Hypothalamic transplantation.

Tissue transplantation aided in formulating the neurohumoral hypothesis of anterior pituitary function. The concept of a hypophysiotropic region within the hypothalamus stemmed from experiments in which pituitary tissue was transplanted into the brain. Restoration of aberrant function of the central nervous system by transplants has been reported in two neuroendocrine models: the antidiuretic hormone-deficient Brattleboro rat and the gonadotropin-releasing hormone-deficient hypogonadal mouse. Neural transplants into the Brattleboro rat result in the survival of axons containing antidiuretic hormone but reversal of the physiological defect has not been confirmed. In the hypogonadal mouse grafts of preoptic area tissue into the third ventricle have restored pituitary hormone synthesis and secretion and gonadal activity, leading to nearly normal reproductive function. The gonadotropin-releasing hormone axons specifically innervate the median eminence of the hypothalamus, their normal target, which raises interesting questions of neurobiological graft/host interactions. The hpg model has been used to investigate factors affecting graft survival; by suitable immunosuppression it has been possible to reverse the hypogonadism with grafts of rat preoptic area tissue. Perhaps the most dramatic recent development has been the restoration of circadian rhythmicity to suprachiasmatic nucleus-lesioned hamsters by grafts of similar tissue. The rhythmicity restored is typical of the donor tissue.

Gonadal steroids influence neurophysin II distribution in the forebrain of normal and mutant mice.

The distribution of arginine vasopressin-associated neurophysin (neurophysin II) immunoreactivity was investigated in normal and mutant house mice during development and after various gonadal steroid manipulations. During postnatal development of normal mice dense networks of neurophysin II immunoreactivity in the lateral septal nucleus and lateral habenular nucleus appeared earlier in male than in female mice, with an adult pattern of immunoreactivity being attained by 8 weeks and 12 weeks of age, respectively. The neurophysin II immunoreactivity in the male was denser than that in female mice. After gonadectomy of adult normal mice there was a gradual loss of neurophysin II immunoreactivity in the lateral septum and lateral habenula over a period of 15 weeks. In hypogonadal mice, a mutant in which gonadal development is arrested postnatally due to a deficiency in hypothalamic gonadotrophin releasing hormone, no immunoreactive neurophysin II could be detected in the lateral septum or lateral habenula. A pattern of neurophysin II immunoreactivity similar to that in normal control mice was observed in hypogonadal mice which had been implanted for 4 weeks with silicone elastomer capsules containing testosterone or oestradiol-17 beta, but not 5 alpha-dihydrotestosterone or progesterone. Stimulation of gonadal development and endogenous steroid production in hypogonadal mice by third ventricular grafts of preoptic area tissue from normal neonatal animals also produced a normal pattern of neurophysin II immunoreactivity in the lateral septum and lateral habenula. In the androgen-insensitive testicular feminized mouse immunoreactive neurophysin II was undetectable in the lateral septum and lateral habenula. Treatment of testicular feminized mice with oestradiol-17 beta, but not progesterone, produced a normal pattern of neurophysin II immunoreactivity. The main immunohistological findings were confirmed by radioimmunoassay of tissue extracts which showed that the concentration of arginine vasopressin in lateral septum was far greater in normal males than females and was undetectable in hypogonadal mice; no oxytocin could be detected in the septum of normal or hypogonadal mice. These results show that the expression of neurophysin II immunoreactivity in the lateral septum and lateral habenula of the mouse brain is dependent on the presence of aromatizeable androgens or oestrogens.

Angioarchitecture of the CNS, pituitary gland, and intracerebral grafts revealed with peroxidase cytochemistry.

Blood vessels of the fetal, neonatal, and adult subprimate and primate CNS, including circumventricular organs (e.g., median eminence, pituitary gland, etc.), and of solid CNS and nonneural (anterior pituitary gland) allografts placed within brains of adult mammalian hosts were visualized with peroxidase cytochemistry applied in three ways: to tissues from animals injected systemically with native horseradish peroxidase (HRP) or peroxidase conjugated to the lectin wheat germ agglutinin (WGA) prior to perfusion fixation; to tissues from animals infused with native HRP into the aorta subsequent to perfusion fixation; and to tissues from animals fixed by immersion and incubated for endogenous peroxidase activity in red cells retained within blood vessels. In neonatal and adult animals receiving native HRP intravascularly, non-fenestrated vessels contributing to a blood-brain barrier were outlined with HRP reaction product when tetramethylbenzidine (TMB) as opposed to diaminobenzidine (DAB) was used as the chromogen; fenestrated vessels of circumventricular organs were not discernible due to the density of extravascular reaction product. Fenestrated and non-fenestrated cerebral and extracerebral blood vessels exposed to bloodborne WGA-HRP were visible when incubated in TMB and DAB solutions. Native HRP infused into the aorta of fixed animals likewise labeled non- fenestrated vessels throughout the brain upon exposure to TMB or DAB but obscured fenestrated vessels of the circumventricular organs. Endogenous peroxidase activity of red cells, seen equally well with TMB and DAB, outlined blood vessels throughout the cerebral gray and white matter and all circumventricular organs in fetal, neonatal, and adult animals. Application of the three peroxidase cytochemical approaches to study the development or absence of a blood-brain barrier in intracerebral allografts demonstrated that the vascularization of day 16-19 fetal/1 day neonatal CNS allografts is not well defined prior to 7 days following intracerebral placement of the grafts. CNS allografts secured from donor sites expected to possess a blood-brain barrier exhibited blood vessels that were not leaky to HRP injected intravenously in the host. Fenestrated blood vessels associated with anterior pituitary allografts were evident prior to 3 days posttransplantation within the host brain and permitted blood-borne HRP in the host to enter the graft and surrounding host brain parenchyma.

Postnatal ovarian follicle development in hypogonadal (hpg) and normal mice and associated changes in the hypothalamic-pituitary ovarian axis.

Significant uterine growth occurred in normal and hypogonadal (hpg) mice between Days 7 and 21 but thereafter no further growth was observed in hpg mice. The ovaries of hpg mice were significantly smaller than those of normals at all ages, but there was no significant difference between the number of non-growing follicles in the ovaries of mutants and their normal littermates at any age studied, and normal and hpg mice showed a marked reduction in the number of non-growing follicles during the first month of life. The size and composition of the growing follicle population in hpg mice, however, differed markedly from those in normal animals and by 21 days of age the number of growing follicles in mutants was significantly reduced. There was no significant difference in the number of Type 3b follicles before 60 days of age, but the number of all other follicle types was significantly less in hpg mice at all ages studied. Follicles in which the antrum is fully developed (Type 7 and 8) were never seen in the ovaries of mutants and corpora lutea were never observed. Interstitial tissue development was also very poor in hpg ovaries. The hypothalamic GnRH content in normal mice remained low until Day 20, before rising sharply to adult levels (approximately 800 pg) between Days 20 and 30. The pituitary FSH content increased over the first 10 days of life to reach a peak of about 5000 ng, before declining to the adult value of about 2000 ng by Day 30, whilst the plasma FSH concentration was high in the first 10 days, but fell to adult levels over the next 20 days. Pituitary LH content increased significantly between Days 5 and 10 to reach the adult level of about 600 ng. Hypothalamic GnRH was undetectable at all ages in hypogonadal mice, but the pituitary content of FSH and LH had risen to the attenuated mutant adult value by Day 15, and unlike normals, plasma FSH concentrations were not elevated during the neonatal period. These results suggest that minimal gonadotrophic stimulation of the ovary from birth has no effect on the total number of follicles but reduces the number of growing follicles and prevents follicle growth beyond the early antral stage. Gonadotrophins therefore appear to have a role in the initiation and continuance of follicle growth in the adult mouse.

Priming effect of luteinizing hormone releasing hormone in the hypogonadal mouse.

We have investigated the LH response to LH releasing hormone (LH-RH) in female hypogonadal (hpg) mice in which the hypothalamus contains no LH-RH and the pituitary gland contains significantly less LH than in normal mice. Both the releasing action and the priming effect of LH-RH were not significantly different in hpg compared with normal mice. Raised plasma concentrations of oestradiol-17 beta reduced pituitary responsiveness to LH-RH in normal but not in hpg mice. These results show that in the mouse neither long-term exposure to normal levels of LH-RH nor a normal pituitary content of LH are necessary for either the releasing or the priming action of LH-RH.

Brain grafts reverse hypogonadism of gonadotropin releasing hormone deficiency.

Hypogonadism in the mutant hpg mouse is characterized by a deficiency of hypothalamic gonadotropin releasing hormone (GnRH). Affected male mice exhibit immature reproductive organs, small abdominal testes and low pituitary and plasma gonadotropin concentrations. Recent studies have demonstrated the potential of fetal brain transplants to establish functional connections with host tissues. We therefore sought to use this approach to correct the hpg deficit. Fetal preoptic area (POA) (a site of GnRH production) from unaffected animals of the hpg strain was transplanted into the anterior third ventricle of adult hpg mice. We report that in such implanted animals, killed 2 months post-implantation, the POA grafts contained GnRH neurones, from which GnRH-positive fibres could be traced to capillaries of the median eminence. Hypothalamic GnRH and pituitary and plasma gonadotropin concentrations were increased compared with levels in untreated (hpg) animals. The testes were enlarged and had descended into the scrotum. Evidence of full spermatogenesis and interstitial cell development was present in testicular sections. No such effects were seen with transplants of cortical tissue.

Effect of copulation on the hypothalamic content of gonadotrophic hormone-releasing hormone in the vole, Microtus agrestis.

There was a drop of 56% in the hypothalamic content of Gn-RH in female voles 5 min after mating compared with that in unmated but receptive animals. This suggests that the surge of LH in vole plasma associated with reflex ovulation is evoked by a massive release of Gn-RH.

Gonadotrophin release in hypogonadal and normal mice after electrical stimulation of the median eminence or injection of luteinizing hormone releasing hormone.

Electrical stimulation of the median eminence, using parameters known to cause the release of LH in normal male mice, failed to elicit any gonadotrophin response in nypogonadal (hpg) male mice. Administration of 40 ng synthetic LH releasing hormone (LH-RH) resulted in release of LH from the pituitary gland of hpg mice, although the response was significantly lower than that of normal mice. These results were consistent with the hypothesis that the hypogonadal state of the hpg mouse results from a functional basence of LH-RH in the hypothalamus rather than from a lack of response of the pituitary gland to the releasing hormone.

The purification of luteinizing-hormone-releasing factor with some observations on its properties.

Bullock median-eminence tissue was used as a source of luteinizing-hormone-releasing factor for small-scale experiments to explore methods for its isolation. The presence of luteinizing-hormone-releasing factor was detected by the ovulation response in rabbits after intrapituitary infusion of the extract. Gel filtration was found to be suitable for the purification of these extracts. The releasing factor appeared to be a basic peptide of molecular weight in the range 1200-2500. On a larger scale, an extract of hypothalamic tissue from sheep was used to establish a multi-stage isolation procedure that resulted in a 200000-fold purification of luteinizing-hormone-releasing factor. After the initial extraction the isolation process consisted of: (1) two cycles of gel filtration; (2) anion-exchange chromatography; (3) gel filtration in a partially organic medium; (4) thin-layer chromatography on cellulose. Stage (3) separated two zones of activity each containing peptides. One of these was purified further by stage (4) to give a preparation that was active at a dose of 6mug. of peptide/animal, although activity diminished seriously during storage. This preparation contained only five or six components, but the small amounts of peptides obtained at this stage of purity were insufficient for full characterization.