Cryptorchid rhesus macaques: long term studies on changes in gonadotropins and gonadal steroids

We studied the relationship of testosterone (T), dihydrotestosterone (DHT), and 27 beta-estradiol (E2) to FSH and LH in the systemic circulation of six rhesus macaques with surgically induced cryptorchidism at selected times over 420 days. We measured these hormones by RIA and compared their concentrations with those of four sham-operated controls. Midway through the experimental procedure the animals were electroejaculated, and on day 425 the testes were removed and analyzed histologically. The cryptorchid monkeys did not have sperm in their ejaculates, and treatment adversely affected spermatogenesis. For the first 30 days after treatment none of the hormones differed significantly between cryptorchid monkeys and controls. Gonadotropins in three castrated males, however, gradually rose to postcastration levels within approximately 2 weeks post operation. After castration, all three steroids declined significantly within 24 h. Beginning on days 60, 150, 215, 320, and 420 post operation the animals were bled for 5 days on a diurnal regimen. The steroids as well as LH concentrations varied diurnally. The concentration of FSH never showed diurnal variation in any of the groups at any of the periods studied. Concentrations of T and LH did not differ significantly between control and cryptorchid groups at any time. With longer time periods (beginning 215 days after cryptorchidism had been induced), DHT in cryptorchid monkeys was significantly higher than in controls (P < 0.01). Although E2 was significantly lower in cryptorchid monkeys on days 60 and 150 post operation, this difference disappeared with time. Changes in steroid concentrations were not associated with the elevations in FSH that occurred in cryptorchid monkeys by days 60 and 420. Therefore, we assumed that they were independent phenomena. Significant FSH elevations in cryptorchid monkeys occurred only in the fall of the year. Testicular homogenates from cryptorchid and control monkeys produced similar quantities of T, DHT, and androstenedione in vitro. Very little E2 or estrone was found. Although significant amounts of progesterone were quantified in the incubation media of control testes, little or no progesterone was found in media from cryptorchid testes. Similar results were obtained when these steroids were measured in plasma collected from the testicular veins. In testicular venous plasma, 20 alpha-dihydroprogesterone concentrations were not elevated after cryptorchidism. These data suggest that there is similar negative feedback control of gonadotropins in crytorchid and control rhesus monkeys. The absence of dramatic differences in systemic concentrations of FSH between the two groups suggests that the seminiferous tubule does not play a major role in the negative feedback control of gonadotropins in this species or that the tubular component of this control system is not perturbed by the cryptorchid condition...     

Incertohypothalamic A13 dopamine neurons: effect of gonadal steroids on tyrosine hydroxylase.

In this study the effect of gonadectomy and steroid treatment on the dorsal component of the incertohypothalamic dopamine system or nucleus A13 was assessed by immunocytochemistry using an antibody raised to tyrosine hydroxylase (TH). A computer graphic system interfaced to a microscope was used to count and measure the diameters of TH-positive neurons and display the data in the three-dimensional space of the nucleus. In males, castration resulted in a dramatic decrease in the reaction product representative of TH. The number of TH-positive cells in the A13 DA nucleus decreased to 25% of intact levels. In females, ovariectomy also caused an impressive loss in the TH-immunostainable material, but this was not indicated by a change in the total number of TH-positive neurons. In both sexes the loss in TH immunostain was confined mainly to the mid-portion of the nucleus. Hormone treatment restored the TH immunostain (cell number and size) to and/or above intact levels in both sexes. These data suggest that A13 TH immunostain is stimulated by gonadal steroids in male and female rats.     

Effects of luteinizing hormone releasing hormone on the ultrastructure of gonadotrophs in the foetus of the rhesus monkey near term.

Eighty micrograms of synthetic luteinizing hormone releasing hormone (LH-RH) were infused systemically into male and female foetuses of rhesus monkeys near term. Control animals were given infusions of saline. Morphologically, the control gonadotrophs varied from cells filled with secretory granules to highly stimulated cells with numerous cytoplasmic vesicles. In the LH-RH-treated animals, however, many cells showed depletion of secretory granules, dilatation of the endoplasmic reticulum and condensed nuclear chromatin. It is concluded, therefore, that foetal gonadotrophs can respond to administration of synthetic LH-RH.     

Sexual dimorphism in the synaptic input to gonadotropin releasing hormone neurons

GnRH neurons form the final common pathway regulating the secretion of gonadotropins from the anterior pituitary. Since the patterns of gonadotropin release display profound sexual dimorphism among mammals including the rodent, we undertook an ultrastructural analysis to determine whether these neurosecretory cells were differentially innervated between the sexes. As a further exploration of the organization of the neurocircuitry integrating GnRH neurons with the central nervous system, we also determined the degree to which GnRH cells and their processes were innervated by terminals containing either the endogenous opiate, beta-endorphin (BE) or GnRH itself. Sections from the diagonal band of Broca and the preoptic area of adult male and diestrus II female rats were immunocytochemically processed for dual localization of GnRH and BE. GnRH neurons cut through the plane of the nucleus were identified in 1 micron sections. Serial ultrathin sections were made and analyzed for 1) total synaptic input to both cell bodies and dendrites; 2) BE input; and 3) input arising from GnRH itself. We report that GnRH neuronal cell bodies in females received approximately twice the number of synapses as did those of males. The input to the GnRH dendrites, when measured as percent of plasma membrane in synaptic contact, also showed a profound sexual dimorphism with the female having a larger proportion of the dendrite in synaptic apposition. BE innervation contributed to this dimorphism at the level of both the cell body and dendrite. In contrast, the distribution and number of GnRH terminals did not differ between the sexes. In both they were confined to the dendritic arbor. We hypothesize that the capacity of the female rodent GnRH system to show neurogenic derived alterations in GnRH output not seen in the male may be due in part to these anatomical differences.     

Sex steroids in the umbilical circulation of fetal rhesus monkeys from the time of gonadal differentiation

Male rhesus monkey fetuses have significantly more testosterone (T) in their circulation than females on days 35--50 of gestation (P less than 0.01; n = 6 males and 6 females). However, we found no sex differences for androstenedione (delta 4). T concentrations remained significantly higher in male fetuses than in females later in gestation, e.g. days 79--84, 100--133, and 140--160. Levels of delta 4 differed between the sexes only on days 79--84, and dihydrotestosterone concentrations were significantly higher in male fetuses than in females on days 100--133 and 140--163. The fact that delta 4 concentrations were not different between the sexes at the earliest period studied (days 35--50) indicates that systemic concentrations of this hormone in the fetus probably are not important for sexual differentiation, especially of the central nervous system. Quantification of three steroids (T, delta 4, and dihydrotestosterone) in umbilical arterial and venous plasma from five male and nine female fetuses (days 35--100) revealed significant arterial/venous differences only for T in males (arterial greater than venous). These data, which suggest that fetal testes secrete T during morphological differentiation, lend credence to the hypothesis that endogenous T partially regulates sexual differentiation.     

Dendritic processing of excitatory synaptic input in hypothalamic gonadotropin releasing-hormone neurons

The activity of hypothalamic GnRH neurons results in the intermittent release of GnRH required for reproductive function. This intermittent neurosecretory activity has been proposed to reflect integration of intrinsic properties of and synaptic input to GnRH neurons. Determining the relative impact of synaptic inputs at different locations on the GnRH neuron is difficult, if not impossible, using only experimental approaches. Thus, we used electrophysiological recordings and neuronal reconstructions to generate computer models of GnRH neurons to examine the effects of synaptic inputs at varying distances from the soma along dendrites. The parameters of the models were adjusted to duplicate measured passive and active electrophysiology of cells from mouse brain slices. Our morphological findings reinforce the emerging picture of a complex dendritic structure of GnRH neurons. Furthermore, analysis of reduced morphology models indicated that this population of cells is unlikely to exhibit low-frequency tonic spiking in the absence of synaptic input. Finally, applying realistic patterns of synaptic input to modeled GnRH neurons indicates that synapses located more than about 30% of the average dendrite length from the soma cannot drive firing at frequencies consistent with neuropeptide release. Thus, processing of synaptic input to dendrites of GnRH neurons is probably more complex than simple summation.     

A potential apulsatile mode of GnRH release in the male rhesus monkey (Macaca mulatta).

The hypothalamic component of the reproductive axis in vertebrates is comprised of a pulse generator that stimulates the release of GnRH. Several lines of evidence are in agreement that the activity of this pulse generator is intermittent and results in the pulsatile pattern of GnRH and LH release. During a recent investigation of the re-initiation of LH secretion in the agonadal, prepubertal male monkey, we observed a daytime profile of LH secretion, which suggests an apulsatile mode of GnRH release. The first purpose of this study was to describe this observation of apulsatile LH release during the peripubertal transition. Furthermore, we have explored the dependence of this form of LH secretion on GnRH release. Five male rhesus monkeys (Macaca mulatta) were castrated prepubertally and were treated with an intermittent infusion of GnRH to prematurely sensitize the juvenile pituitary to endogenous GnRH release. Alternate daytime (1100-1800 h) and nighttime (1900-0200 h) assessments of LH release were performed at 10-day intervals throughout the peripubertal transition with samples taken every 12 min. In a second experiment, four agonadal males which demonstrated an apulsatile profile of LH release were maintained on an infusion of physiological saline and were treated with the GnRH antagonist Nal-Glu (i.m., 500 microgram/kg). Circulating levels of LH were determined 22 h after antagonist treatment. In peripubertal animals, circulating levels of LH were similar between morning and evening assessments. However, pulse frequency was significantly lower during the daytime. GnRH antagonist reduced LH levels by 72% and a similar reduction in response to an exogenous GnRH test stimulus occurred. These findings suggest an apulsatile mode of GnRH release.     

Effects of nitrogen dioxide and ozone on monkey lung ultrastructure

Pulmonary ultrastructural abnormalities were studied in squirrel monkeys exposed to 30 ppm NO₂ or 3 ppm O₃ for three hours with intermittent exercise. The lung tissues were processed either immediately after exposure or 1, 4 or 7 days later. Both the transmission and scanning electron microscopes were used for observation. Initial changes caused by the NO₂ were the presence of fibrin in the alveoli, blebbing of the alveolar epithelium and the removal of cilia from the terminal bronchiolar epithelium. Seven days after exposure the tissues appeared to be recovering. Three ppm O₃ was more damaging to alveoli than the 30 ppm NO₂. The epithelial lining was degenerating or completely gone in many loci. Where intact, the width of the blood-air barrier increased with exposure and further with delay of sacrifice. Abnormal porosity of the alveolar walls was generally observed and remained through seven days after exposure. An unusual number of “paired” type 2 epithelial cells were present and contained a few to many homogeneous spherical inclusions of medium density. The results observed indicate that quantitation of morphological changes will be possible even at ambient exposures of atmospheric pollutants.     

Effects of cytokines on gonadotropin-releasing hormone (GnRH) gene expression in primary hypothalamic neurons and in GnRH neurons immortalized conditionally

Various cytokines produced during the immune reaction can modulate the neuroendocrine reproductive axis, probably by inducing changes in the activity of hypothalamic GnRH neurons. However, the precise cellular and molecular effects of cytokines on these neurons have not been reported yet. To gain a better insight into these regulations, we first examined the pattern of expression of cytokine receptors in a novel neuronal cell line expressing GnRH (Gnv-4 cells). Among others, gp130 is expressed in Gnv-4 cells, together with the ligand receptor subunits specific for IL-6 as well as oncostatin M (OSM). Consistent with the latter observation, we show that OSM stimulates the expression of the immediate early genes c-fos and early growth response-1 in Gnv-4 cells, an effect dependent upon the activation of the MAPK Erk1/2 intracellular signaling pathway. Functional studies performed in parallel in Gnv-4 cells and in primary hypothalamic neuronal cell cultures show that OSM, although devoid of any effect of its own on GnRH gene expression, can inhibit dose-dependently the stimulation of GnRH expression by N-methyl-d-aspartic acid. In conclusion, these data demonstrate that a GnRH-expressing neuronal cell line can be modulated in vitro by cytokines implicated in the regulation of the reproductive axis. Moreover, they provide the first evidence of an involvement of OSM in these regulations.     

Structural interactions between kisspeptin and GnRH neurons in the mediobasal hypothalamus of the male rhesus monkey (Macaca mulatta) as revealed by double immunofluorescence and confocal microscopy

Kisspeptin is recognized to play a critical role in eliciting the pubertal resurgence of pulsatile GnRH release, the proximal trigger of puberty in higher primates. Expression of the kisspeptin receptor (GPR54) by GnRH neurons indicates a direct action of kisspeptin on the GnRH neuronal network. The purpose of the present study was to examine the distribution of kisspeptin cell bodies in the monkey hypothalamus and to assess the structural basis for the stimulatory action of kisspeptin on the GnRH neuronal network. Three castrated male rhesus monkeys, 39-51 months of age, were deeply anesthetized and their brains perfused transcardially with 4% paraformaldehyde in PBS. Serial 25-microm coronal sections throughout the hypothalamus were prepared, and immunopositive neurons identified using a cocktail of specific primary antibodies (sheep anti-kisspeptin at 1:120,000, and rabbit anti-GnRH at 1:100,000) detected with fluorescently tagged secondary antibodies (antisheep, Alexa Fluor 488; antirabbit, Cy3) in combination with confocal microscopy. Kisspeptin perikarya were found only in the mediobasal hypothalamus (MBH) almost exclusively in the posterior two-thirds of the arcuate nucleus. Surprisingly, kisspeptin-beaded axons made only infrequent contacts with GnRH neurons (kisspeptin and GnRH profiles abutting in a 0.5- to 1.0-mum optical section) in the MBH. In the median eminence, kisspeptin and GnRH axons were found in extensive and intimate association. GnRH contacts on kisspeptin perikarya and dendrites were observed. These findings indicate that nonsynaptic pathways of communication in the median eminence should be considered as a possible mechanism of kisspeptin regulation of GnRH release, and provide an anatomical basis for reciprocal control of kisspeptin neuronal activity by GnRH.     

Effects of gonadal steroids on somatotroph function in the rat: analysis by the reverse hemolytic plaque assay

The mechanism by which gonadal steroids modulate GH secretion is not known. We have used the reverse hemolytic plaque assay to examine whether gonadal steroid-induced modulation of GH secretion is effected by changes in the population of somatotrophs and/or alterations in their secretory properties. Two groups of Sprague-Dawley rats were studied: group 1 (n = 6) comprised male (M), castrate (Cx), and testosterone-replaced castrate male (Cx + T) rats and group 2 (n = 5) consisted of male (M), female (F), and 17 beta-estradiol-replaced castrate male (Cx + E) rats. The number of plaque-forming cells (expressed as both absolute number and a percentage of all cells) was determined, and secretory status was assessed by measuring plaque areas in response to 0, 0.01, 0.1, 1, 10, and 100 nM GHRH. While mean basal GH plaque areas were similar among the treatment groups of group 1, the maximal GH plaque area was significantly decreased in Cx [16.8 +/- 2.4 vs. 26.4 +/- 3.9 X 10(6) microns2 (mean +/- SEM); P less than 0.05], but not in Cx + T (27.5 +/- 4.1 microns2) rats. The GHRH EC50 was unaffected by castration or T replacement. The percentage and absolute population of somatotrophs were reduced in Cx, but not in Cx + T, rats, while the numbers of lactotrophs remained unchanged in these treatment groups. For group 2, the mean peak GH plaque area was reduced in Cx + E (16.5 +/- 2.9 microns2; P less than 0.001) compared to that in M rats (36.2 +/- 2.3 microns2), but was not significantly different from that in F (13.0 +/- 1.5 microns2) rats. The EC50 was significantly (P less than 0.025) greater in Cx + E (10.9 +/- 2.3 nM) and F (7.9 +/- 1.6 nM) compared to M rats (2.8 +/- 0.7 nM). The absolute somatotroph and lactotroph populations were increased in Cx + E compared to M and F rats, as were the populations of other pituitary cell types. Testosterone enhances GH secretion by increasing the secretory capacity, but not the sensitivity, of somatotrophs to GHRH and by recruiting the function of a subpopulation of somatotrophs. Estradiol reduces the secretory capacity and sensitivity of somatotrophs to GHRH, but increases the population of somatotrophs, lactotrophs, and non-GH- and non-PRL-secreting cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of gonadal steroids on clearance of growth hormone at steady state in the rat

Gonadal steroids have been implicated in the modulation of GH secretory patterns in the rat. We have studied the effects of testosterone (T) or estradiol (E2) on the steady state clearance (Clss) and plasma half-life (t1/2) of GH in male and female rats (n = 4-6/group). A femoral and a jugular cannula were surgically implanted into adult Sprague-Dawley rats. At the time of cannulation some rats were orchidectomized, and a Silastic capsule containing E2, T, or nothing was implanted sc. After recovery from surgery, either purified rat GH or a crude extract of rat pituitary was infused iv to attain steady state plasma GH concentrations. Blood samples were taken every 30 min for 4 h during the infusion, and nine samples were taken at 2.5-min intervals immediately after stopping the infusion. The mean Clss for GH in female rats were significantly (P less than or equal to 0.001) less than that in males, whereas the t1/2 did not differ between the two groups. Neither the Clss nor the t1/2 was affected by castration in males or females. The Clss of GH in E2-treated castrated males was significantly less (P less than 0.001) than that for intact males, but the t1/2 did not differ between the two groups. The Clss for GH was greater in T-treated ovariectomized rats than in intact females, but the t1/2 did not differ with T treatment. These results suggest that 1) the Clss for GH is lower in female rats than in males; 2) 4 weeks of gonadectomy has no effect on the Clss in males or females; 3) under experimental conditions, E2 decreases and T increases the Clss for GH; and 4) the t1/2 for GH is not different in males or females. The steroid-induced changes in Clss in the absence of detectable effects on t1/2 suggest that factors affecting the volume of distribution at steady state (i.e. plasma GH-binding proteins or GH heterogeneity) are involved in the effects of gonadal steroids on GH clearance in the rat.     

Effects of gonadal steroids on gonadotropin secretion in males: studies with perifused rat pituitary cells

The feedback effects of testosterone (T) and estradiol (E2) on FSH and LH secretion were compared in dispersed pituitary cells from adult male rats perifused with pulses of GnRH. Cells were stimulated with 10 nM GnRH for 2 min every 1 h. T (10 nM) pretreatment for 24 h reduced the amplitude of FSH and LH pulses to 77 +/- 4% (mean +/- SE) and 47 +/- 3% of control values, respectively (P less than 0.01), whereas 6-h T treatment was without effect. By contrast, interpulse secretion of FSH was increased after 24 h T to 184 +/- 7% of the control value (P less than 0.01), but interpulse LH release was unchanged (104 +/- 5%). E2 (0.075 nM) treatment of pituitary cells reduced GnRH-stimulated FSH and LH release within 2 h to 75 +/- 2% and 73 +/- 3% of control values, respectively (P less than 0.01). E2 pretreatment for 24 h stimulated (P less than 0.025) GnRH-induced FSH (136 +/- 10%) and LH (145 +/- 8%) release and also increased (P less than 0.01) interpulse FSH (127 +/- 5%) and LH (145 +/- 8%) secretion. These data indicate that the suppression of FSH and LH secretion by T in males is due in part to a direct effect on the pituitary. The findings that T suppresses GnRH-stimulated FSH less than LH, and that T stimulates interpulse FSH, but not LH, provide evidence for differential regulation of FSH and LH secretion by T. The dissimilar actions of T on GnRH-stimulated pulses and interpulse gonadotropin secretion suggest that interpulse secretion is unrelated to stimulation by GnRH, although its physiological significance is unknown. Since E2, in physiological levels for males, increased pituitary FSH and LH secretion, the suppression of gonadotropin secretion by E2 in vivo in males may result from an effect on the hypothalamic pulse generator; however, additional studies are needed before extending these conclusions to higher mammals and men.