Regulation of GHRH receptor gene expression in the neonatal and adult rat pituitary.

The growth hormone releasing hormone (GHRH) receptor gene is essential for normal growth, and its expression is developmentally regulated. The factors that control GHRH receptor expression in the neonatal pituitary are not well understood. This study focuses on the regulation of GHRH receptor gene expression by thyroid hormone, glucocorticoids, insulin-like growth factor-I (IGF-I) and IGF-II in rat pituitary cell cultures. In newborn pituitaries, both T3 and hydrocortisone (24 h) caused a dose-dependent increase in GHRH receptor mRNA abundance, reaching levels 4.8-fold (P<0.001) and 6.5-fold (P<0.001) over corresponding controls. T3 and hydrocortisone also stimulated GHRH receptor expression in adult (70 day) pituitary cell cultures, consistent with our earlier findings. IGF-I treatment suppressed the inductive effects of T3 (P<0.02) and hydrocortisone (P<0.03) on GHRH receptor expression in adult pituitaries but not in neonatal pituitaries. Unlike IGF-I, IGF-II treatment had no effect on T3-induced or hydrocortisone-induced GHRH receptor expression in either neonates or adults. Taken together, these results indicate that (1) thyroid hormone and hydrocortisone act directly at the neonatal pituitary as potent stimulators of GHRH receptor gene expression, (2) IGF-I, but not IGF-II, acts at the pituitary to suppress GHRH receptor mRNA expression and (3) the effects of IGF-I on GHRH receptor gene expression are developmentally determined.

A novel mechanism of body mass regulation.

While significant attention has been devoted to the identification of hormonal factors that control body mass, little attention has been paid to the role of mechanical loading on animal mass. Here, we provide evidence that intraperitoneal implantation of metabolically inert mass results in a compensatory reduction in tissue mass. Deer mice (Peromyscus maniculatus) were surgically implanted with weights of 1, 2 or 3 g. There was a resulting loss of tissue mass (total body mass minus implant mass) that was proportional to the mass of the implant. This reduction in tissue mass followed a reduction in food intake in animals with 3 g implants. Evaluation of body composition failed to identify any single component that contributed to the loss of tissue mass. Removal of implants led to a transient restoration of body mass to levels similar to the total body mass of those control animals in which the implant had not been removed. However, within 12 days of implant removal, body mass again declined to the level seen before implant removal. These results suggest the existence of a set point that is sensitive to changes in the perception of mass and that is transduced via neural pathways.

Differential GH-releasing hormone regulation of GHRH receptor mRNA expression in the rat pituitary.

To understand the capacity of growth hormone-releasing hormone (GHRH) to regulate expression of the GHRH receptor, we studied the effects of GHRH on GHRH receptor mRNA expression in immature and adult rats by use of pituitary cell culture and immunoneutralization approaches. Pituitary cell cultures from neonatal (2-day-old) and adult (70-day-old) rats were treated with GHRH for 4, 24, or 72 h. The effect of GHRH on GHRH receptor mRNA expression depended on the duration of GHRH exposure in both age groups; short-term (4 h) GHRH treatment significantly reduced GHRH receptor mRNA expression (P < 0.05), whereas intermediate treatment (24 h) restored GHRH receptor mRNA to basal levels, and long-term treatment (72 h) stimulated GHRH receptor mRNA expression (P < 0.02). The long-term stimulatory effect of GHRH on GHRH receptor mRNA expression required the presence of serum in the culture medium, and, in the absence of serum, the stimulatory effect was completely abolished. Moreover, the capacity of the pituitary to increase GHRH receptor mRNA expression in response to 72-h GHRH treatment was age dependent, with neonatal pituitaries exhibiting a much greater stimulatory effect than adult pituitaries (P < 0.025). Immunoneutralization of endogenous GHRH significantly reduced GHRH receptor mRNA expression in neonatal (P < 0.004), juvenile (P < 0.003), and mature (P < 0.004) pituitaries compared with age-matched controls. Taken together, these results indicate that GHRH is a potent regulator of GHRH receptor gene expression in immature and mature pituitaries; however, the nature and direction of GHRH regulation of its receptor depend significantly on several variables, including the duration of GHRH exposure, the presence of permissive components in serum, and the developmental stage of the pituitary.

Pituitary somatostatin receptor (sst)1-5 expression during rat development: age-dependent expression of sst2.

The capacity of the pituitary to suppress hormone secretion in response to somatostatin (SRIF) is markedly age dependent. Immature pituitaries are relatively resistant to SRIF effects, and increasing sensitivity to SRIF with advancing age is believed to cause characteristic developmental changes in pituitary hormone secretion in mammals. However, the cellular mechanism(s) underlying this developmental pattern of response to SRIF are not understood. Because somatostatin receptors (ssts) are critical mediators of SRIF's actions on target tissues, we investigated the expression of sst1, sst2, sst3, sst4, and sst5 messenger RNA (mRNA) in pituitaries of developing and mature rats. Animals were studied at embryonic day 19.5, and at postnatal days 2, 12, 30, 45, 70, and 1 yr; these ages correspond to major changes in circulating GH levels and pituitary responsiveness to SRIF. Pituitary levels of sst2 mRNA increased strikingly and progressively with advancing age after birth (F = 30.92, P < 0.0001). Compared with 2-day-old pituitaries, sst2 mRNA abundance rose 3.25-fold by 12 days of age and 6-fold by 70 days of age. Moreover, Western blot analysis indicated a marked increase in pituitary expression of sst2A protein with advancing age. By contrast, pituitary abundance of sst1, sst3, sst4, and sst5 mRNAs did not differ with age. To assess the role of endogenous SRIF in regulating perinatal sst2 gene expression, we also administered a well-characterized SRIF antiserum (or NSS as controls; 10 microl/10 g) sc daily from postnatal days 2 to 12 of life. Treatment with SRIF antiserum raised GH levels but did not alter pituitary sst2 mRNA abundance, compared with controls. Taken together, these data indicate that 1) the perinatal rat pituitary expresses the same complement of ssts as the adult pituitary; 2) expression of ssts is developmentally regulated in a highly subtype-specific manner; 3) pituitary sst2 mRNA and sst2A protein increase markedly and progressively with advancing age after birth; and 4) the perinatal rise in sst2 mRNA levels is unlikely to be regulated by endogenous SRIF. The finding of subtype-specific, developmentally determined sst expression indicates a novel and potentially fundamental mechanism of sst regulation, and suggests a molecular mechanism underlying developmental maturation in the capacity of the pituitary to respond to SRIF.

Pituitary expression of protein kinase C isotypes during early development.

Protein kinase C (PKC) is a critical regulator of signal transduction and cell function in many tissues, including pituitary. Although PKC influences pituitary hormone secretion in adults, its role in determining characteristic perinatal patterns of hormone secretion and synthesis is not known, and the expression of major PKC isotypes in perinatal pituitary is poorly defined. We therefore determined the developmental, cell-specific expression of the major PKC isotypes, using Western analysis and double label immunohistochemistry, in pituitaries of perinatal and mature rats. Expression of specific PKC isotypes was strikingly age-dependent. Pituitary expression of PKC alpha was particularly high in neonates and declined significantly with age, with levels in adult rats approximately half those of neonates as assessed by Western analysis. Similarly, immunohistochemistry indicated that PKC alpha was less abundant in adult than in neonatal pituitaries; the most intensely staining cells of both age groups were identified as somatotrophs and gonadotrophs. In contrast to PKC alpha, pituitary expression of PKC epsilon increased approximately two-fold with advancing age as assessed by Western analysis; this age-dependent pattern was confirmed by immunohistochemistry. Perinatal pituitaries expressed PKC epsilon in some somatotrophs and in all gonadotrophs, whereas PKC epsilon expression was limited to gonadotrophs in the mature pituitary. Pituitary expression of PKC betaII, delta, and zeta did not differ with age, and PKC gamma was not detected in pituitaries of any age group. These results indicate that expression of PKC isotypes within the pituitary is developmentally regulated in a cell-specific and isotype-specific manner, and are consistent with the concept that PKC contributes to the regulation of pituitary function during early development.

Phenotypic differences in the GnRH neuronal system of deer mice Peromyscus maniculatus under a natural short photoperiod.

The neural mechanisms by which short photoperiod induces gonadal regression among seasonally breeding mammals are not well understood. One hypothesis suggests that the proximate cause of seasonal gonadal regression is a photoperiod-induced modification in GnRH secretion. This hypothesis is indirectly supported by our recent findings using immunocytochemistry which identified specific photoperiod-induced adjustments in the number and morphology of GnRH containing neurones between reproductively competent and reproductively regressed laboratory housed male deer mice. Herein, we report that the GnRH neuronal system is similarly affected in reproductively responsive and nonresponsive wild male deer mice Peromyscus maniculatus exposed to a natural short photoperiod. The distribution of immunoreactive (IR)-GnRH neurones was nearly identical in field caught animals and those housed under artificial photoperiod in the laboratory. Compared with reproductively nonresponsive males, reproductively responsive mice from the field population possessed a greater total number of IR-GnRH neurones, a greater number of IR-GnRH neurones within the lateral hypothalamus, and a greater proportion of bipolar IR-GnRH neurones. Each of these distributional and morphological characters was consistent with our findings in laboratory housed male deer mice exposed to an artificial short photoperiod. Taken together, these data underscore the validity of using an artificial photoperiod to evaluate seasonal adjustments in reproductive function in the laboratory.

Phenotypic variation in seasonal adjustments of testis size, body weight, and food intake in deer mice: role of pineal function and ambient temperature.

We investigated pineal function as well as reproductive and energetic characteristics in male deer mice (Peromyscus maniculatus) that differentially respond to short photoperiod with full, partial or no gonadal regression. In mice at both high (23 degrees C) and low temperature (1 degree C), these phenotypic differences in reproductive responses to short days were not reflected by differences in urinary excretion of 6-sulphatoxy-melatonin, the main metabolite of pineal melatonin. Neither duration nor amplitude or phase-angle of nocturnal peaks in 6-sulphatoxymelatonin significantly differed between reproductive phenotypes at either temperature. Differences in testis size were, however, associated with different energy requirements. In gonadally regressed males only, food intake and body weight were significantly (P < 0.01) reduced by up to 29% and 13% respectively. Chronic cold exposure (5 degrees C) had no effect on the proportion of males undergoing testicular regression under short days, but caused a general elevation in body weights among all mice (P < 0.05). Phenotypic differences in body weight and food intake were maintained in the cold. Together, these results suggest that within-population variation of reproductive responses in male deer mice is based on post-pineal differences in the regulation of gonadal function, and that phenotypic characteristics in reproductive and energetic responses to short days are largely unaffected by ambient temperature.

Photoperiod and steroid-dependent adjustments in hypothalamic gonadotropic hormone-releasing hormone, dopamine, and norepinephrine content in male deer mice.

The purpose of this study was twofold: 1) to examine photoperiod-dependent and steroid-dependent adjustments in hypothalamic GnRH and catecholamine content in male deer mice, Peromyscus maniculatus, and 2) to evaluate whether the commonly used measure of GnRH accumulation serves as an accurate indicator of altered reproductive function in short-day-housed rodents. Deer mice provide a unique animal model in which to evaluate the effect of short day length on the neural substrates directing reproductive function, since individual deer mice respond to short photoperiod with a range of neuroendocrine, endocrine, metabolic, and thermoregulatory adjustments. These adjustments, in part, result in two distinct reproductive phenotypes; reproductively competent "nonresponsive" mice, and gonad-regressed "responsive" mice. Reproductively mature males were maintained on either long (16L:8D) or short (8L: 16D) photoperiod for 8 wk. Thereafter, mice were phenotyped and either remained intact, castrated, or castrated and provided with an s.c. testosterone-filled silicone elastomer capsule releasing long-day or twice long-day levels of testosterone; mice were then housed for an additional 4 wk. At autopsy, plasma levels of LH and testosterone, hypothalamic GnRH content, and hypothalamic dopamine and norepinephrine concentrations were determined. Short photoperiod caused a similar increase in GnRH content in both short-day phenotypes, and this increase was primarily localized to the medial basal hypothalamus (MBH). Castration decreased GnRH content within the MBH of mice housed on both a long and short photoperiod. However, castration also reduced GnRH content within the medial preoptic area (MPOA) only among the short-day phenotypes. Steroid replacement at levels similar to those of intact long-day males restored GnRH content in all brain regions to levels of intact mice. On the other hand, steroid replacement at twice long-day levels restored GnRH content among long-day mice but reduced GnRH content among both short-day reproductive response groups, specifically within the MBH and MPOA. There were no dramatic effects of photoperiod or steroid treatment on catecholamine concentrations. But our results do suggest that the lateral preoptic area (LPOA) and MBH may be important sites of steroid feedback among mice exposed to short photoperiods and that the LPOA and diagonal band of Broca-septal regions may specifically be associated with catecholamine-directed photoperiod-induced adjustments of reproductive responsive mice. Taken together, these data show that GnRH accumulation is not the proximate signal directing seasonal changes in pituitary-gonadal function and cast doubt on the use of this factor as an accurate indicator of altered reproductive function.

Thyroid hormone and glucocorticoid regulation of pituitary growth hormone-releasing hormone receptor gene expression.

The GH-releasing hormone receptor (GHRH-R) is a critical link between hypothalamic GH-releasing hormone (GHRH) and pituitary GH secretion. However, the factors that regulate GHRH-R are not well understood. Despite the importance of thyroid hormone and glucocorticoids in influencing the GH axis in vivo, it is not known whether these hormones act directly at the pituitary to regulate expression of GHRH-R. We tested the effects of T3 and hydrocortisone on GHRH-R gene expression in primary pituitary cell cultures of adult male rats. Pituitary cells were treated for 24h with increasing concentrations of T3 (0.06-60 nM) or hydrocortisone (2.8 nM-2.8 microM). GHRH-R mRNA levels were assessed by ribonuclease protection assay. T3 caused a striking dose-dependent increase in GHRH-R mRNA, reaching levels 5.1 +/- 0.5 fold over controls (P < 0.001). Hydrocortisone also stimulated a marked dose-dependent increase in GHRH-R mRNA, reaching levels 5.6 +/- 0.7 fold over controls (P < 0.001). Combined treatment with both hormones did not cause further augmentation of GHRH-R mRNA levels. These data indicate that T3 and hydrocortisone act directly at the pituitary as potent regulators of GHRH-R gene expression.

Developmental regulation of pituitary growth hormone-releasing hormone receptor gene expression in the rat.

The mechanisms governing age-dependent patterns of GH secretion are not well understood. Studies have shown that pituitaries of fetal and neonatal mammals are highly responsive to the stimulatory effect of GH-releasing hormone (GHRH) compared to those of mature mammals. Differential pituitary responsiveness to GHRH may, therefore, contribute to the elevated circulating GH concentrations characteristic of the perinatal period and the subsequent decline in circulating GH late in life. Age-dependent expression of GHRH-receptor (GHRH-R) would provide a cellular mechanism to direct differential somatotroph responsiveness to GHRH. Therefore, we determined the ontogeny of rat GHRH-R gene expression. We studied rats at ages that correspond to major changes in circulating GH levels: embryonic day 19.5 (of 21.5-day gestation period); postnatal days 2, 12, 30, 45, and 70; and 1 yr of age. We found that GHRH-R messenger RNA (mRNA) expression was markedly age dependent (P < 0.0003). The concentration of GHRH-R mRNA was highest on day 19.5 of gestation, the earliest age studied, and declined during the perinatal period to reach a nadir at 12 days of age. GHRH-R mRNA levels increased at 30 days of age, at time corresponding to the onset of sexual maturation, and then declined later in life. In addition, we assessed the expression of GH and Pit-1 mRNAs in pituitaries of the same age groups; we found age-dependent patterns for these transcripts that did not parallel the ontogenic pattern observed for GHRH-R mRNA. Together, these data indicate that expression of rat pituitary GHRH-R mRNA is developmentally regulated and suggest that maturation of GHRH-R may be an important determinant of somatotroph function during early development.

Differential testicular responses to short daylength in deer mice are reflected by regional and morphological differences in the GnRH neuronal system.

Male deer mice (Peromyscus maniculatus) respond to short photoperiod with a range of spermatogenic responses. About one-third of all short day exposed mice exhibit a complete cessation of spermatogenesis (reproductive responsive), while about an equal number remain reproductively competent (reproductive nonresponsive). These differential spermatogenic responses are mirrored by endocrine measures; reproductive responsive males exhibit reduced circulating levels of testosterone and luteinizing hormone, while reproductive nonresponsive males exhibit long day levels of both hormones. A variety of evidence indicates that despite individual differences in reproductive responses, all short day exposed mice detect and respond to the change in photoperiod at the level of the hypothalamus, irrespective of gonadal response. We investigated whether deer mice exhibiting disparate gonadal responses to short days differed in aspects of the GnRH neuronal system, as revealed by immunocytochemistry. Reproductively mature males were maintained on either long (16L:8D) or short (8L:16D) photoperiod for 8 weeks. Thereafter, regional and morphological differences in GnRH-containing neurons were compared among long day (n = 8) and short day reproductive responsive (n = 8) and nonresponsive (n = 8) deer mice. Results demonstrate both an effect of photoperiod and reproductive state on the number, location and morphology of immunoreactive-GnRH neurons. Short days caused immunoreactive GnRH cell number to increase in both short day phenotypes relative to long day mice. Significant differences among treatment groups in the numbers of cells per region were confined to the lateral hypothalamus and lateral preoptic area. Both short day phenotypes exhibited an increase in cell number in the lateral preoptic area, compared to long day mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of gonadal steroids and inhibitory photoperiod in regulating body weight and food intake in deer mice (Peromyscus maniculatus).

We investigated the role of declining daylength and gonadal steroids on body weight and food intake in male deer mice (Peromyscus maniculatus). This species was chosen for study because individual males display different reproductive responses to inhibitory daylength. About one-third of all mice exposed to short days undergo testicular regression and exhibit reduced circulating levels of luteinizing hormone and testosterone (reproductive responsive males). In contrast, testicular function and circulating levels of both these hormones remain unaffected in an equal number of mice (reproductive nonresponsive males). Previous studies have shown that each phenotype exhibits a distinct set of metabolic responses to short days, including adjustments in body weight. These characteristics make deer mice a useful animal model in which to study the interactive effects of gonadal steroids and photoperiod on neural substrates regulating body weight. A gonadectomy/steroid replacement experimental model was used to test the role of testosterone in regulating body weight and food intake in short day-housed male deer mice. Among gonad-intact males, short daylength caused a decline in body weight in both reproductive responsive and nonresponsive individuals. However, reproductive responsive mice lost significantly more body weight than did nonresponsive mice. Furthermore, while the weight loss was accompanied by a significant reduction in food intake in responsive mice, the relatively minor weight loss in nonresponsive mice was not accompanied by a change in food intake. Because changes in body weight and food intake (data not shown) occurred nearly simultaneously during the 8-week exposure to short daylength, results suggest that modifications in body weight are not responsible for the decline in food consumed, and vice versa. Gonadectomized reproductive responsive mice lost the same amount of weight as intact responsive mice but ate significantly more food. Among nonresponsive males, gonadectomy led to significantly greater weight loss, relative to intact mice, but caused an increase in food intake per gram body weight. Steroid replacement prevented weight loss and increased food intake in both gonadal phenotypes. Despite the observations that food intake was steroid dependent and the magnitude of the effect differed between reproductive phenotypes, changes in food consumption do not fully explain the inhibitory effects of short days on body weight in either phenotype. Taken together with previous studies, these results suggest that reproductive quiescence confers significant metabolic benefits to individual deer mice by reducing the amount of daily energy requirements via a reduction in body weight.(ABSTRACT TRUNCATED AT 400 WORDS)