Cytochemical characterization of pituitary target cells for biotinylated gonadotropin releasing hormone

These studies describe the application of new cytochemical stains that co-localize a biotin-labeled gonadotropin releasing hormone (GnRH) analog and FSH or LH in the same field or cell. Pituitary monolayer cells were stimulated with the [D-Lys6] GnRH analog or the same analog labeled with biotin. Biotinylated [D-Lys6] GnRH exhibited a higher affinity and was 7-10 X more potent than unlabeled [D-Lys6] GnRH. The avidin-biotin peroxidase complex technique (ABC) was applied to localize the biotinylated GnRH on the cells with the use of a dense black peroxidase substrate. Specificity tests showed that the stain could be eliminated by competition with unlabeled [D-Lys6] GnRH. The GnRH stain was followed by immunocytochemical stains for LH beta, FSH beta or 25-39ACTH with a different peroxidase substrate (amber or orange-red). Stain for GnRH was found on the surfaces of 16% of the cells and 60-90% of the GnRH stained cells also stained for one of the gonadotropins. Most (90-100%) of the gonadotropes showed stain for GnRH. Our studies demonstrate that a potent biotinylated GnRH analog binds cells that can be identified specifically as gonadotropes.     

Application of dual pre-embedding stains for gonadotropins to pituitary cell monolayers with avidin-biotin (ABC) and peroxidase-antiperoxidase (PAP) complexes: light microscopic studies

Double stains for gonadotropins and gonadotropin-releasing hormone were developed for fixed whole pituitary cells from cycling female rats. Monolayer cells were stimulated with [D-Lys6]GnRH, fixed in 2.5% glutaraldehyde, and then stained for luteinizing hormone (LH) (1:50,000-12 h) or follicle stimulating hormone (FSH) (1:60,000-12 h) and the avidin-biotin-peroxidase complex technique (ABC) with a jet-black substrate (nickel intensified diaminobenzidine-DAB). This was followed by a stain for the other gonadotropin with either ABC or peroxidase-antiperoxidase complex (PAP) techniques and amber (DAB) or red (3-amino-9-ethyl-carbazole) substrates. Additional monolayers were stimulated with biotinylated [D-Lys6]GnRH and stained with the ABC technique and the black (nickel-DAB) substrate. These monolayers were then stained immunocytochemically for LH or FSH with either ABC or PAP methods and orange or red substrates. The controls showed that the omission of the second primary antiserum abolished the stain indicating that the second staining solutions did not react with components in the first group. The addition of the second peroxidase substrate in sequence after the first stain indicated that no residual peroxidase activity remained from the first stain. Our tests also showed that saponin was not needed to aid reagent or antibody penetration. The dual stains demonstrated that 50-60% of the gonadotropes stored LH and FSH together, often in separate regions of the same cell. Some cells contained only one hormone (20-22%). The dual stains for GnRH and gonadotropins demonstrated that 80-90% of the GnRH bound cells are gonadotropes. These techniques allow a study of storage sites for multiple hormones in or on whole cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of Dual Pre-Embedding Stains for Gonadotropins to Pituitary Cell Monolayers with Avidin-Biotin (ABC) and Peroxidase-Antiperoxidase (PAP) Complexes: Light Microscopic Studies

Double stains for gonadotropins and gonadotropin-releasing hormone were developed for fixed whole pituitary cells from cycling female rats. Monolayer cells were stimulated with [d-Lys⁶]GnRH, fixed in 2.5% glutaraldehyde, and then stained for luteinizing hormone (LH) (1:50,000-12 h) or follicle stimulating hormone (FSH) (1:60,000-12 h) and the avidin-biotin-peroxidase complex technique (ABC) with a jet-black substrate (nickel intensified diaminobenzidine—DAB). This was followed by a stain for the other gonadotropin with either ABC or peroxidase-antiperoxidase complex (PAP) techniques and amber (DAB) or red (3-amino-9-ethyl-carbazole) substrates. Additional monolayers were stimulated with biotinylated [d-Lys⁶]GnRH and stained with the ABC technique and the black (nickel-DAB) substrate. These monolayers were then stained immunocytochemically for LH or FSH with either ABC or PAP methods and orange or red substrates. The controls showed that the omission of the second primary antiserum abolished the stain indicating that the second staining solutions did not react with components in the first group. The addition of the second peroxidase substrate in sequence after the first stain indicated that no residual peroxidase activity remained from the first stain. Our tests also showed that saponin was not needed to aid reagent or antibody penetration. The dual stains demonstrated that 30-60% of the gonadotropes stored LH and FSH together, often in separate regions of the same cell. Some cells contained only one hormone (20-22%). The dual stains for GnRH and gonadotropins demonstrated that 80-90% of the GnRH bound cells are gonadotropes. These techniques allow a study of storage sites for multiple hormones in or on whole cells. The studies agree with and augment the results from the use of serial sections.     

Similar effects of inhibin and cycloheximide on gonadotropin release in superfused pituitary cell cultures

The actions of two inhibin preparations and cycloheximide on gonadotropin release were investigated in superfused pituitary cell cultures. Pituitary cells isolated from 18-day-old male rats were grown in Matrigel-coated superfusion chambers in chemically defined medium. After stationary culture for 4 days, the cell monolayers were superfused at a constant speed (0.25 ml/min) and were intermittently stimulated (6 min/h) with 10 nM gonadotropin-releasing hormone (GnRH). Groups of cultures were exposed to the test substances for varying time periods during stationary culture and/or during superfusion. Inhibitory effects of both inhibin preparations on the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in response to GnRH pulses were observed after 2 h of exposure and became maximal after about 6 h. Basal secretion of FSH between GnRH pulses was also suppressed, whereas the basal interpulse secretion of LH was not changed. When exposure to inhibin was discontinued, the secretion of both FSH and LH progressively increased and returned to control values by approximately 6 h. Cycloheximide (500 ng/ml) affected gonadotropin release with dynamics similar to those observed for the inhibin preparation. These data support the hypothesis that inhibition of gonadotropin synthesis may be an important step in the molecular mechanism of action by which inhibin regulates gonadotropin release.     

Preparation and characterization of photoreactive derivatives of GnRH. Persistent activation of function by photoaffinity labeling

A photoreactive derivative of the highly potent gonadotropin releasing hormone (GnRH) agonist, D-Lys6-GnRH(1-9)-ethylamide, was prepared by selective modification of the epsilon-amino group with 2-nitro-4-azidophenyl sulfenyl chloride (2,4-NAPS C1). The modified peptide [D-Lys(NAPS)]6-GnRH-(1-9)-ethylamide was found to be a full agonist of LH release from rat pituitary cells with a relative potency 23 compared to GnRH. Covalent attachment of the photoreactive analog to rat pituitary cells resulted in prolonged activation of LH secretion which could not be inhibited by a potent GnRH antagonist. Persistent stimulation of pituitary gonadotrophs caused by covalently bound hormone led to desensitization of the LH releasing mechanism.     

Differential actions of corticosterone on luteinizing hormone and follicle-stimulating hormone biosynthesis and release in cultured rat anterior pituitary cells: interactions with estradiol

Previous in vivo studies from our laboratory suggested that glucocorticoids antagonize estrogen-dependent actions on LH secretion. This study investigated whether corticosterone (B) may have similar actions on gonadotropin biosynthesis and secretion in vitro. Enzymatically dispersed anterior pituitary cells from adult female rats were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse serum with or without 0.5 nM estradiol (E2). The cells were then cultured for 24 h with or without B in the presence or absence of E2. To evaluate hormone release, 5 x 10(5) cells were incubated with varying doses of GnRH (0, 10(-11)-10(-7) M) or pulsatile GnRH (10(-9) M; 20 min/h) for 4 h. Cell and medium LH and FSH were measured by RIA. To evaluate LH biosynthesis, 5 x 10(6) cells were incubated for an additional 24 h with 10(-10) M GnRH, 60 microCi 3H-glucosamine (3H-Gln), 20 microCi 35S-methionine (35S-Met), and the appropriate steroid hormones. Radiolabeled precursor incorporation into LH subunits was determined by immunoprecipitation, followed by SDS-PAGE. Continuous exposure to GnRH stimulated LH release in a dose-dependent manner, and this response was enhanced by E2. B by itself had no effect on LH release, but inhibited LH secretion in E2-primed cells at low concentrations of GnRH (10(-10) M or less). Total LH content was not altered by GnRH or steroid treatment. Similar effects of B were observed in cells that were given a pulsatile GnRH stimulus. In contrast to LH, E2 or B enhanced GnRH-stimulated FSH release at the higher doses of GnRH, while the combination of E2 and B increased basal and further augmented GnRH-stimulated release. Total FSH content was also increased in the presence of B, but not E2 alone, and was further augmented in cells treated with both steroids. There were no effects of the steroids on the magnitude of FSH release in response to GnRH pulses, but the cumulative release of FSH was greater in the E2 + B group compared to controls, indicating an increased basal release. Independent of E2, B suppressed the incorporation of 3H-Gln into LH by more than 50% of control, with only subtle effects on the incorporation of 35S-Met.(ABSTRACT TRUNCATED AT 400 WORDS)     

Active immunization of intact mares against gonadotropin-releasing hormone: differential effects on secretion of luteinizing hormone and follicle-stimulating hormone

Five lighthorse mares were actively immunized against gonadotropin releasing hormone (GnRH) to determine the relative importance of this hypothalamic hormone in the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Five mares immunized against the conjugation protein served as controls. Mares were initially immunized in November and received secondary immunizations 4 wk later, and then at 6-wk intervals until ovariectomy in June. All mares immunized against GnRH exhibited an increase (p less than 0.01) in the binding of tritiated GnRH by plasma, an indication that antibodies against this hormone had been elicited. Concentrations of LH, FSH and progesterone in weekly blood samples were lower (p less than 0.05) in GnRH-immunized mares than in controls after approximately 4 mo of immunization. However, the LH concentrations were affected to a greater degree than were FSH concentrations. All five control mares exhibited normal cycles of estrus and diestrus in spring, whereas no GnRH-immunized mare exhibited cyclic displays of estrus up to ovariectomy. All mares were injected intravenously with a GnRH analog (which cross-reacted less than 0.1% with the anti-GnRH antibodies) in May, after all control mares had displayed normal estrous cycles, to characterize the response of LH and FSH in these mares; two days later, the mares were injected with GnRH. The LH response to the analog, which was assessed by net area under the curve, was lower (p less than 0.01) by approximately 99% in mares immunized against GnRH than in control mares. In contrast, the FSH response to the analog was similar for both groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basal and GnRH-induced secretion of FSH and LH in anestrous versus ovariectomized bitches

The basal and gonadotropin releasing hormone (GnRH)-induced plasma concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were studied in four anestrous and four ovariectomized (OVX) bitches. Blood samples were obtained via jugular venipuncture 40min before and 0, 10, 20, 30, 60, 90, and 120min after the i.v. administration of synthetic GnRH in a dose of 10microg/kg body weight. The basal plasma FSH and LH concentrations were significantly higher in the OVX bitches than in the anestrous bitches. In the anestrous bitches, the plasma FSH concentration was significantly higher than the pretreatment level at 10, 20, and 30min, whereas the plasma LH concentration was significantly elevated at 10 and 20min. The maximal GnRH-induced plasma FSH concentration in the anestrous bitches did not surpass the lowest plasma FSH concentration in the OVX bitches, whereas the GnRH-induced plasma LH concentrations in the anestrous bitches overlapped with the basal plasma LH concentrations in the OVX bitches. In the OVX bitches, GnRH administration did not induce a significant change in the plasma FSH concentration, whereas the plasma LH concentration increased significantly at 10 and 20min. In conclusion, the results of the present study indicate that in anestrous bitches GnRH challenge results in increased plasma levels of both FSH and LH, whereas in the OVX bitches, in which the basal plasma FSH and LH concentrations are higher, only a rise in the plasma LH concentration is present after GnRH stimulation. The results also suggest that a test to measure plasma concentration of FSH in single samples appears to have potential in verification of neuter status in bitches.     

Photoaffinity labeling of gonadotropin releasing hormone receptors in control and desensitized pituitary cells

Gonadotropin releasing hormone (GnRH), preincubated with cultured rat pituitary cells, induced down regulation of GnRH receptors in a time- and dose-dependent manner. The specific binding was inhibited by 50% after 30 min and maximal inhibition (70%) was obtained after 75 min preincubation with 1 microM GnRH. Preincubation of the cells for 2 h with 10 nM GnRH inhibited the specific binding by 20%, reaching a plateau of 70% inhibition with 0.1 microM GnRH. Concomitantly, exposure of the cells to GnRH caused a time- and dose-dependent desensitization of LH release. The responsiveness of the desensitized cells was not parallel to the binding capacity and was inhibited to a greater extent (93%). Photoactivation of GnRH receptors with iodinated [azidobenzoyl-D-Lys6]GnRH in control and desensitized cells resulted in the identification of a single specific band with the same apparent molecular weight of 60K daltons. These results indicate that structural alterations of GnRH receptors are not associated with GnRH-induced desensitization. Therefore, desensitization may involve conformational changes in the receptor or more likely a post-receptor mechanism.     

Receptor-binding properties of gonadotropin-releasing hormone derivatives. Prolonged receptor occupancy and cell-surface localization of a potent antagonist analog

The receptor-binding properties and in vitro biological effects of a highly active gonadotropin-releasing hormone (GnRH) antagonist, [N-acetyl-D-p-chloro-Phe1,2D-Trp3,D-Lys6,D-Ala10]GnRH, were compared with those of the GnRH superagonist analog, [D-Ala6] des-Gly10-GnRH-N-ethylamide. In rat pituitary particles and isolated pituitary cells, the 125I-labeled GnRH antagonist showed saturable high-affinity binding (Ka v 8.4 +/- 1.4 X 10(9) M-1) to the same receptor sites which bound the GnRH agonist. The rate of dissociation of the receptor-bound antagonist from pituitary particles and cells was extremely slow in comparison with that of the agonist ligand. Also, dissociation of the antagonist analog was incomplete, with a residual fraction of tightly bound ligand that was proportional to the duration of preincubation. The [D-Lys6]GnRH antagonist prevented GnRH-induced luteinizing hormone release during static incubation and superfusion of cultured pituitary cells, but in contrast to the agonist did not cause desensitization of the gonadotroph. Although the antagonist caused a prolonged reduction in available GnRH receptor sites, this was attributable to persistent occupancy by the slowly dissociating ligand rather than to receptor loss. Autoradiographic analysis of [D-Lys6]GnRH-antagonist uptake by cultured pituitary cells revealed that the peptide remained bound at the cell membrane for up to 2 h, in contrast with the rapid endocytosis of GnRH agonists. The slow dissociation of receptor-bound antagonist was consistent with its ability to cause sustained blockade of GnRH actions, and its prolonged cell-surface location suggests that receptor activation is necessary to initiate the rapid internalization of hormone-receptor complexes that is a feature of the agonist-stimulated gonadotroph.     

Phorbol ester-induced LH release in pituitary gonadotrophs: effects of antagonists of calmodulin and GnRH.

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of Ca(2+)- and phospholipid-dependent protein kinase (C kinase), stimulates luteinizing hormone (LH) release from rat pituitary cells. The actions of TPA upon LH release were compared with those of the GnRH superagonist [D-Ala6] des-Gly10-GnRH N-ethylamide (GnRHa) in cultured pituitary cells. LH release was stimulated by 0.1 nM TPA and the maximum response at 10 nM TPA was 50% of the LH response to GnRHa. The ED50 values for TPA and GnRHa were 1.2 and 0.037 nM, respectively, and the maximum stimulatory effects of TPA and GnRHa on LH release were not additive. GnRHa-stimulated LH release was decreased by calmodulin (CaM) antagonists including pimozide, trifluoperazine, W5 and W7, being most effectively reduced (by 70%) by 10 microM pimozide. In contrast to their inhibition of GnRH action, these antagonists enhanced TPA-stimulated LH release, so that 10 microM pimozide and W7 doubled the maximum LH response. The potent GnRH antagonist [Ac-D-p-Cl-Phe1.2, D-Trp3, D-Lys6, D-Ala10]GnRH, which completely inhibited GnRHa-stimulated LH release with ID50 of 6.8 nM, also reduced maximum TPA-stimulated LH release by about 50%. These results suggest that both Ca2+/CaM and C kinase pathways are involved in the LH release mechanism, and indicate that C kinase plays a major role in the action of GnRH upon gonadotropin secretion. The synergism between CaM antagonists and TPA suggests that blockade of CaM-mediated processes leads to enhanced activation of the C kinase pathway, possibly by removal of an inhibitory influence. Furthermore, the partial inhibition of TPA-stimulated LH release by a GnRH antagonist suggests that the pathway(s), specifically connected with LH release in the diverse effects of C kinase, might be locked by the continuous receptor inactivation by antagonist and indicates the complicated pathways which diverge from the receptor and converge into specific cellular response.     

Effect of vitamin A deficiency upon gonadotropin response to gonadotropin-releasing hormone

There is a monotypic change in basal serum gonadotropin levels following retinol treatment of chronically vitamin A-deficient (VAD) male rats. The present study was undertaken to investigate the hypothesis that the specific increase in serum follicle-stimulating hormone (FSH) represents a change in gonadotrope responsiveness to gonadotropin-releasing hormone (GnRH). To this end, a test dose of GnRH was given to VAD rats pre-, 5 days post-, and 10 days postreplacement of vitamin A (PVA). In VAD rats, basal serum FSH and luteinizing hormone (LH) levels were higher than those of controls. Increased LH/testosterone ratios, both in basal levels and in the secretory response to GnRH, suggested Leydig cell hyporesponsiveness in VAD animals. Both the FSH and LH responses to GnRH were maximal at 1 h, declining thereafter. Although the absolute increments in FSH and LH 1 h after GnRH in VAD rats were greater than in controls, the percent increase in FSH tended to be lower in VAD rats and to increase after vitamin A replacement. The specific enhancement of FSH release PVA became evident only when assessing total secretion of FSH and LH after GnRH. Luteinizing hormone response to GnRH increased PVA, but not significantly, while FSH secretion after GnRH increased both 5 and 10 days PVA, times during which basal FSH levels were also increasing. These changes in FSH secretion could not be attributed either to increases in endogenous GnRH or to changes in testosterone or estradiol levels. Basal serum androgen binding protein levels, elevated in VAD animals, did not respond to the acute increases in FSH after GnRH and remained high PVA, suggesting no acute change in Sertoli cell function. Thus, the PVA increase in FSH secretion unmasks a partial inhibition of the gonadotrope present in the retinol-deficient, retinoic acid-fed male rat.     

Interaction of fluorescent gonadotropin-releasing hormone with receptors in cultured pituitary cells

A fluorescent derivative of the gonadotropin-releasing hormone (GnRH) agonist analog, [D-Lys6]GnRH, was synthesized for receptor studies and shown to be biologically active. The rhodamine-derivatized peptide (Rh-GnRH) retained 40% of the receptor binding activity of [D-Lys6]GnRH, and 50% of the luteinizing hormone-releasing activity assayed in cultured pituitary cells. The fluorescent analog was employed to visualize the distribution of GnRH receptors in cultured pituitary cells, using the technique of video-intensified fluorescence microscopy. The binding of Rh-GnRH was confined to the large gonadotrophs which comprised 15% of the cell population. The specificity of the binding was shown by the absence of significant fluorescence in the presence of a 100-fold excess of [D-Lys6]GnRH, or when Rh-GnRH was incubated with choriocarcinoma, neuroblastoma, or 3T3 cell lines devoid of GnRH receptors. The interaction of Rh-GnRH with living pituitary cells was characterized by an initial diffuse distribution, followed by the formation of polar aggregates that later appeared to be internalized. These observations emphasize the value of fluorescent derivatives of GnRH for elucidating the course of the interaction with specific receptors on pituitary gonadotrophs. The initial results indicate that GnRH-receptor complexes undergo aggregation during stimulation of luteinizing hormone release, and are later internalized for subsequent degradation and/ or intracellular actions.     

Visualization of binding sites for bovine parathyroid hormone (PTH 1-84) on cultured kidney cells with a biotinyl-b-PTH (1-84) antagonist

Parathyroid hormone (PTH) receptors have been found in a subpopulation of kidney cells. In this report, we investigated the feasibility of techniques that apply a partial antagonist of PTH conjugated to biotin to localize receptors cytochemically on bovine kidney cortical cells in monolayer culture at the light microscopic level. Biotinylated bovine PTH (1-84) (biotinyl-PTH) was bound to the cultured cells for 1-30 min at 37 degrees C in the amounts of 10(-5) -10(-10) M. In a different set of experiments, the cells were also exposed to a solution containing 10(-6) M biotinylated PTH and an excess of unlabeled PTH, insulin, adrenocorticotropin, or calcitonin for 10 and 30 min at 37 degrees C to test the specificity of the binding. The cells were then fixed in 2.5% glutaraldehyde and stained with the avidin-biotin peroxidase complex (ABC) technique. Diffuse labeling was evident on 30% of the cells in 10 min with concentrations of biotinyl-PTH as low as 10(-8) M. The stain was diffuse, but more intense after 1-10 min in higher concentrations (10(-6) M). If a 15-1500-fold excess of unlabeled PTH was added to the biotinyl-PTH, no staining was observed. The other peptides (insulin, ACTH or calcitonin) had no effect on binding. Longer times in biotinyl-PTH (10(-6) M for 10-30 min) resulted in intense patches of label on the cells resembling caps (in addition to the pale diffuse label). The percentage of labeled cells in the monolayer (30%) did not change with time. These studies show that a partial antagonist of PTH can be used as a cytochemical probe for specific PTH receptors in a subpopulation of cultured cortical kidney cells.     

Characterization of gonadotropin-releasing hormone (GnRH) receptors in the ovary of common carp (Cyprinus carpio).

Gonadotropin-releasing hormone (GnRH) binding sites have been characterized in the fully mature common carp ovary, using an analog of salmon GnRH ([D-Arg6,Trp7,Leu8,Pro9-NEt]-GnRH; sGnRH-A) as a labeled ligand. Binding of sGnRH-A to carp follicular membrane preparation was found to be time-, temperature-, and pH-dependent. Optimal binding was achieved after 40 min of incubation at 4 degrees C at pH 7.6; binding was found to be unstable at room temperature. Binding of radioligand was a function of tissue concentration, with a linear correlation over the range of 8.0-40.0 micrograms membrane protein per tube. Incubation of membrane preparations with increasing levels of [125I]sGnRH-A revealed saturable binding at radioligand concentrations greater than 400 nM. The binding of [125I]sGnRH-A to the carp ovary was also found to be reversible; addition of unlabeled sGnRH-A (10(-6) M) after reaching equilibrium resulted in complete dissociation of [125I]sGnRH-A within 30 min, and the log dissociation plot indicated the existence of a single class of binding sites. Addition of unlabeled sGnRH-A displaced the bound [125I]sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis suggested the presence of one class of high affinity GnRH binding sites. Bound [125I]sGnRH-A was also found to be displaceable by other GnRH peptides, including sGnRH ([Trp7,Leu8]-GnRH), cGnRH-II ([His5,Trp7,Tyr8]-GnRH) and a GnRH antagonist ([D-pGlu1,D-Phe2,D-PTrp3,6]-GnRH; GnRH-ANT) in a parallel fashion, indicating that these peptides bind to the same class of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activin stimulates secretion of follicle-stimulating hormone from pituitary cells desensitized to gonadotropin-releasing hormone

The secretion of follicle-stimulating hormone (FSH) by pituitary cells is stimulated by activin and gonadotropin-releasing hormone, GnRH. To examine the possible interrelationships between the intracellular actions of these secretagogues, responsiveness to activin was tested following pretreatment with 0, 0.1, or 10 nM GnRH. In cells pretreated with 0 or 0.1 nM GnRH, FSH secretion was increased approximately 2-fold during a subsequent challenge with either activin or GnRH. In contrast, in cells pretreated with 10 nM GnRH, FSH secretion became unresponsive to GnRH but could still be stimulated 2-fold by activin. These results demonstrate that activin is able to stimulate FSH secretion in cells that have undergone desensitization to GnRH.     

Effect of treatment with cortisol in vivo on secretion of gonadotropins in vitro

To investigate the site of action of glucocorticoids in modulating secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from pituitaries of male rats, we implanted intact male rats with 250-mg pellets of cortisol (F) or cholesterol (C). Four days later, we collected and enzymatically dispersed the pituitaries. After the dispersed pituitaries had been in culture for 2 days, we treated the cells with gonadotropin-releasing hormone (GnRH) (0-150 nM) and determined the concentrations of LH and FSH in the medium after 6 h of incubation. Cells from donor animals pretreated with F secreted 30-60% more LH approximately 75% more FSH than cells from donor animals pretreated with C. This increase occurred regardless of the presence of F or C in the incubation medium in vitro. The slopes and ED50s of the GnRH dose-response curves were not altered. These data show that glucocorticoids have stimulatory effects on both LH and FSH. The inhibitory effects observed in vivo must be exerted by some mechanism that is not carried over to the in vitro model, and perhaps involve sites of action in addition to the pituitary.     

Effectiveness of an antagonist to gonadotrophin releasing hormone on the FSH and LH response to GnRH in perifused equine pituitary cells,and in seasonally acyclic mares

We wish to use a gonadotrophin-releasing hormone (GnRH) antagonist in the mare as a tool for investigating the control of the oestrous cycle. The aim of this study was to test the effectiveness of the antagonist cetrorelix by testing both in vitro, using perifused equine anterior pituitary cells, and in vivo in seasonally acyclic mares. Pituitary cells were prepared and after 3-4 days incubation, loaded onto columns and given four pulses of GnRH (at 0, 30, 60 and 90 min; dose-response study). After the second GnRH pulse, infusion of cetrorelix began (0, 100, 1000 and 2000 pmol/l) and continued until the end of the experiment. To mimic luteal phase conditions, cells were pre-incubated and perifused with progesterone (25 nmol/l) and GnRH pulses given at 0, 90, 180 and 270 min. Cetrorelix (0 or 1000 pmol/l) began after the second GnRH pulse. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations were measured in 5 min fractions. Both FSH and LH response areas (above baseline) after GnRH were inhibited by 1000 pmol/l cetrorelix (P < 0.01, P < 0.01, respectively) but not by 100 pmol/l cetrorelix. Similarly, in the presence of progesterone, cetrorelix inhibited the FSH (P < 0.001) and LH (P = 0.0002) response area. Seasonally acyclic mares, pre-treated for 3 days with progesterone (150 mg i.m. per day) were given cetrorelix as (i) a loading dose of 1 microg/kg then infusion at 2.2 ng/(kg min) for 90 min, (ii) a s.c. injection at 20 microg/kg, (iii) infusion at 2.2 ng/(kg min) for 48 h, and (iv) no cetrorelix (control mares). At 90 min, 6, 24 and 48 h after cetrorelix was first administered, mares were given a bolus injection of GnRH (22.2 ng/kg i.v.) and the FSH and LH responses measured. All doses of cetrorelix inhibited the FSH response at 90 min. The response was no longer suppressed at 6 h in the 90 min infusion group, showing a rapid recovery from inhibition. At 24 h, the FSH responses in the injected and 48 h infusion group were suppressed. The LH concentrations were low and showed no significant changes. This study has defined the time course and dose of cetrorelix with respect to its effect on FSH in the horse. It is concluded that cetrorelix could be used to elucidate the role of FSH in follicular development in cyclic mares.     

Stimulation and inhibition of Leydig cell steroidogenesis by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate: similarity to the effects of gonadotropin-releasing hormone

To explore the mechanism of gonadotropin-releasing hormone (GnRH) action on Leydig cell steroidogenesis the effects of a GnRH analog (GnRHa) were compared to those of 12-O-tetradecanoylphorbol 13-acetate (TPA). Both compounds acutely stimulated androgen production 2-4 fold with EC50's of 9 nM (TPA) and 0.2 nM (GnRHa). The effects of TPA and GnRHa were not additive and neither compound acutely altered the luteinizing hormone (LH) concentration-response relationship. After 24 h of exposure to TPA or GnRHa the ability of LH to stimulate androgen production was impaired. The parallel effects of TPA and GnRHa on Leydig cell steroidogenesis suggest that they are acting via similar mechanisms; presumably the activation protein kinase C.