showed that 10 nM GnRH induced [Ca2+]i oscillations. We conclude that Ca2+ derived from intracellular pools is involved in producing the membrane potential oscillations. The [Ca2+]i fluctuations may activate the apamin-sensitive, Ca(2+)-dependent SK-type K+ channel, and entrain TTX-sensitive action potentials to a bursting pattern of generation following GnRH stimulation. In the absence of T-type currents, the Na+ current spikes may be crucial for activation of the nifedipine- and CGTX-sensitive high-voltage-activated Ca2+ channels.

Effect of adenylate cyclase inhibitor and protein kinase C inhibitor on GnRH-induced LH release and LH beta subunit biosynthesis in rat anterior pituitary cells.

According to our previous studies together with others, GnRH, a hypothalamic decapeptide, has been known to be a major regulator for LH release and its subunit biosynthesis in anterior pituitary gonadotropes. But the precise mechanisms by which GnRH exerts stimulatory effects on LH release and its subunit biosynthesis have not been clearly understood. In the present study we examined the effect of GnRH on protein kinase C (PKC) activity and intracellular cAMP content in cultured anterior pituitary cells of rat to clarify whether PKC or cAMP are involved in GnRH action. Moreover, we examined the effects of staurosporine (ST), a PKC inhibitor and 2',3'-dideoxyadenosine (2',3'-DDA), an adenylate cyclase inhibitor, on LH release and steady state LH beta subunit mRNA levels in cultured anterior pituitary cells of rat. PKC activity was rapidly increased within 30 min after GnRH treatment whereas intracellular cAMP level was elevated 18 h after GnRH treatment. ST significantly inhibited GnRH-induced LH release and LH beta subunit mRNA levels in a dose-dependent manner, showing an half maximal response at 50 nM ST. 2',3'-DDA inhibited GnRH-induced LH release and LH beta subunit mRNA levels in a dose-dependent manner in pituitary cells. From these results, it is suggested that GnRH stimulates LH beta subunit mRNA level as well as LH release in anterior pituitary cells and this GnRH action might be mediated by PKC activation and cAMP stimulation.

Normal Reproductive Neuroendocrinology in the Female

The reproductive axis in women comprises a number of components that must function in a highly orchestrated manner for reproductive potential to be optimal. The neuroendocrine components of this axis, including the hypothalamus and the pituitary gland, are central to this system. Within the hypothalamus, the specialized neuronal system responsible for synthesizing and secreting gonadotropin-releasing hormone (GnRH) is itself modulated by a number of peptide and biogenic amine neurotransmitters that mediate feedback signals of ovarian origin. The luteinizing hormone and follicle-stimulating hormone secreting anterior pituitary gonadotropes perceive and transduce neural input in the form of GnRH, but are themselves also modulated by the ambient gonadal hormone concentrations. The authors review the physiologic relevance of the pulsatile nature of the GnRH signal, and some proposed mechanisms through which these signals are stimulated and modulated and subsequently perceived and transduced by gonadotropes.

Gonadotropin-releasing hormone modifies action potential generation in sheep pars distalis gonadotropes.

Cell-intact patch-clamp recording was used to determine the electrophysiological responses of sheep anterior pituitary gonadotropes to stimulation with gonadotropin-releasing hormone (GnRH). Cells were identified prior to recording by reverse haemolytic plaque assay (RHPA), or using morphological criteria in preparations enriched in gonadotropes by Percoll density gradient centrifugation. Most cells identified by RHPA did not generate action potentials, and responses to GnRH were inconsistent. The majority of gonadotropes in enriched preparations however spontaneously generated action potentials requiring the entry of both extracellular Na+ and Ca2+, and involving tetraethylammonium-sensitive K+ channels. Two-minute GnRH application (10(-7) M) evoked a characteristic sequence of changes in action potential generation. The immediate response was an inhibition of action potentials, followed by a recovery of these events, with a progressive decline in amplitude over about 10 min. The cells then remained quiescent for up to 1 h. The results indicate that GnRH may evoke an initial hyperpolarization involving Ca(2+)-dependent K+ channels, followed by a sustained depolarizing response, with consequent inactivation of action potential generation.

Electrophysiological properties of a cell line of the gonadotrope lineage.

The role of ion channels in the secretion of gonadotropins from anterior pituitary gonadotropes has been difficult to study at the single cell level because the cells are difficult to distinguish from other pituitary cell types. Recently, a cell line, alpha T3-1, has been generated that makes and secretes the alpha-subunit of gonadotropins. These cells have GnRH receptors, but not TRH receptors, and are, thus, specific to the gonadotrope lineage. We have used the patch clamp technique to investigate the types of ion channels expressed in alpha T3-1 cells and to test for electrophysiological responses to GnRH and a phorbol ester. These cells express TTX-sensitive sodium channels with rapid kinetics, several types of potassium channels, including Ca2(+)-sensitive ones, and two types of calcium channels. The currents through calcium channels are augmented by application of 100 nM GnRH or 10 nM phorbol 12-myristate 13-acetate, a phorbol ester. The augmentation by GnRH and phorbol 12-myristate 13-acetate is consistent with other reports that a portion of stimulated gonadotropin release is dependent on external calcium and sensitive to block by dihydropyridine antagonists. Thus, this cell line may be useful for studies of mechanisms underlying responses to GnRH.

Is cellular disruption the mechanism of release of basic fibroblast growth factor from anterior pituitary gonadotropes?

Adult female Fischer 344 (F344) and Sprague-Dawley (SD) rats, intact and ovariectomized (10–30d), have been used for immunolocalization of basic fibroblast growth factor (FGF). Tissues were selected from three specific sites (postero-lateral, lateral wing, and anterior wedge) of the periphery of the anterior pituitary (AP) carefully maintaining the association between the gland and the highly vascular meningeal connective tissue which envelops it. In both rat strains, most of the periphery of the AP was characterized by intact parenchymal cells delimited from the meningeal connective tissue by an intact basal lamina. However, foci also were evident in which parenchymal cells projected directly into the connective tissue without a basal lamina intervening. These zones, designated the Non-Delimited Peripheral Parenchyma (NDPP), were present minimally in control rats, but were more numerous in ovariectomized rats. Profiles of focally disrupted gonadotropes were evident within the NDPP of 20–30d ovariectomized rats juxtaposed against intact, granulated parenchymal cells. Partially disrupted gonadotropes also were evident within the peripheral parenchyma within approximately 100μ of the edge, and occasionally the disruptions resulted in an association of neighboring gonadotropes as a syncytium. FGF was localized only within the cytosol of gonadotropes, i.e., cells immunopositive for LH-β and FSH-β subunits. Gonadotropes nearer to the edges of the AP, especially the postero-lateral edge, were the most intensely stained. Electron microscopy and immunostaining for S-100 protein, a marker for folliculostellate cells (FSC), demonstrated that in intact and ovariectomized SD rats FSC were present in all peripheral zones of the AP, whereas portions of the postero-lateral periphery of the AP of intact and ovariectomized F344 rats often lacked FSC. We propose that FGF may be released from the cytosol of gonadotropes by a mechanism of cellular disruption. FGF released at peripheral sites of the AP would be well-positioned to stimulate angiogenesis from systemic blood vessels within the meninges. Since FSC are known phagocytes within the AP, their consistent presence in the periphery of the AP of SD rats may help regulate the effects of the released FGF, and by contrast, their absence in F344 rats may intensify or prolong the effects of released FGF. Such differences may underlie the higher incidence of pituitary tumors in F344 rats.

Gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) secretion from perifused equine pituitaries

In vitro responsiveness of the horse anterior pituitary (AP) gonadotropes to single and multiple GnRH challenges was examined. The pituitaries were collected from reproductively sound mares in estrus (n=5) and diestrus (n=5). Uniform 0.5 mm AP slices were subdivided using a 3 mm biopsy punch and then bisected for use in the perifusion chamber. Four bisected sections per chamber were perifused at 0.5 ml/min at 37 C for 560 min in Medium 199 saturated with 95% 0 2/5% CO 2. Ten minute fractions were collected after an initial 2 hr equilibration period. Four different treatment regimes of GnRH (10 −10 M) were evaluated: (A) three consecutive 10 min GnRH pulses separated by 80 and 100 min, respectively; (B) a single 120 min GnRH infusion; (C) a 10 min GnRH pulse followed 80 min later by a 120 min GnRH infusion and (D) two 10 min GnRH pulses separated by 60 min followed 80 min later by a 120 min GnRH infusion. Estimated total pituitary LH content was higher in estrous than diestrous mares (p<0.05). The total amount of LH released in response to GnRH tended to be greater in estrus than diestrus (p<0.1), whereas the percentage of LH released in estrus and diestrus was similar. An increase in the area under the LH response curve was noted with each successive 10 min pulse of GnRH during both estrus and diestrus (p<0.05), demonstrating a self-priming effect of GnRH. In addition, a significant increase in the peak LH amplitude (p<0.05) and the slope to peak amplitude (p<0.05) were observed for the 120 min GnRH pulse in regime C and D indicating that prior exposure to short-term pulses of GnRH increased the acute LH secretory response. These results suggest that in the cycling mare (1) the responsiveness of the pituitary (amount of LH released as percent of total LH) is similar in both estrus and diestrus, however, the magnitude of the LH response (total microgram amount of LH released) differs with the stage of the estrous cycle, being highest in estrus, and appears to be related, in part, to pituitary LH content and (2) GnRH self-priming occurs independently of the stage of the estrous cycle. Furthermore, we have demonstrated that the pulsatile mode of GnRH can act directly on the anterior pituitary to dictate the pulsatile release pattern of LH in the cycling mare.

Cell Lines of the Pituitary Gonadotrope Lineage Derived by Targeted Oncogenesis in Transgenic Mice

Study of the molecular and cellular biology of the gonadotropin hormones would be greatly facilitated by the availability of immortalized anterior pituitary gonadotrope cell lines. We directed expression of the simian virus-40 (SV40) T-antigen (Tag) oncogene to specific cells in the anterior pituitary of transgenic mice using the promoter/enhancer region from the human glycoprotein hormone alpha-subunit gene. Transgenic mice carrying this fusion gene developed anterior pituitary tumors. Clonal cell lines established from these tumors express the endogenous mouse alpha-subunit gene and synthesize and secrete alpha-subunit protein. However, they do not express beta-subunit genes. Alpha-subunit mRNA is induced by GnRH in a dose- and time-dependent manner, but is not regulated by TRH. Thus, we have targeted tumorigenesis in transgenic mice to anterior pituitary cells of the gonadotrope lineage to immortalize this specific endocrine cell while maintaining several highly differentiated functions unique to gonadotropes.

Gonadotropin-releasing hormone-induced Ca2+ transients in single identified gonadotropes require both intracellular Ca2+ mobilization and Ca2+ influx.

We examined the effects of gonadotropin-releasing hormone (GnRH) on the intracellular free Ca2+ concentration ([Ca2+]i) in single rat anterior pituitary gonadotropes identified by a reverse hemolytic plaque assay. Concentrations of GnRH greater than 10 pM elicited increases in [Ca2+]i in identified cells but not in others. In contrast, depolarization induced by 50 mM K+ increased [Ca2+]i in all cells. Ca2+ transients induced by GnRH exhibited a complex time course. After an initial rapid rise, the [Ca2+]i fell to near basal levels only to be followed by a secondary extended rise and fall. Analysis of the Ca2+ transients on a rapid time base revealed that responses frequently consisted of several rapid oscillations in [Ca2+]i. Removal of extracellular Ca2+ or addition of the dihydropyridine Ca2+-channel blocker nitrendipine completely blocked the secondary rise in [Ca2+]i but had no effect whatsoever on the initial spike. Nitrendipine also blocked 50 mM K+-induced increases in [Ca2+]i in identified gonadotropes. The secondary rise induced by GnRH could be enhanced by a phorbol ester in a nitrendipine-sensitive fashion. Multiple spike responses to GnRH stimulation of the same cell could only be obtained if subsequent Ca2+ influx was permitted either by allowing a secondary rise to occur or by producing a Ca2+ transient by depolarizing the cells with 50 mM K+. It therefore appears that the response to GnRH consists of an initial phase of Ca2+ mobilization, probably mediated by inositol trisphosphate, and a subsequent phase of Ca2+ influx through nitrendipine-sensitive Ca2+ channels that may be activated by protein kinase C. The relative roles of these phases in the control of gonadotropin secretion are discussed.

Slice cultures of LHRH neurons in the presence and absence of brainstem and pituitary.

Luteinizing hormone releasing hormone (LHRH) neurons from the preoptic area (POA)/hypothalamus of the postnatal rat were cultured for up to 7 weeks using a slice explant roller culture technique. The slices thinned to quasi-monolayers, but maintained organotypic distributions of large numbers of immunocytochemically identifiable LHRH, neurotensin, tyrosine hydroxylase, neurophysin and corticotropin releasing hormone-containing neurons. The distribution, survival and morphology of LHRH cells in co-cultures with brainstem and anterior pituitary was quantitated, and found to be similar to that observed in single cultures. LHRH fibers grew into either pituitary or brainstem tissue, however when all three tissues were co-cultured, LHRH fibers preferentially invaded the pituitary. LH immunoreactive anterior pituitary gonadotropes were maintained only in co-cultures containing POA/hypothalamic slices, and addition of an LHRH antagonist in such cultures, inhibited LH immunoreactivity in the gonadotropes. This slice explant roller culture method effectively maintains the cyto- and chemoarchitecture and functional properties of the LHRH system for long periods in vitro and should provide excellent models for studying the interactive and molecular characteristics of postnatal LHRH neurons.

Clinical applications of gonadotropin-releasing hormone and gonadotropin-releasing hormone analogs

Investigations have proved the clinical importance of hypothalmic gonadotropin-releasing hormone (GnRH) and its agonistic and antagonistic analogs. A pulsatile pattern of stimulation of specific receptors in the anterior pituitary gonadotrope has been shown to activate pituitary-gonadal function; continuous administration inhibits it. Clinical research has shown promising results in the induction of ovulation using the pulsatile pattern of GnRH administration in patients with hypogonadotropic amenorrhea. Attempts to use GnRH and its agonists to activate the pituitary-gonadal system in patients with idiopathic delayed puberty has proved logistically impractical because of the duration of treatment required to achieve sexual maturation. Treatment of cryptorchidism by intranasal administration 6 times daily for 4 weeks resulted in complete descent in 38% of the 48 boys and partial descent in 28%. For GnRH nonresponders sequential treatment with human chorionic gonadotropin produced an 86.2% success rate. Because of the ability of GnRH agonists to reversibly suppress the pituitary-gonadal axis without side effects it can be used in diseases mediated by gonadal steroids. Successful halting of precocious puberty through the administration of GnRH to suppress the nocturnal release of gonadotropin has been demonstrated. Unlike treatment with progestational agents no side effects are discerned. Continuous administration of GnRH agonist in patients with endometriosis reduces ovarian estrogens and androgens to castration levels mimicking an ovariectomy. This castration effect highlights the potential use of GnRH agonists in the treatment of metastatic breast cancer. The use of GnRH agonists for the treatment of androgen-dependent prostatic carcinoma has induced clinical improvement with nonmetastatic stage 3 disease and pain relief in metastic stage D disease. In polycystic ovary syndrome administration of GnRH agonist shows promising results. As a potential contraceptive GnRH agonists have not yet demonstrated practical advantages. The actions under study include: ovulation inhibition luteolysis induction induction of luteal phase defect and reduction of testosterone production. Numerous antagonistic analogs of GnRH have been synthesized and have shown some potential contraceptive effects in animal studies.