Richard W. Steger

NEUROENDOCRINE AND REPRODUCTIVE CONSEQUENCES OF OVEREXPRESSION OF GROWTH HORMONE IN TRANSGENIC MICE

Abstract Availability of recombinant growth hormone (GH) and development of long-acting formulations of this material will undoubtedly lead to widespread use of GH in animal industry and in medicine. GH can act, directly or indirectly, on multiple targets, but its influence on the reproductive system and on the hormonal control of reproduction is poorly understood. Overexpression of GH genes in transgenic animals provides a unique opportunity to study the effects of long-term GH excess. Transgenic mice overexpressing bovine, ovine, or rat GH (hormones with actions closely resembling, if not identical to, those of endogenous [mouse] GH), exhibit enhancement of growth, increased adult body size, and reduced life-span as well as a number of endocrine and reproductive abnormalities. Ectopic overexpression of bovine GH (bGH) driven by metallothionein or phosphoenolpyruvate carboxykinase promoters is associated with altered activity of hypothalamic neurons which produce somatostatin, loss of adenohypophyseal GH releasing hormone (GHRH) receptors, and suppression of endogenous (mouse) GH release. Elevation of plasma levels of GH (primarily bGH) and insulin-like growth factor (IGF-I) in these transgenic mice leads to increases in the number of hepatic GH and prolactin (PRL) receptors, in the serum levels of GH-binding protein (GHBP), in the percent of GHBP complexed with GH, and in the circulating insulin levels. In addition, plasma adrenocorticotropic hormone (ACTH) and corticosterone levels are elevated. Plasma levels of luteinizing hormone (LH), as well as its synthesis and release, are not consistently affected, but follicle-stimulating hormone (FSH) levels are suppressed, apparently due to pre-and post-transiational effects. Pituitary lactotrophs exhibit characteristics of chronic enhancement of secretory activity, and plasma PRL levels are elevated. Prolactin responses to mating or to pharmacological blockade of dopamine synthesis are abnormal. Reproductive life span and efficiency are reduced in both sexes, with the severity and frequency of reproductive deficits being related to plasma bGH levels. Most transgenic females expressing high levels of bGH are sterile due to luteal failure. Overexpression of human GH which, in the mouse, interacts with both GH and PRL receptors leads to additional endocrine and reproductive abnormalities including stimulation of LHβ mRNA levels and LH secretion, loss of responsiveness to testosterone feedback, overstimulation of mammary glands, enhanced mammary tumorigenesis, and hypertrophy of accessory reproductive glands in males. Results of these studies indicate that GH can exert a variety of direct and indirect actions at the hypothalamic, pituitary, gonadal, and reproductive tract levels, and that the consequences of prolonged exposure to supraphysiological levels of GH cannot always be predicted from the known or the presumed physiological actions of this hormone.

Presence of tyrosine-hydroxylase activity in interior pituitary adenomas and ectopic anterior pituitaries in male rats

There is evidence for local regulatory effects of dopamine (DA) and norepinephrine (NE) on the release of prolactin (PRL) and other hormones from the anterior pituitaries transplanted to an ectopic site. In order to further explore these suspected regulatory mechanisms and to determine if they may exist also in pituitary tumors, we have examined the activity of tyrosine-hydroxylase (TH) in ectopic pituitaries and in hyperplastic pituitaries of estrogen-treated rats. Adult male rats with a pituitary graft in each kidney and sham-operated controls were examined 5 weeks after surgery. Rats implanted for 3 months with Silastic capsules containing diethylstilbestrol (DES) or with empty capsules were used 10 weeks after the capsules were removed. TH activity in ectopic anterior pituitaries of grafted rats was significantly higher than that measured in the eutopic anterior pituitaries of control animals. Similarly, TH activity was significantly higher in DES-induced pituitary tumors than in control pituitaries. These data support the possible existence of local catecholaminergic mechanism(s) that could be modulating PRL secretion from pituitary tumors and from ectopic pituitaries.

Plasma microdialysis. A technique for continuous plasma sampling in freely moving rats.

Microdialysis provides a means of continuous plasma sampling without repeated blood drawing. We report here the use of a specially designed and constructed microdialysis probe to sample plasma glucose, protein, and luteinizing hormone from the right atrium of a freely moving rat. Our probe has a unique side-arm tubing, which can be used to draw blood for in vivo probe calibration and infuse heparin continuously to prevent blood clotting. Glucose recovery rate (18%) of the probe remained relatively stable in continuously heparinized rats over 24 hr, but it dropped rapidly to 1% in nonheparinized rats. The concentration of plasma glucose was significantly underestimated, when it was converted from the perfusate concentration based on the in vitro, but not in the in vivo, recovery rate of the probe. The recovery of plasma protein was only 0.07% initially and rapidly declined to about 0.03%. Luteinizing hormone was not detected in the perfusates from either normal or luteinizing hormone-releasing hormone-stimulated rats. These results indicate that continuous heparinization and in vivo probe calibration are essential for successful plasma microdialysis, and our current dialysis membrane can be used to sample non-protein-bound molecules in the plasma.

Neuroendocrine Function in Transgenic Male Mice with Human Growth Hormone Expression

The neuroendocrine effects of human growth hormone (hGH) secretion were studied in adult male mice into which an hGH gene fused with mouse metallothionein 1 (mMT-1) promoter had been introduced. Intact transgenic mice had significantly greater plasma luteinizing hormone (LH) levels than did normal littermate controls. Castration increased LH levels in normal mice but was without effect on plasma LH levels in the transgenic mice. In vitro LH secretion and pituitary LH content were higher in the intact transgenic mice than in intact controls, while there was no significant difference in pituitary LH levels and in vitro LH secretion between the 2 groups of castrate animals. Intact transgenic mice exhibited a greater median eminence (ME) norepinephrine (NE) turnover than control animals, but ME NE turnover did not increase after castration in the transgenic animals as was the case in control mice. Castrate mice expressing the hGH gene had plasma levels of prolactin (PRL) similar to those seen in castrate controls, which was unexpected based on a previous study showing greatly attenuated PRL levels in intact hGH mice when compared to intact controls from the same line. Dopamine (DA) turnover in the ME was not significantly affected by the presence of the hGH gene, suggesting that the difference in plasma PRL levels between normal and transgenic mice is mediated through changes in PRL-regulating factors other than DA. In conclusion, the expression of the mMT-1/hGH hybrid gene in male mice leads to major alterations in LH secretion and lesser changes in PRL secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged Neuroendocrine Effects of a Brief Exposure of Adult Male Rats to Diethylstilbestrol

Eleven weeks of exposure to diethylstilbestrol (DES) releasing capsules caused marked changes in neuroendocrine function in male rats sacrificed 22 months later. Plasma prolactin levels were increased and plasma LH levels were decreased in the DES-exposed as compared to the control animals. DES-treated animals had significantly decreased levels of dopamine (DA) in the median eminence and lower rates of DOPA accumulation after blockade of aromatic amino acid decarboxylase than did the control animals. Norepinephrine and serotonin content and 5-hydroxytryptophan content in the ME, the medial basal and the anterior hypothalamus were not significantly affected by DES treatment. Basal prolactin release in vitro was reduced in the DES animals while prolactin responsiveness to inhibitory effects of 10(-7) M DA was enhanced. From these data it appears that a relatively brief exposure to DES in early adulthood causes marked and persistent changes in tuberoinfundibular DA function that are responsible for continued hyperprolactinemia. Associated reductions in LH secretion also appear to be due to hypothalamic as opposed to pituitary factors.

Influence of Photoinhibition, Photostimulation and Prolactin on Pituitary and Hypothalamic Nuclear Androgen Receptors in the Male Hamster

Testosterone (T) feedback sensitivity is markedly altered in adult male golden hamsters following exposure to short photoperiods (SD). Using a technique which measures total androgen receptors within the cell nucleus, the present study examined pituitary and hypothalamic nuclear androgen receptor levels in animals exposed to (1) long days (LD) or SD in the presence and absence of a constant T level supplied via a Silastic implant, (2) photostimulation following SD-induced testicular regression and (3) short-term prolactin injections following SD-induced testicular regression. Short photoperiods were associated with a drop in nuclear androgen receptor levels which was correlated with a decline in circulating T. When constant T was supplied to gonadectomized hamsters, those exposed to SD possessed higher pituitary and similar hypothalamic nuclear androgen receptor levels than those exposed to LD. As expected, plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were greatly reduced in SD-exposed, castrated, T-treated hamsters as compared to LD-exposed, castrated, T-treated animals. Photostimulation of intact SD-exposed hamsters for 5 or 10 days was associated with a decline in pituitary nuclear androgen receptors. Prolactin treatment caused no noticeable change in pituitary or hypothalamic androgen receptors even though plasma LH and FSH levels were significantly increased. These results support the hypothesis that altered T feedback mechanisms controlling LH and FSH release following chronic exposure to SD may be related to an alteration in the amount of androgen receptors present in the anterior pituitary.

Growth Hormone (GH) Binding and Effects of GH Analogs in Transgenic Mice

Abstract Overexpression of human (h) or bovine (b) growth hormone (GH) in transgenic mice is associated with marked (2- to 12-fold) and significant increase in hepatic binding of GH and prolactin (PRL). This is due to an increase in the number of GH and PRL receptors (GHR, PRLR) per mg of microsomal protein without changes in binding affinity. Comparison of results obtained in transgenic animals expressing bGH with a mouse metallothionein (MT) or a rat phosphoenolpyruvate carboxykinase (PEPCK) promoter suggests that effects of bGH on hepatic GHR and PRLR do not require GH overexpression during fetal life and, within the dose range tested, the effects on PRLR are not dose dependent. The increase in hepatic GHR was accompanied by significant increases in plasma GH-binding protein (GHBP) and in mean residence time of injected GH. Thus life-long elevation of peripheral GH levels alters the availability of both free GH and GHR. Site-directed in vitro mutagenesis was used to produce hGH and bGH analogs mutated within one of the sites involved in binding to GHR and PRLR. Mutating hGH to produce amino acid identity with bGH at Position 11, 18 (within Helix 1), 57, or 60 (within the loop between Helix 1 and 2) did not affect binding to GHR in vitro, or somatotropic activity in transgenic mice in vivo but reduced lactogenic activity in Nb2 cells by 22%-45%. Mutations of bGH designed to produce amino acid identity with hGH at one to four of the corresponding positions in the bGH molecule did not interfere with binding to GHR or somatotropic activity in vivo, and failed to produce significant binding to PRLR but resulted in alterations in the effects on the hypothalamic and anterior pituitary function in transgenic mice. Apparently region(s) outside the domains examined are essential for lactogenic activity of hGH, and different portions of the GH molecule are responsible for its diverse actions in vivo.

Possible Involvement of Hypothalamic Monoamines in Mediating the Action of Alpha-2u-Globulin on the Pituitary-Testicular Axis in Rats

A major androgen-dependent urinary protein of male rodents, alpha 2u-globulin, has been shown to influence adenohypophyseal hormone release. In an attempt to elucidate the mechanisms of its action, we have examined several parameters of hypothalamic and pituitary function in adult male rats treated for 2 weeks with two injections daily of 0.75 mg alpha 2u-globulin and sacrificed 16 h after the last injection. This treatment led to an increase in plasma luteinizing hormone levels, a decrease in plasma prolactin levels, an increase in testosterone concentrations in both plasma and testicular tissue, and increases in testicular weights. The norepinephrine turnover in median eminence and anterior hypothalamus was increased in alpha 2u-globulin-injected animals, while the norepinephrine turnover in the remaining medial basal hypothalamus was reduced. alpha 2u-Globulin-treated animals had a significantly decreased dopamine turnover in the anterior hypothalamus, while in the medial basal hypothalamus the dopamine metabolism was increased. These data suggest that alpha 2u-globulin-induced changes in gonadotropin and prolactin secretion are mediated by changes in catecholamine metabolism in several hypothalamic regions. Increased testosterone secretion appears to be due to increased secretion of gonadotropins from the pituitary.

Role of Prolactin in the Regulation of Sensitivity of the Hypothalamic-Pituitary System to Steroid Feedback

During sexual maturation, pituitary gonadotropins stimulate the gonads to produce increasing amounts of biologically active steroids and yet gonadotropin release does not become suppressed until concentrations of sex hormones, LH and FSH, in peripheral circulation stabilizes at a higher adult level. There is a substantial amount of evidence that in many mammals, this transition from prepubertal to adult level of activity of the pituitary-gonadal axis is associated with a reduction in the sensitivity of the hypothalamic-adenohypophyseal system to negative feedback of gonadal steroids. In the female, these changes are accompanied by the appearance of positive estrogen feedback on gonadotropin release. In seasonal breeders, annual transitions between the periods of gonadal activity and quiescence are associated with corresponding shifts in the sensitivity to steroid feedback. Peripheral levels of pituitary prolactin (PRL) typically increase during sexual maturation and exhibit large seasonal fluctuations in response to changes in photoperiod and ambient temperature. We propose that PRL is one of the factors which regulate the sensitivity of gonadotropin release to gonadal steroid feedback. In hyperprolactinemic women, responsiveness to negative estrogen feedback increases, while LH response to positive estrogen feedback is reduced or absent. In hyperprolactinemic men, both LH and testosterone levels are reduced, implying increased sensitivity of LH release to negative testosterone feedback. In the male rat, both physiological amounts of PRL and experimentally-induced hyperprolactinemia increase the ability of exogenous testosterone to suppress LH and FSH release. Different regulatory mechanisms appear to operate in the seasonally breeding male golden hamster, in which short photoperiod causes concomitant suppression of PRL, LH, FSH and testosterone release. In this species, pharmacologic suppression of PRL release leads to increased responsiveness of plasma gonadotropin levels to negative feedback effects of testosterone, while PRL-secreting ectopic pituitary transplants exert an opposite effect. We have examined some of the suspected mechanisms of PRL modulation of testosterone feedback in male golden hamsters. In immature animals, the amount of cytoplasmic androgen receptors in the anterior pituitary was decreased by mild hyperprolactinemia and increased by treatment with bromocriptine, an inhibitor of PRL release. Bromocriptine increased pituitary androgen binding also in adult hamsters. These findings would imply that PRL modulates the responsiveness to negative steroid feedback at the pituitary level.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of Estrogen and Ectopic Pituitary Transplants on the Responsiveness of Pituitary Prolactin Release to Luteinizing Hormone-Releasing Hormone and Dopamine

The responsiveness of prolactin release to regulatory inputs depends on the functional state of the lactotrophs. In the present study, we have examined the effects of luteinizing hormone‐releasing hormone (LHRH; 10–7 or 10–6 M) on the release of prolactin in vitro from hyperplastic pituitaries of estrogen‐treated male Fischer rats, ectopic pituitary transplants and in situ pituitaries of grafted and control rats. The effects of dopamine (10–8 or 10–7 M) in this system were also examined. The extent of inhibition of prolactin release by dopamine was not related to the amounts of prolactin secreted under basal conditions or to plasma prolactin levels. LHRH significantly suppressed prolactin secretion in all groups but its effect was most pronounced in the ectopic pituitary transplants and in the hyperplastic pituitaries of animals after chronic exposure to estrogen followed by a period of recovery. Thus, the effects of LHRH on prolactin release appear to be related to the secretory activity and/or to the absolute or relative number of the lactotrophs.