Michael D. Culler

Passive immunoneutralization of endogenous inhibin: sex-related differences in the role of inhibin during development.

The purpose of these studies was to ascertain the extent to which endogenous inhibin regulates follicle-stimulating hormone (FSH) secretion at different intervals during development in the male and female rat. This was determined by examining the changes in plasma FSH that resulted from immunoneutralizing endogenous inhibin in male and female rats at different ages during development and into adulthood. Passive immunoneutralization of endogenous inhibin was achieved using specific, high titer ovine antiserum, generated against the alpha-subunit of the recently described inhibin molecule. Optimal antiserum volumes and time after injection required to observe maximal changes in FSH secretion were determined in initial experiments. No clear effect of immunoneutralizing endogenous inhibin could be demonstrated on FSH secretion in female rats until 20 days of age, after the completion of the endogenous rise in FSH which occurs between days 5 and 20. Thereafter, injection of the anti-alpha-inhibin serum (anti-alpha IN) produced a progressively marked increase in plasma FSH as the age of the females increased. In male rats, injection of the anti-alpha IN serum caused an increase in FSH secretion as early as 5 days of age, although the response was more delayed at this age than at later times. The ability of the anti-alpha IN serum to increase plasma FSH was observed through 20 days of age. At 30 days of age, during the peak of the endogenous rise in plasma FSH, injection of the anti-alpha IN serum failed to further increase the already elevated levels of plasma FSH. As the endogenously high levels of FSH gradually decreased, the ability of anti-alpha IN serum to increase FSH secretion returned (40 days of age) but was diminished by 50 days of age and was completely lost by 60 days of age. The results of the present study indicate that inhibin plays an increasingly important role as a regulator of FSH secretion in the female from at least 20 days of age into adulthood. In the male, however, the role of inhibin in regulating FSH secretion, which is clearly present during early postnatal development, is apparently lost at the time of puberty.

Immunocytochemical localization of inhibin in rat and human reproductive tissues

Recently, the structures of two forms of inhibin present in human follicular fluid were elucidated from the corresponding cDNA sequences. Using specific antisera generated against the alpha-chain common to both forms, we have examined the cellular localization of inhibin in the male and female rat gonads and in human placental tissue. Specific alpha-inhibin immunoreactivity was localized within the Sertoli cells of a number of tubules in each testes section. However, other adjacent tubules were unstained suggesting a stage-specific production of inhibin. Intense immunostaining was observed in the granulosa cells of ovarian follicles at various stages but not in the thecal cells. Immunostaining was present in the human placenta and limited to the cytotrophoblast cells, suggesting a role of inhibin during pregnancy. The present study demonstrates the probable site of production of inhibin in the gonads and placenta and further implicates this important factor as a key regulator of reproductive functions.

Immunocytochemical Localization of the Gonadotropin-Releasing Hormone-Associated Peptide of the LHRH Precursor

Using specific rabbit anti-GAP (gonadotropin-releasing hormone-associated peptide) serum, we have immunocytochemically localized GAP in the rat brain. Immunostaining of neuronal perikarya, fibers and terminals was demonstrated with GAP antiserum under conditions of tissue preparation which make immunostaining with LHRH antisera difficult or undetectable. GAP-immunoreactive perikarya were observed in sections of perfused or nonperfused brains without colchicine pretreatment. Using a double immunoperoxidase staining method, both GAP and LHRH immunoreactivities were shown to coexist in the same neurons. The common distribution of LHRH and GAP immunoreactivity in the rat brain is strongly supportive of GAP representing the non-LHRH portion of the LHRH precursor. The use of GAP antisera that can distinguish between LHRH and the remaining portion of its prohormone represents a valuable tool for studies of LHRH-prohormone processing and distribution.

Destruction of testicular leydig cells reveals a role of endogenous inhibin in regulating follicle-stimulating hormone secretion in the adult male rat

It has previously been demonstrated that passive immunoneutralization of endogenous inhibin results in a dramatic elevation in follicle-stimulating hormone (FSH) secretion in the adult female rat but not in the adult male. The purpose of the present study was to investigate whether the effects of immunoneutralizing endogenous inhibin on FSH secretion in the adult male rat might be masked by the presence of additional, compensating, FSH-suppressing factors. This was determined by examining the individual and combined effects of removing the testicular influences provided by the Leydig cells using the selective toxicant, ethane dimethane sulfonate (EDS), and passive immunoneutralization of endogenous inhibin. Within 24 h of a single i.p. injection of EDS, plasma testosterone levels were lowered to near assay limits and by 3 days were undetectable. Plasma FSH levels were significantly elevated 3 and 7 days after EDS treatment, but not to the levels observed in rats castrated for similar periods of time. Castration of rats, treated 3 days earlier with EDS, resulted in a further significant increase in FSH secretion as compared with EDS-treated, sham-operated controls, indicating that the testes were providing an additional FSH-suppressing factor(s) other than those originating in the Leydig cells. Injection of anti-inhibin serum, into rats treated 3 or 7 days earlier with EDS, induced a further significant increase in FSH secretion that raised plasma FSH to a level comparable to that observed in male rats castrated for similar periods of time. Plasma LH secretion was also dramatically elevated by EDS treatment to levels that equaled or exceeded those observed in similarly timed castrates. Pituitary sensitivity, as tested by the injection of an exogenous challenge of luteinizing hormone-releasing hormone (LHRH), was significantly increased 3 or 7 days after either EDS treatment or castration in terms of LHRH-stimulated LH release, but not in terms of LHRH-stimulated FSH release. Immunoneutralization of endogenous inhibin induced no further observable changes in pituitary sensitivity to LHRH. These results demonstrate that in the absence of the Leydig cells a secondary role is revealed for endogenous inhibin in suppressing FSH secretion that, in combination with the Leydig cell influence(s), accounts for the postcastration increase in FSH. The need to remove the Leydig cell influence(s) to reveal an effect of endogenous inhibin on FSH secretion in the adult male rat may suggest that the inhibin effect is normally masked by the presence of the comparatively larger suppressive influence(s) derived from the Leydig cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of specific antisera and a radioimmunoassay procedure for the gonadotropin-releasing hormone associated peptide (GAP) of the LHRH prohormone

Recently, the structure of the cDNAs encoding the human and rat LHRH prohormones were determined and the corresponding peptide sequences deduced. In addition to LHRH, both cDNAs encoded a 56 amino acid sequence which has been designated gonadotropin-releasing hormone associated peptide (GAP). In order to initiate studies on the LHRH prohormone, three antisera (MC-1, MC-2 and MC-3) were generated using the human GAP (hGAP) 25-53 fragment as immunogen and a corresponding radioimmunoassay procedure was developed. The binding of 125I-labeled hGAP 1-56 to all three antisera was displaced completely by unlabeled hGAP 1-56. Acid extracts of rat median eminence produced displacement curves that were parallel to the hGAP 1-56 curve. Conversely, extracts of rat cortex and a number of brain and pituitary peptides, including LHRH and LHRH analogs, did not affect tracer binding. Several fragments of the human and rat GAP (rGAP) sequences (hGAP 25-53, hGAP 27-40 and rGAP 20-43) produced tracer displacement curves that were non-parallel with the hGAP 1-56 curve and/or gave only incomplete tracer displacement when all three antisera were tested. This suggests that the antisera contain multiple antibody populations directed toward several different antigenic determinants within the mid portion of the hGAP and rGAP sequences. The rGAP fragments, rGAP 1-11 and rGAP 39-53, failed to displace the tracer, further supporting that the antigenic sites occur within the mid portion of the rGAP sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Transmembrane signals and intracellular messengers mediating LHRH and LH secretion.

The gametogenic and endocrine functions of the gonads are both under the integrative control of the central nervous system. The primary regulatory component in this system is the hypothalamic peptide luteinizing hormone-releasing hormone (LHRH), a decapeptide produced by neurons located in the hypothalamic-preoptic region. After synthesis, LHRH is transported to nerve terminals located in the median eminence, where it is released in close proximity to portal capillaries. The decapeptide is then transported to the anterior pituitary gland through the hypophyseal portal circulation, where, acting on specific membrane receptors, it stimulates LH and FSH secretion. These gonadotropins, in turn, profoundly affect gonadal function.

Immunocytochemical localization of the gonadotropin-releasing hormone-associated peptide portion of the LHRH precursor in the hypothalamus and extrahypothalamic regions of the rat central nervous system

SummaryThe gonadotropin-releasing hormone-associated peptide (GAP) of the LHRH precursor and the decapeptide LHRH were localized in the rat brain by immunocytochemistry in 12 to 18-day-old animals, by use of thick Vibratome sections and nickel intensification of the diaminobenzidinereaction product. Our results indicate that the GAP portion of the LHRH precursor is present in the same population of neurons that contain LHRH in the rat brain. An important difference observed was that the GAP antiserum, in contrast to LHRH antisera, stained several perikarya in the medial basal hypothalamus. GAP-immunoreactive perikarya were observed in the following regions: the olfactory bulb and tubercle, diagonal band of Broca, medial septum, medial preoptic and suprachiasmatic areas, anterior and lateral hypothalamus, and several regions of the hippocampus. In addition to the preoptico-terminal and the septopreoptico-infundibular pathways, we also observed GAPimmunopositive processes in several major tracts and areas of the brain, including the amygdala, stria terminalis, stria medullaris thalami, fasciculus retroflexus, stria longitudinalis medialis, periventricular plexus, periaqueductal gray of the mesencephalon and extra-cerebral regions, such as the nervus terminalis and its associated ganglion. These results confirm the specificity of previous immunocytochemical results obtained with antisera to LHRH. The presence of GAP immunoreactivity in nerve terminals of the rat brain indicates that GAP or a GAP-like peptide is located in the proper site to serve as a hypophysiotropic substance and/or as a neurotransmitter or neuromodulator.

Adenosine receptor-dependent modulation of inhibin secretion in cultured immature rat Sertoli cells

Inhibitory (A1) adenosine receptors that attenuate adenylate cyclase activity are present in cultured Sertoli cells. To investigate the possible effect of activating these receptors on the secretion of inhibin by the Sertoli cell, immature rat Sertoli cells were incubated for 24 h with follicle-stimulating hormone (FSH) in the absence or presence of the non-metabolizable, adenosine agonist phenyl-isopropyl-adenosine (PIA), and the accumulation of alpha-inhibin immunoreactivity was measured in the medium. Although devoid of effects when added alone, PIA inhibited the FSH-dependent secretion of alpha-inhibin in a concentration-dependent manner (ED50 = 1-1.5 nM). PIA treatment of the Sertoli cells also rendered the cells less sensitive to FSH in terms of alpha-inhibin secretion. The concentration-response curve to FSH was shifted to the right when cells were incubated in the presence of 100-1000 nM PIA. In contrast, dibutyryl cAMP stimulation of alpha-inhibin accumulation was unaffected by treatment with PIA, indicating that the site of PIA action is at the level of cAMP synthesis. These data provide experimental evidence of adenosine modulation of inhibin secretion by the Sertoli cell and suggest that adenosine may act as a local modulator within the pituitary-testicular axis.

Differential secretion of proLHRH fragments in response to [K+], prostaglandin E2 and C kinase activation.

ProLHRH contains the luteinizing hormone-releasing hormone (LHRH) decapeptide and a 56 amino acid peptide designated gonadotropin-releasing hormone-associated peptide (GAP). We studied the effects of various known secretagogues of LHRH on the in vitro release of proLHRH fragments from the median eminence (ME) and subsequently characterized these immunoreactive products according to molecular weight (MW). GAP- and LHRH-like immunoreactive (LI) materials were secreted simultaneously into the media under basal conditions. Prostaglandin E2 stimulated release of both peptides by approximately 2-fold. Both phorbol ester and [K+] stimulated release of GAP-LI by 4-fold and LHRH-LI by 9-fold over baseline levels. When materials from [K+]-stimulated media were separated according to MW by high performance size-exclusion chromatography, a single peak eluted at 1300 MW in the same position as synthetic LHRH. Two GAP-LI peaks were observed. One eluted in the void volume, while the predominant peak co-eluted with synthetic rat GAP1-56 at approximately 6500 MW. These results indicate that GAP and LHRH are co-secreted, that they are released as intact peptides, and that activation of different intracellular pathways may cause their differential secretion. These results emphasize the importance of using both in vitro and chromatographic methodologies to evaluate the changes which may occur in LHRH prohormone processing and secretion.

Peptide-steroid interactions in brain regulation of pulsatile gonadotropin secretion

Reproductive function is regulated by an intricate system of peptide, steroid and amine factors interacting within the brain, pituitary and gonads. At no point is the complexity of the reproductive system better exemplified than in the exquisite interplay of factors required to produce and modulate pulsatile gonadotropin secretion. By extension, analysis of the pulsatile secretory pattern of the gonadotropins, as a means of assessing the contribution of these various factors, can reveal interactions too subtle to be detected by the conventional examination of mean gonadotropin concentration. Analysis of the pulsatile secretory patterns of both LH and FSH reveals striking differences between the two gonadotropins in their response to inhibitory, gonadal peptide and steroid factors, suggesting divergent paths of brain-pituitary regulation. Further studies to clarify this disparity in regulation have demonstrated that neutralization of endogenous LHRH completely abolishes pulsatile LH secretion without affecting pulsatile FSH secretion, suggesting the existence of another, as of yet unknown, brain factor which regulates FSH secretion. The feedback signals provided by gonadal steroids can induce both inhibition and facilitation of LHRH and LH secretion. Neurons of the central opiatergic system exert a tonic inhibitory influence on the catecholaminergic neurons regulating LHRH secretion, and are believed to mediate the inhibitory actions of the gonadal steroids on the LHRH system. Withdrawal of the gonadal steroids has been reported to cause a rapid loss of the tonic inhibitory control of the opiate system on LHRH secretion as revealed by a lack of response to naloxone. Reassessment of this system by analyzing the pulsatile pattern of LH secretion, however, reveals that the loss of naloxone effect after gonadectomy occurs very gradually and that an effect can still be obtained up to 2 weeks after the removal of gonadal steroids. These studies provide excellent examples of the complex interplay observed just between selected factors regulating pulsatile gonadotropin secretion. The use of pulsatile gonadotropin analysis is a powerful model, not only for providing greater clarity of known regulatory interactions, but also for revealing new and more subtle levels of control in the brain-pituitary-axis.