The neurobiology of reproductive development.

This brief review has highlighted some of the major advances in the last decade or so in understanding the central control of puberty. These include the discovery that GnRH-I neurons develop in the olfactory placode and migrate into the forebrain, the recognition that puberty is a reactivation of GnRH secretion, the identification of leptin as a metabolic signal which may permit puberty to occur, unraveling the molecular basis of the circadian clock which underlies photoperiodic control of puberty in seasonal species, the identification of the structure of pheromones in urine, and the discovery of other populations of GnRH neurons in mammals expressing the GnRH-II gene. Such advances generate further questions: what regulates the migratory pathways of GnRH neurons, and what controls axon outgrowth and targeting to the median eminence? What is the mechanism which causes GnRH secretion to decline between the neonatal and pubertal phase of development? How do leptin and other sensory inputs finally communicate to the GnRH neuron? How do GnRH neurons communicate with each other such that co-ordinated pulsatile release of GnRH occurs? What is the function of GnRH-II? Some of these issues may be better addressed using the transgenic technologies which allow the identification and thus the recording, sampling and observation of GnRH neurons in living tissue, but in order to understand how internal and external cues influence puberty it will also be important to study a variety of other mammalian models in which the relative importance of such inputs differs.

Estrogenic induction of spermatogenesis in the hypogonadal mouse.

Abnormal sperm production and reduced fertility have been reported in transgenic male mice lacking the alpha-subtype of the estrogen receptor (ER)alpha or aromatase. The aim of this study was to investigate the role of estrogen in male reproductive function, by determining the effect of estradiol on testicular function in hypogonadal (hpg) mice congenitally lacking gonadotropin; and thus, sex steroid production. hpg mice were treated, at 2-3 months of age, with slow-release estradiol implants, which achieved circulating estradiol concentrations of approximately 40 pg/ml. Treatment for 35 days reliably induced a 4- to 6-fold increase in testicular weight, compared with the vestigial testes in the untreated or cholesterol-treated controls. The degree of testicular growth after 35 days was similar to that in hpg mice receiving an intrahypothalamic graft of preoptic area tissue taken from neonatal mice on the day of birth, a procedure known to induce testicular development in hpg mice by activation of the pituitary gland. Histological analysis revealed that the testes contained elongated spermatids after 35 days of estradiol treatment, whereas germ cell development never progressed beyond the pachytene stage in control hpg mice. Treatment for 70 days induced full qualitatively normal spermatogenesis in hpg mice. Testis weight increased 5-fold, reflecting a 5-fold increase in total seminiferous tubule volume and a 4- to 5-fold increase in the total volume of the seminiferous epithelium. In all experiments, spermatogenesis proceeded in the absence of measurable androgen concentrations, but circulating FSH concentrations were slightly (but significantly) elevated, relative to cholesterol-treated control hpg mice. This stimulatory action of estradiol on FSH secretion was unexpected, particularly because identical estradiol treatments significantly decreased serum FSH levels in wild-type littermates. These results indicate that estrogens may play a role in spermatogenesis, via stimulatory effects on FSH secretion. An alternative or complementary explanation, given the recent identification of estrogen receptors (ERalpha and ERbeta) and aromatase within various cell types in the testis, is that estrogens exert paracrine actions within the testis to promote spermatogenesis. The identification of effects of estradiol on testicular function provides a conceptual basis to reexamine the speculative link between increased exposure to environmental estrogens and reduced fertility in man.

Seasonal regulation of food intake and body weight in the male Siberian hamster: studies of hypothalamic orexin (hypocretin), neuropeptide Y (NPY) and pro-opiomelanocortin (POMC).

The aim of these experiments was to investigate the relationship between hypothalamic expression of orexin (also called hypocretin), neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) mRNA and seasonal cycles of body weight and food intake in the Siberian hamster. Adult males were transferred from long days of 16 h light and 8 h dark to short days of 8 h light and 16 h dark, a procedure known to induce major reductions in food intake and body weight in this species. After 8 weeks of exposure to short days, while body weight was declining, hypothalamic NPY mRNA levels as assessed by in situ hybridization were slightly lower (P < 0.05) than in age-matched controls exposed to long days. After 12 weeks with short days, when body weight would be expected to have reached its seasonal nadir, POMC mRNA levels were lower (P < 0.05) than in hamsters under long days. At no stage did orexin mRNA levels in hamsters under short days differ significantly from levels in those under long days. To investigate further the role of these peptide systems in seasonal changes in body weight and food intake, two provocative tests were carried out. Firstly, a 48-h fast induced a significant increase (P < 0.025) in hypothalamic NPY mRNA levels in both long- and short-day conditions, but did not change hypothalamic POMC or orexin mRNA levels. Secondly, systemic (intraperitoneal) treatment with recombinant murine leptin (5 mg/kg body weight) significantly decreased (P < 0.01) food intake over a 6-h post-treatment period in both long- and short-day conditions. However, this acute leptin treatment did not induce significant changes in hypothalamic orexin, NPY or POMC mRNA abundance. The increase in NPY expression in both long- and short-day conditions following food restriction and the suppression of food intake by leptin in both conditions suggests that acute homeostatic mechanisms operate in both long-day (obese) and short-day (nonobese) conditions. The lack of major changes in orexin, NPY and POMC in such different metabolic states suggest that other central systems must play a greater role in generating these states. Such findings are consistent with the 'sliding set-point' hypothesis, that is, seasonal cycles in food intake and fat metabolism are brought about by as yet unknown central mechanisms that chronically alter the level ('set point') around which homeostasis occurs, rather than resulting from changes in the potency of the acute feedback mechanisms themselves.

The pharmacology of gene therapy.

The objective for human gene therapy is to express exogenous DNA at a site in vivo for long enough, and at sufficient levels to produce a therapeutic response. The obstacles to this objective are numerous and include the formulation or packaging of the DNA, in vivo delivery, penetration of biological barriers, DNA elimination within the cell and from the tissue compartments of the whole body, control of product expression and overt toxicity. The current challenge is to resolve each of these obstacles to produce a practical and efficient gene therapy. In doing so, it is vital to understand the disposition of DNA vectors in vivo, and to know how conventional medicines may be used to modulate this disposition and to enhance the therapeutic effect of these vectors. Many of the general concepts of human gene therapy have been reviewed extensively in the literature. This review discusses some of the pharmacological aspects of gene delivery and the fate of vectors in vivo, and then highlights how drugs are being used to modulate gene therapy.

Seasonal neuroendocrine rhythms in the male Siberian hamster persist after monosodium glutamate-induced lesions of the arcuate nucleus in the neonatal period.

The aim of these experiments was to examine the role of the arcuate nucleus in the control of seasonal cycles of body weight, feed intake, moulting and reproduction in the Siberian hamster. The arcuate nucleus has previously been implicated as a central site where systemic feedback signals (e.g. leptin) might act to regulate feed intake and body weight, so it was predicted that hamsters with lesions of this structure would be unable to display the inhibitory effects of short days on these parameters. In the first series of studies, lesions that destroyed approximately 80% of the cells in the arcuate nucleus were produced by treating hamsters neonatally with monosodium glutamate (MSG; 4 mg/g body weight sc), and vehicle- and MSG-treated males were raised from birth in long days (LD) or short days (SD). In hamsters raised in LD, the initial gain in body weight and testicular growth were significantly reduced by MSG treatment, however, growth rate and testis weight were still significantly greater than in vehicle- or MSG-treated hamsters raised in SD. In the second study, hamsters treated neonatally with vehicle or MSG were raised in LD for 8 weeks and, subsequently, approximately half in each group were transferred to SD for 18 weeks. As expected, vehicle-treated hamsters showed a characteristic decline in body weight when exposed to SD, while those remaining in LD continued to increase body weight. Feed intake decreased in parallel with the decline in body weight in SD, a complete moult to the white winter pelage occurred by 16 weeks in SD, and testicular regression occurred. Responses to SD also occurred in the MSG-treated hamsters: body weight decreased in SD but increased in their lesioned litter mates remaining in LD, and feed intake paralleled body weight changes in these groups. The moult to winter pelage was significantly retarded in MSG-treated hamsters transferred to SD. The testes were completely regressed in sham- and MSG-treated hamsters exposed to SD, whereas testes weights in MSG-treated hamsters maintained in LD were intermediate between those in vehicle-treated hamsters in SD and LD. Thus, despite initial effects on growth, the MSG-treated hamsters bearing substantial lesions of the arcuate nucleus were able to show appropriate responses to photoperiod, although not always of the same magnitude as the unlesioned controls. We conclude that feedback mechanisms operating via the arcuate nucleus are not the major regulators of seasonal cycles of body weight, feed intake, pelage and reproduction.

Manipulations of glutamatergic (N-methyl-D-aspartate receptor) neurotransmission alter the rate of photoperiodically regulated sexual maturation in the male Siberian hamster.

This study investigated whether central glutamatergic pathways are involved in sexual maturation in the Siberian hamster, a species in which puberty is regulated by photoperiod. The aim of the initial experiments was to determine whether exogenous activation of N-methyl-D-aspartate (NMDA) receptor-mediated glutamatergic pathways could induce premature testicular development in photoinhibited hamsters. Male hamsters raised in an inhibitory short-day photoperiod (8L:16D) received systemic injections of the glutamate agonist NMDA. Twice-daily treatment with 20 or 40 mg/kg BW caused significant increases in testicular weight within 4 wk, but this was not accompanied by a significant increase in circulating testosterone. Subsequent experiments revealed that the degree of testicular development induced by NMDA was comparable to that induced by daily treatment with GnRH (1 microg). This study demonstrates the potential for increased glutamatergic activity to induce testicular development. The aim of the second series of experiments was to determine whether blockade of endogenous NMDA receptor-mediated glutamatergic pathways could prevent testicular development in photostimulated hamsters. Males were transferred from short (SD) to long days (LD; 16L:8D) at 80-100 days of age to induce rapid testicular growth and were concurrently treated with MK801, a noncompetitive NMDA receptor antagonist (2 mg/kg BW per day), or CGP40116, a competitive NMDA receptor antagonist (5 or 10 mg/kg BW per day) for 4 wk. LD photoperiod caused a rapid increase in testicular weight in vehicle-treated hamsters within 4 wk. Both antagonists significantly reduced the LD-stimulated testicular growth. MK801 treatment induced a degree of sedation and a loss of body weight, suggesting that the reduced testicular development in this group may have been secondary to decreased growth rates; but no behavioral changes or loss of body weight was observed in the hamsters treated with CGP40116. These observations in a photoperiodic species provide support for the hypothesis that activation of NMDA receptor-mediated glutamatergic pathways contributes to sexual maturation.

Resistance of gonadotropin-releasing hormone neurons to glutamatergic neurotoxicity.

Although many studies provide evidence that glutamatergic pathways regulate the secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, it is controversial as to whether they act directly upon GnRH neurons. The aim of the current study was to determine whether GnRH neurons are susceptible to the neurotoxic actions of specific glutamate agonists (N-methyl-D-aspartate [NMDA] and kainic acid), the rationale being that neurotoxic loss of GnRH neurons would provide evidence that the perikarya possess specific classes of glutamate receptor. Unilateral 1-microl injections of NMDA (12-120 mM), kainic acid (0.5-2.5 mM), or vehicle were stereotaxically directed at the preoptic area (mPOA)/diagonal band of Broca (dbB) in the region of the organum vasculosum of the lamina terminalis (OVLT) of male adult hamsters (Phodopus sungorus). The number and appearance of GnRH neurons were determined by immunocytochemistry 3-8 days later. The morphology of GnRH neurons in the vicinity of the injection sites appeared normal after both kainic acid and NMDA treatment, and there was no significant decrease in the numbers of GnRH perikarya identified following these treatments. Both agonists caused massive cellular loss when injected directly into cortical areas and striatum. In the experimental studies, there was little neuronal loss within the mPOA or dbB after either toxin, despite clear neuronal loss in areas adjacent to the injection sites, including ventral striatum and olfactory cortex. In follow-up studies, immunocytochemical and in situ hybridisation analysis of the NMDAR1 and NMDAR2 glutamate receptor subunits confirmed their widespread distribution in regions containing GnRH perikarya, but no colocalization within GnRH neurons was observed. The susceptibility of neural areas to NMDA neurotoxicity did not correlate with any difference in the regional expression of these glutamate receptor subunits. The resistance of GnRH neurons to the neurotoxic actions of two different glutamate agonists and the failure to detect colocalisation of NMDAR1 or NMDAR2 subunits within GnRH perikarya are consistent with the notion that the effects of glutamate upon GnRH secretion are not exerted directly upon GnRH cell bodies.