Preoptic Area Grafts Research Articles

Restoration of Bone Mass in Hpg Mouse by Preoptic Area Grafting

Hereditary hypogonadism in the hpg mouse, caused by a deletion mutation in the gonadotropin-releasing hormone (GnRH) gene, is associated with sterility, absent ovarian development, and undetectable circulating sex steroids. Eight-month-old female hpg mice had a significantly reduced bone mineral density (BMD) at the lumbar spine, femur, and tibia. In addition, the mice showed significant reductions in liver and kidney weight, with virtually nonexistent ovaries. Successfully transplanted hpg mice with preoptic area grafts contained GnRH-positive neurons, consistent with our previous experience, and the host median eminence was innervated by GnRH immunoreactive fibers. A return of reproductive function was evident from increased ovarian weight and vaginal cornification. Of note was that grafted hpg mice showed a complete reversal to baseline of their BMD measured at all three sites. This establishes that the low bone mass that occurs in old hpg mice can be fully and rapidly ameliorated by preoptic area grafting.

Increased galanin synapses onto activated gonadotropin-releasing hormone neuronal cell bodies in normal female mice and in functional preoptic area grafts in hypogonadal mice.

Galanin synaptic input onto gonadotropin-releasing hormone (GnRH) neuronal cell bodies was analysed in female mice using the presynaptic vesicle-specific protein, synaptophysin (Syn) as a marker. In the first experiment, forebrain sections from normal ovariectomized ovarian steroid-primed mice exhibiting a surge of luteinizing hormone were processed for immunohistochemical labelling for GnRH, synaptophysin, galanin and Fos. Two representative sections from each brain, one passing through the anterior septum (anterior section) and the other through the organum vasculosum lamina terminalis-preoptic area (posterior section), were analysed under the confocal microscope. None of the GnRH cells analysed in the anterior sections were Fos immunoreactive (IR) or received input from galanin-IR fibres. In contrast, the majority of GnRH cells in the posterior sections analysed were Fos-positive. The number of galanin synapses onto the Fos-positive GnRH cells was significantly higher than that in the Fos-negative cells in this area of the brain, even though the number of Syn-IR appositions was comparable to each other. Transplantation of preoptic area (POA) into the third cerebral ventricle of hypogonadal (HPG) mice corrects deficits in the reproductive system. In the second experiment, synaptic input to GnRH cells was compared between HPG/POA mice with (functional graft) or without (nonfunctional graft) gonadal development. The mean numbers of Syn-IR appositions and galanin synapses per GnRH cell and the proportion of GnRH cells with galanin input were significantly higher in the functional than in the nonfunctional grafts. The results suggest that galanin can act directly on the GnRH cell bodies and may have an important regulatory role on the GnRH system.

Increased synaptic input to gonadotropin releasing hormone cells in preoptic area grafts that support reproductive development in female hypogonadal mice.

Ultrastructural studies have established that gonadotropin releasing hormone (GnRH) neuronal cell bodies receive sparse synaptic input compared to other neuronal cell types. In the present studies, immunocytochemistry for the presynaptic marker synaptophysin, coupled with confocal microscopy, was employed to evaluate whether there was a difference in synaptic input to GnRH cells within preoptic area grafts (hypogonadal, HPG; preoptic area, POA) in hypogonadal female mice that did or did not show ovarian development. GnRH cells in HPG/POA mice with ovarian development exhibited significantly higher numbers of synaptophysin immunoreactive (syn-IR) appositions as compared with HPG/POA mice without ovarian development. This suggests that synaptic input to the grafted GnRH cells is important for the correction of reproductive functions in HPG/POA mice. Following mating, Fos immunoreactivity was present in several GnRH cells in HPG mice with successful POA grafts, indicating the establishment of neuronal projections conveying somatosensory information to the GnRH cells in these mice. The presence of a higher number of syn-IR appositions to GnRH cells in the successful grafts supports this hypothesis.

Preoptic Area Grafts Implanted in Mammillary Bodies of Hypogonadal Mice: Patterns of GNRH Neuronal Projections

Gonadotropin-releasing hormone (GnRH) axons project to the median eminence, where the peptide is released to stimulate pituitary gonadotrophs. Hypogonadal mice (hpg) do not synthesize GnRH due to a deletion in the gene. When neonatal preoptic area (POA) tissue from normal mice containing GnRH neurons is transplanted into the third ventricle of hpg mice, GnRH axons exit the graft and specifically project to the median eminence, where the release of GnRH in the portal circulation induces the stimulation of the pituitary-gonadal axis. To test the hypothesis that the median eminence region is critical to targeting, we placed POA grafts in the region of the mammillary bodies, which never contains GnRH cell bodies, but is nevertheless close to the median eminence. Control mice received bilateral grafts into the anterior hypothalamus. GnRH axons innervated the median eminence in animals with grafts in the mammillary bodies and posterior hypothalamus. Mice with such grafts for 4–5 months had gonadal development, while those with grafts for shorter periods did not. Anterior hypothalamic grafts merged into the third ventricle and, consistent with previous studies, this resulted in GnRH innervation of the median eminence and gonadal development. However, when grafts were located within dorsal regions such as the thalamus, no median eminence innervation was seen. In these cases, GnRH axons borrowed other bundles of fibers to travel within the host brain. The pattern of innervation from grafts within ventro-caudal regions of the hypothalamus vs. that from dorsal regions supported the hypothesis that the median eminence releases diffusible substances directing GnRH outgrowth.

Comparison of afferent and efferent competence of transplanted GnRH cells in the brains of hypogonadal female mice.

A deletion in the gene encoding gonadotropin-releasing hormone (GnRH) induces hypogonadism in mice caused by the deficiency of GnRH. Activation of the reproductive axis can be achieved in these hypogonadal (hpg) mice by third cerebro-ventricular transplantation of preoptic area (POA) containing GnRH neurons, obtained from normal fetal mice. The present study was carried out in female hpg mice with POA grafts (hpg/POA) to investigate anatomical integration of the GnRH cells required for the functional activation of the reproductive system. Ovarian development was present only in mice in which the graft tissue was located close to the median eminence (ME). The total lack of ovarian development in individuals with grafts containing GnRH cells located elsewhere in the brain suggests that the mere presence of GnRH cells does not guarantee ovarian development, but that the location of the graft may be important. Activation of the grafted GnRH cells following mating, as evidenced by the induction of Fos immunoreactivity, was observed in hpg/POA mice in which there was no ovarian development or detectable GnRH immunoreactive fiber innervation of the ME. Although ovarian development was evident in individuals with grafts located close to the ME, release of luteinizing hormone (LH) in response to mating was apparent in only some of these mice. The occurrence of mating and pregnancy only in hpg/POA mice with ovarian development and the reflex release of LH in response to mating suggests that both the efferent and afferent connections of the GnRH system are important for the full functioning of the system.

What Nature's Knockout Teaches Us about GnRH Activity: Hypogonadal Mice and Neuronal Grafts

The hypogonadal mouse is one of “nature's knockouts,” bearing a specific deletion in the gene for gonadotropin-releasing hormone (GnRH), with the result that no GnRH peptide is detectable in the brain. The lack of reproductive development after birth provides an animal model that has proved fruitful in clarifying the role of GnRH in reproductive behavior and physiology. Behavioral studies with hypogonadal mice convincingly demonstrate that although GnRH may facilitate the appearance of sexual behavior, this peptide is not essential for either male or female sexual behavior in the mouse. Administration of GnRH to hypogonadal mice with regimens mimicking GnRH pulsatility initiates reproductive development. Surprisingly, continuous exposure to GnRH stimulates remarkable ovarian and uterine growth and increased FSH release, although pituitary content of LH and FSH remains unchanged. In contrast, when brain grafts of normal fetal preoptic area (POA), containing GnRH cells, are implanted in the third ventricle of adult hypogonadal mice, both pituitary and plasma gonadotropin levels increase. Grafted GnRH neurons innervate the median eminence of the host and support pulsatile LH secretion in the majority of animals with graft-associated gonadal development. Studies of hypogonadal mice with POA grafts demonstrate that distinct components of reproductive function are dissociable: hosts may demonstrate reflex but not spontaneous ovulation; others may show positive but not negative feedback. Activation of grafted GnRH cells in response to sensory input to the host, as revealed in Fos expression studies, is an example of the integration of the graft with the host brain that underlies such capabilities. A goal of these studies is to elucidate the specific connectivity underlying discrete aspects of reproductive function.

FOS expression in grafted gonadotropin-releasing hormone neurons in hypogonadal mouse: Mating and steroid induction

We used FOS expression, widely accepted as a marker for neuronal activation, to evaluate physiologically induced activation of gonadotropin-releasing hormone (GnRH) neurons within intraventricular preoptic area grafts in hypogonadal (hpg) female mice. Hpg mice lack endogenous GnRH due to a mutated gene, but can respond to grafted GnRH neurons with reproductive development. The purpose of this study was to determine the degree to which the host brain regulates grafted GnRH neurons. FOS expression in grafted GnRH neurons was induced in progesterone-primed female mice paired with sexually active males. The degree of sexual activity did not affect the outcome, with 40.9 +/- 12.2% of the grafted GnRH cells expressing FOS when male partners performed intromissions, and 47.5 +/- 10.2% when they also ejaculated. There was little or no FOS expression in the grafts of unprimed hpg mice paired with sexually active males, in unpaired mice primed with progesterone or sequential estradiol benzoate and progesterone, or in controls. The pattern of FOS expression in the brains of the female hpg mice engaged in mating behavior was similar to that reported in other species, with moderate to high expression in the medial preoptic area, ventromedial nucleus, and medial amygdala in females paired with males that ejaculated. The present results support the hypothesis that host-derived activation of grafted GnRH neurons underlies aspects of reproductive responses seen in hpg mice with grafts, and further, that at least a portion of the host-graft connectivity is steroid sensitive.

Neuromodulation of transplanted gonadotropin-releasing hormone neurons in male and female hypogonadal mice with preoptic area brain grafts.

Implantation of normal preoptic area (POA) tissue into the third ventricle of adult hypogonadal (HPG) mice provides a source of GnRH neurons that innervate the host median eminence and stimulate reproductive development in the sterile mutants. To further evaluate graft-host integration, the effects of N-methyl-D,L-aspartic acid (NMA) and opiate antagonists on LH secretion in HPG mice with POA transplants (HPG/POA) were tested. NMA challenges significantly stimulated LH secretion in 10 of 11 HPG/POA females. Only 5 of 12 HPG/POA males responded to the same treatment. Administration of the opiate antagonists naloxone or naloxone methiodide was ineffective in stimulating LH release in any mice, but opiate antagonist pretreatment significantly potentiated the LH secretory response to NMA in female, but not male, HPG/POA mice. A potential anatomical substrate for this facilitation may be the beta-endorphin-immunoreactive innervation of the POA grafts in all HPG/POA brains examined. beta-Endorphin fibers were also present in the median eminence in the vicinity of GnRH outgrowth from the grafts. However, similar innervation patterns in HPG/POA males that did not respond to opioid antagonism suggests that this is not sufficient. We tested whether the sex difference in HPG/POA responsivity to neuromodulation is related to the steroid milieu in the hosts. 17 beta-Estradiol (E2) treatment facilitated the LH secretory response of male HPG/POA to NMA challenges whether animals were castrated and given an E2 capsule prior to graft implantation or one week before testing two months after graft surgery. Intact or vehicle (sesame oil)-treated, castrated HPG/POA males rarely responded to NMA challenges, yet graft-derived GnRH innervation of the hosts' median eminence was comparable in all treatment groups. GnRH challenge testing indicated that pituitary sensitivity of the HPG/POA males was not significantly altered by E2 treatment, suggesting that estrogen acted centrally. These results indicate that the activity of grafted GnRH neurons may be modulated by endogenous opioids of host origin as well as by the hormonal milieu.

Neuroendocrine brain grafts

Neuroendocrine grafts have been used to ameliorate symptoms associated with genetic or imposed deficiencies. In the course of these studies, it has become evident that the technique may be used to study some basic questions in neurobiology, including targeting, plasticity and integration of neurons. Among the genetic models employed, the hypogonadal mouse, deficient in gonadotropin-releasing hormone (GnRH), has provided an excellent opportunity to address certain of these issues. GnRH cells present in preoptic area grafts derived from normal mice show directed axonal outgrowth to the median eminence of the host, and GnRH neurons may migrate from the graft into the host hypothalamus. The adult host brain sends identified neuronal processes, into the graft and modulates physiological functioning of the grafted cells.

HUMAN MIDSIZED NEUROFILAMENT EXPRESSION IN TRANSGENIC MOUSE-DERIVED GRAFTS FACILITATES STUDY OF GRAFT-HOST INTERACTIONS IN HYPOGONADAL MICE.

Our previous studies established that targeted axonal outgrowth into the host median eminence (ME) from grafted gonadotropin-releasing hormone (GnRH) neurons is essential for stimulation of reproductive function in hypogonadal (HPG) mice homozygous for a deletion in the GnRH gene. In the current experiments transgenic mice expressing the human midsized neurofilament NF(M) were used as sources of grafts to clarify the extent of transplant-derived innervation of the host that accompanies this dramatic recovery process. Preoptic area (POA) tissue from 1- or 2-dayold transgenic pups was implanted in the third ventricle of adult male HPG mice. Polymerase chain reaction (PCR) analysis verified that each donor bore the transgene. Immunohistochemistry using a monoclonal antibody specific for human NF(M) revealed intensely immunoreactive parvicellular soma and proximal dendrites in the grafts. Interestingly, human NF(M)-positive neurons also migrated out of the graft into the host hypothalamus. In the animal with the most dramatic increase in testicular and seminal vesicle weight, human NF(M)-positive cells were observed within the host ME. In some cases, human NF(M)-positive axonal bundles were present within the wall of the third ventricle of the host as well as in the host ME. These were GnRH negative. More extensive anatomical studies will delineate the degree and patterning of axonal outgrowth. In these early studies, however, it is apparent that neuronal fiber outgrowth into the host brain is not exclusive to GnRH fibers from the preoptic area grafts. This is important information in regard to our studies of the specificity of the signals derived from the ME that may be involved in GnRH fiber targeting.

Targeting of gonadotropin-releasing hormone axons from preoptic area grafts to the median eminence.

Implantation of normal GnRH neurons can reverse many of the reproductive deficiencies that characterize hypogonadal (hpg) mice. Since the GnRH axons follow a stereotyped trajectory to their target we investigated the possibility that host brain regions adjacent to the graft might provide signals that induced this directional growth. The role of the adenohypophysis in GnRH axonal outgrowth was studied in mice with co-grafts of fetal preoptic area (POA) and pituitary and in hypophysectomized hosts. When fetal pituitaries were grafted together with the POA, immunoreactive GnRH fibers did enter the glandular tissue but they also grew into the host median eminence. Surgical removal of the pituitary of hpg hosts prior to POA graft placement was also compatible with GnRH innervation of the host median eminence although in some individuals that innervation pattern was confined to the more caudal aspects. The results of these two experiments suggest that the anterior pituitary gland may be an attractive target for GnRH axons but that this tissue is not essential for directed GnRH axonal outgrowth to its target. To determine if the median eminence itself could direct the growth of GnRH axons, co-grafts of POA and a fetal medial basal hypothalamic (MBH) block, which was predominantly median eminence, were made. Immunocytochemistry showed that an intragraft mini-median eminence was formed with a highly organized and robust GnRH innervation. Ultrastructural analysis indicated that these axons terminated near fenestrated capillaries. However, even under these conditions some GnRH axons exited into the host median eminence. It now seems likely that a cellular component of the median eminence can provide a signal to attract GnRH axons. Whether this signal is produced by the specialized ependymal cells, by the endothelia, or by meningeal (pial) components must now be tested.

Application of a fluorescent dye to study connectivity between third ventricular preoptic area grafts and host hypothalamus.

The mutant hypogonadal (hpg) mouse lacks a functioning gene for the neurohormone gonadotropin releasing hormone (GnRH). Previous studies from our laboratory had indicated that the initiation and maintenance of reproductive function in these mice could be brought about by the implantation of normal fetal grafts into adult hosts. Testicular or ovarian growth and other indicators of normal neurosecretory output were always accompanied by survival of GnRH neurons and growth of GnRH axons into the host median eminence where such axons terminate on the hypophysial portal capillaries. To determine if other connections exist between graft and the host hypothalamus, small crystals of the carbocyanine dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) were applied to either graft or host after fixation of the brain. Tissue sections were analyzed for retrograde and and anterograde movement of the dye. When crystals were placed on the graft, labeled axons were found in the host median eminence or in the host hypothalamus taking an arching trajectory toward the median eminence. Retrogradely labeled neurons in the host were few in number and largely confined to the host arcuate nucleus. With DiI crystals applied to the basal hypothalamus, labeled axons were distributed widely in the host but much sparser in the graft. Axons appeared to enter primarily at sites where the graft and host interface lacked an ependymal lining. Small numbers of retrogradely labeled neurons were also seen in the graft. Most were cells of very simple morphology and were distributed randomly in the graft. When double label experiments were carried out most DiI positive cells in the graft contained GnRH. These results indicate the connectivity between host hypothalamus and the third ventricular preoptic area grafts exists but is limited in nature.

Effects ofN-Methyl-D,L-Aspartic Acid on Luteinizing Hormone Secretion in Normal Mice and in Hypogonadal Mice with Fetal Preoptic Area Implants*

Many aspects of reproductive function are corrected in hypogonadal mice with preoptic area grafts (HPG/POA). Gonadotropin release and gonadal development are dependent on the presence of GnRH cells within the grafts and GnRH innervation of the median eminence. This study examined the effect of a known modulator of GnRH secretion, N-methyl-D,L-aspartic acid (NMA), in adult normal and HPG/POA male and female mice. All HPG/POA males had significant testicular development after graft surgery, and most HPG/POA females were in constant vaginal estrus and showed ovarian and uterine development; a few also demonstrated ovulatory cyclicity after pregnancies initiated by reflex ovulation. Groups of normal and HPG/POA males that were intact (INT) or castrated (CX) 7 days before testing were challenged with saline, NMA (20 mg/kg), and GnRH (100 ng/0.1 ml). Sequential blood samples from awake animals were obtained via intracardiac catheters for evaluation of plasma LH. There were significant increases in plasma LH after NMA challenge in normal INT [n = 15; 0 min, 0.26 +/- 0.02 (mean +/- SE); 10 min, 1.20 +/- 0.10 ng/ml; P less than 0.05] and normal CX (n = 13; 0 min, 0.36 +/- 0.06, 10 min, 3.25 +/- 0.27). Plasma LH secretion in response to NMA was significantly correlated (r = 0.786; P less than 0.001) with plasma LH release after the GnRH challenge in normal males. In contrast, only 3 of 17 HPG/POA (1 INT and 2 CX) showed increased circulating LH after NMA challenge, despite heightened pituitary sensitivity to GnRH. Normal and HPG/POA female mice were ovariectomized (OX) or OX and estrogen primed (OXE2) 7 days before testing. Intact cycling normal and cycling HPG/POA mice were tested in estrus (EST). There was a greater response to NMA in normal OX (n = 8; 0 min, 0.39 +/- 0.02; 10 min, 1.44 +/- 0.28) than in OXE2 (n = 13; 0 min, 0.29 +/- 0.01; 10 min, 0.52 +/- 0.07) despite similar gonadotroph sensitivity to GnRH. There was also a significant increase in plasma LH in response to NMA in HPG/POA-OX (n = 7; 0 min, 0.50 +/- 0.10; 10 min, 1.62 +/- 0.22) and HPG/POA-OXE2 (n = 12; 0 min, 0.39 +/- 0.04; 10 min, 1.31 +/- 0.26). Plasma LH levels after NMA treatment were significantly correlated with responses to GnRH in female HPG/POA (r = 0.58; P less than 0.03), but not in normal females. Neither normal-EST nor HPG/POA-EST had increased LH release after NMA challenge, perhaps due to the low gonadotroph sensitivity in this state.(ABSTRACT TRUNCATED AT 400 WORDS)

Pulsatile Luteinizing Hormone Secretion in Normal Female Mice and in Hypogonadal Female Mice with Preoptic Area Implants*

Pulsatile LH secretion is driven by GnRH, the hypothalamic hormone that is lacking in the hypogonadal mutant mouse. Preoptic area grafts containing GnRH neurons correct many reproductive deficits in hypogonadal mice. In this study we evaluated the pattern of LH secretion in hypogonadal female mice with preoptic area grafts (hpg/POA) and in normal female mice. Normal females were ovariectomized at 10 weeks of age, and hpg/POA mice were ovariectomized 4 months after graft surgery. Three weeks later, all mice received intracardial catheters. The next day, sequential blood samples were obtained every 10 min for 4 h from the awake, freely moving mice. At ovariectomy, normal and hpg/POA ovarian weights were 8.6 +/- 0.9 and 7.1 +/- 1.2 mg, respectively. Significant LH pulses were detected in 9 of 10 normal mice and in 9 of 13 hpg/POA mice. Pulse frequency (normal, 0.86 +/- 0.13; hpg/POA, 0.61 +/- 0.13 pulse/h) and interpeak interval (normal, 81.7 +/- 20.3; hpg/POA, 93.2 +/- 24.0 min) were not significantly different (P greater than 0.2), but mean plasma LH levels (normal, 1.07 +/- 0.16 ng/ml; hpg/POA, 0.49 +/- 0.08 ng/ml; P less than 0.005) and mean LH pulse amplitude (normal, 1.92 +/- 0.53; hpg/POA, 0.63 +/- 0.28; P less than 0.05) were significantly lower in the hpg/POA mice. The lower mean LH level and LH pulse amplitude in ovariectomized hpg/POA mice are consistent with the inability of most of these mice to show increased LH secretion after castration. The findings indicate that preoptic area brain grafts are capable of supporting episodic LH release in the hypogonadal mouse and suggest the presence of a functional GnRH pulse generator in the majority of mice with grafts.

An intracellular study of grafted and in situ preoptic area neurones in brain slices from normal and hypogonadal mice.

1. Intracellular recordings have been obtained from forty-one preoptic area (POA) neurones at times up to 14 months after they were grafted into the third ventricle of the mouse. Thirty-one neurones were in grafts from hypogonadal (hpg) mice in which a reversal of the hypogonadism was seen (responders), six were in grafts from hpg mice in which no such reversal occurred (non-responders) and four were in grafts from normal mice. 2. The grafted neurones had a mean resting potential (Em) of -57 mV, a mean apparent input resistance (Rm) of 136 M omega and a mean membrane time constant (tau m) of 7.7 ms. The slopes of the current-voltage (I-V) relations were linear. Approximately a quarter of neurones in responders fired action potentials spontaneously either singly or in bursts. Such activity could underlie the release of gonadotrophin hormone-releasing hormone (GnRH) which is known to occur from such grafts. 3. Two types of response were seen when these neurones were depolarized to firing threshold from Em, in one group a single action potential was discharged; in the other group one or more action potentials arising from a transient, slowly rising and falling depolarization (low-threshold response, LTR) was recorded. Some cells in the former category exhibited a LTR when depolarized from a potential more negative than Em. 4. The commonest response to stimulation of the median eminence in responders was an EPSP either alone or in combination with an IPSP. Antidromic action potentials were seen in four neurones and in two of these cells excitatory synaptic inputs could be demonstrated when the host hypothalamus adjacent to the graft was stimulated. It is suggested that these responses may represent activation of an afferent input from the host to neurones in the graft. 5. The morphology of neurones in POA grafts was determined by intrasomatic injection of horseradish peroxidase (HRP). A variety of profiles were seen and although some neurones extended over distances of up to 635 microns and branched extensively only one appeared to enter the host tissue at the ventrolateral edge of the graft. 6. A comparison was made between grafted POA neurones and cells in the medial preoptic area (MPOA), a region which constituted a significant component of the grafted tissue. No significant difference was noted between neurones in the graft and neurones in the MPOA in terms of their passive membrane properties. With regard to the active properties MPOA neurones could also be classified according to whether or not a LTR was elicited when the neurone was depolarized from Em. The major difference between the grafted neurones and those in the MPOA lay in the proportion of cells which exhibited a LTR under such conditions, being significantly greater in the latter group.

Identification of gonadotropin releasing hormone (GnRH) neurons projecting to the median eminence from third ventricular preoptic area grafts in hypogonadal mice

Functional gonadotropin releasing hormone (GnRH) neurosecretory activity can be restored in genetically hypogonadal ( hpg) adult mice with grafts of GnRH-containing fetal or neonatal septal-preoptic area (S/POA) tissue. Neurons implanted into the third ventricle of the host brain survive and send out axons which innervate one of the normal targets of these neurons, the median eminence (ME). Fibers terminate near primary portal vessels where GnRH is available for release into the vasculature, and this axonal outgrowth is essential for the stimulation of gonadotropin secretion, gonadal and accessory sex structure growth, gametogenesis, and fertility. Although it is known that GnRH axons reach their target, it is not known if all such neurons in a graft contribute to the projection. Taking advantage of the fact that axons in the ME, the sole host target, are outside the blood-brain barrier (BBB), long-term grafted animals were injected intraperitoneally with a retrograde tracer, Fluorogold (FG). Normal male mice were injected for comparison. Animals were sacrificed 5 days after injection and brain sections in the area of the graft were stained immunocytochemically for GnRH. In the normal male mice, two-thirds of the GnRH neurons were double-labeled with FG. In grafted individuals which slowed increased gonadal growth, the percentage of labeled cell ranged ffrom 17 to 75%. The results indicate that despite tissue injury, ectopic location, and a vastly reduced population, transplanted fetal GnRH neurons recapitulate a pattern seen in normal intact mice where some but not all neurons were capable of capturing a peripherally delivered tracer.

Effects of gonadectomy and treatment with gonadal steroids and luteinizing hormone secretion in hypogonadal male and female mice with preoptic area implants.

The ability of male and female hypogonadal (hpg) mice with preoptic area (POA) grafts to show negative feedback was studied. GnRH neurons within POA grafts send axons into the median eminence of the hpg host (HPG/POA), resulting in increased gonadotropin production and gonadal development in the mice that are genetically unable to produce GnRH. The present studies evaluated whether negative feedback, an aspect of normal reproductive function, is present in male and female HPG/POA mice. In normal male mice plasma LH was increased 1 or 5 months after castration and returned to baseline after testosterone propionate treatment. In contrast, no alterations in plasma LH were measured in similarly treated HPG/POA males. HPG/POA female mice were ovariectomized 6 weeks or 3 or 6 months after graft surgery and received sc 17 beta-estradiol (E2) implants 3 months later. Normal mice were studied when 6 weeks old and 8 months old (age-matched to HPG/POA mice ovariectomized 3 months after graft surgery). Further, to determine whether the mice were capable of positive feedback, 1 week after receiving E2 implants, mice in the 6-week and 3-month postgraft surgery groups were challenged with sequential administration of estradiol benzoate and progesterone. The significant increase in plasma LH after ovariectomy or decrease after E2 implant in normal female mice was not present in most HPG/POA female mice. Just 2 of the 24 HPG/POA females studied had increased plasma LH after gonadectomy, and in only 1 of these was plasma LH suppressed by E2 treatment. The ability of an individual HPG/POA mouse to show positive feedback did not predict the ability to show negative feedback, nor did the ability to show negative feedback predict positive feedback capability. Among the mice that failed to respond to ovariectomy with increased LH release were some that had elevated LH in response to steroid challenge or had spontaneously ovulated. On the other hand, neither mouse that had increased LH release after ovariectomy had shown positive feedback to a steroid challenge. Immunocytochemical evaluation revealed GnRH cells within the grafts and GnRH fiber innervation of the host's median eminence, but there was no correlation between numbers of GnRH cells or extent of innervation with the ability to show either negative or positive feedback, nor was the presence of vasoactive intestinal peptide cells within the grafts of predictive value. The failure of negative feedback in most of the HPG/POA mice tested may be due to the failure to establish as yet unidentified but essential afferents to the grafted GnRH cells and/or their axonal processes.

Neurotropic effects of estrogen on the neonatal preoptic area grafted into the adult rat brain.

The preoptic area (POA) or cerebral cortex taken from newborn female rats were transplanted into the third ventricle of ovariectomized adult rats. From the day of transplantation, estradiol-17 beta in a silastic capsule was implanted subcutaneously into host animals for 4 weeks. The POA or cerebral cortex transplants were examined at light- and electron-microscopic levels 4 weeks after transplantation. All of the POA or cortical grafts showed an appearance similar to normal neural tissue. Estrogen exposure for 4 weeks via the host induced a significant increase in the volume of the POA grafts. The neuronal population of the POA grafts exposed to estrogen was not significantly different from that of the POA grafts without estrogen treatment. However, the number of axodendritic shaft and spine synapses of the POA grafts exposed to estrogen was significantly greater than that of the POA grafts without estrogen treatment. In contrast, there was no significant difference in the volume of the cortical tissues transplanted into the brain between the control and estrogen-treated groups. These results suggest that estrogen has a stimulatory effect on the development of neuronal substrates in the intraventricular POA graft, increasing its volume and synaptic population.

Quantitative analysis of synaptic input to gonadotropin-releasing hormone neurons in normal mice and hpg mice with preoptic area grafts

Mutant hypogonadal ( hpg) mice with a truncated gene for the precursor to gonadotropin-releasing hormone (GnRH) show certain aspects of recovery of reproductive function after receiving grafts of normal preoptic area into the third ventricle. We have previously shown that GnRH neurons from within the grafts can innervate the appropriate neural-hemal target in the host. To determine if in turn these exogenously derived neurons receive a synaptic input comparable to the GnRH neurons in the normal aminal we ahve now carried out a quantitative ultrastructural analysis to compare the synaptic input to GnRH neurons in the normal preoptic area and in the grafts. In almost all cases GnRH cells or dendrites in normal brains and within the grafts received a synaptic input. In normal animals, input to GnRH dendritic profiles was significantly greater ( P < 0.001) than to the somatic plasma membrane and this trend was also observed within the grafts though the difference was not statistically significant. In addition, no statistically significant difference was found between the input to GnRH structures within the grafts and in normal preoptic area. However, a substantial variability in input among grafted animals was evident which was not observed in normal animals. The sources of variability within the grafts are discussed and we suggest that the deficiencies and differences that exist in regulation of gonadotropin secretion among grafted hpg animals may be reflected in aberrant synaptic input.

Chapter 15 Functional recovery from neuroendocrine deficits: studies with the hypogonadal mutant mouse

This chapter discusses the effect on the reproductive physiology of the hypogonadal male host of grafts, containing GnRH cells that were obtained from a region not known to project to the median eminence, and the effect of preoptic area grafts placed in the lateral ventricle far from the median eminence. The chapter also presents measurement of plasma luteinizing hormone (LH) in relation to mating in hypogonadal females with preoptic area grafts to determine the presence of an ovulatory LH surge in mutant animals with preoptic area grafts. The ability of preoptic area grafts to correct deficiencies in reproductive development is an important example of successful neural transplants, demonstrating that grafted neuropeptide-secreting cells may survive, develop axonal projections into the appropriate region of the host brain, and secrete their products in a physiological manner. Graft tissue derived from neonatal mouse brain appears to result in as frequent and vigorous correction of reproductive deficiencies as does tissue derived from fetal brain. Anatomical studies confirm that there are synapses onto gonadotropin hormone-releasing hormone (GnRH) perikarya and dendrites within the grafts, although whether the source is intrinsic or extrinsic to the grafts is not yet defined.