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

Hormones and Behavior 31, 212–220 (1997) Article No. HB971387

What Nature’s Knockout Teaches Us about GnRH Activity: Hypogonadal Mice and Neuronal Grafts Marie J. Gibson,*,1 T. J. Wu,† Gregory M. Miller,†,2 and Ann-Judith Silverman‡ *Department of Medicine and †Fishberg Center for Neurobiology, Mount Sinai School of Medicine, New York, New York 10029; and ‡Department of Anatomy and Cell Biology, Columbia College of Physicians & Surgeons, New York, New York 10032

The hypogonadal mouse is one of ‘‘nature’s knockouts,’’ bearing a specific deletion in the gene for gonadotropinreleasing 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 1

To whom correspondence should be addressed at Box 1055, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York, NY 10029. 2 Present address: Neuroendocrine Unit, Massachusetts General Hospital, Boston, MA 02114.

underlies such capabilities. A goal of these studies is to elucidate the specific connectivity underlying discrete aspects of reproductive function. q 1997 Academic Press

Molecular biologists are increasingly using gene ‘‘knockout’’ techniques to produce animals with a specific deletion. These elegant studies have their precedent in nature, where a mutation may result in a limited genetic defect, as seen for example in the vasopressin deficiency of the Brattleboro rat (Schmale and Richter, 1984) and in the gonadotropin-releasing hormone (GnRH) deficit of the hypogonadal mouse (Cattanach, Iddon, Charlton, Chiappa, and Fink, 1977). The defect in hypogonadal mice was later characterized by Mason, Hayflick, Zoeller, Young, Phillips, Nikolics, and Seeburg (1986) as being due to a 33.5-kb deletion in the gene for GnRH. Two of the four exons coding for the GnRH prohormone are included in the deletion. Transcription of the remaining portion of the gene occurs, permitting localization of the cells using a site-specific probe (Mason et al., 1986; Livne, Gibson, and Silverman, 1993). However, there is no GnRH peptide detectable in the brain of the hypogonadal mouse, whether assessed by radioimmunoassay or immunocytochemistry. In the normal mouse brain GnRH-containing neurons number about 800, scattered throughout the rostral forebrain. A majority of these neurons project to the median eminence (Silverman, Jamandas, and Renaud, 1987), where the GnRH peptide is released into the pituitary-portal circulation to stimulate gonadotropin production. The lack of GnRH hormone in the hypogonadal mouse results in greatly reduced levels of pitu0018-506X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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itary and plasma gonadotropins (Cattanach et al., 1977) and a failure of reproductive development after birth. This animal model has been fruitful in clarifying the role of GnRH in behavior and in reproductive physiology. The specific ‘‘knockout’’ of GnRH peptide has permitted a wide range of replacement paradigms, whether by single or multiple injection (Charlton, Halpin, Iddon, Rosie, Levy, McDowell, Megson, Morris, Bramwell, Speight, Ward, Broadhead, Davey-Smith, and Fink, 1983; Saade, London, and Clayton, 1989), chronic administration via implanted pump (Gibson, Kasowski, and Dobrjansky, 1994), gene therapy (Mason et al., 1986), or neural implants of tissue obtained from normal brain containing GnRH neurons (Krieger, Perlow, Gibson, Davies, Zimmerman, Ferin, and Charlton, 1982; Gibson, Charlton, Perlow, Zimmerman, Silverman, and Krieger, 1984a) or of immortalized GnRH cells (Silverman, Roberts, Dong, Miller, and Gibson, 1992a; Miller, Silverman, Roberts, Dong, and Gibson, 1993). This review primarily focuses on insights gained in regard to reproductive behavior, as well as the evidence for specific anatomic integration of successfully grafted GnRH cells.

GnRH AND SEXUAL BEHAVIOR IN THE MOUSE The role of GnRH in sexual behavior has received considerable attention over the years. Initial reports that GnRH significantly increases lordosis behavior in ovariectomized, estrogen-primed rats (Moss and McCann, 1973; Pfaff, 1973) or mice (Luttge and Sheets, 1977) were followed by studies showing that neither the adrenal gland nor the pituitary mediate this effect. The infusion of GnRH antiserum or antagonists into the ventricle (Dudley, Vale, Rivier, and Moss, 1981) or the central gray matter (Sakuma and Pfaff, 1983) results in severely decreased lordotic responses in the rat. To evaluate whether GnRH is required for normal female sexual behavior in mice, Ward and Charlton (1981) studied normal and hypogonadal estrogen-primed ovariectomized females. Their finding that both normal and hypogonadal estrogen-primed animals respond to GnRH with a significant increase in lordotic behavior is consistent with a role for GnRH in mediating sexual behavior. However, estrogen-primed hypogonadal female mice treated with progesterone also show lordotic behavior comparable to that shown by similarly treated normal female mice, indicating that while GnRH may play a facilitatory role, it is not essential in the mouse.

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GnRH has also been implicated in male sexual behavior, for example by shortening ejaculation latencies in rats (Moss, 1979). Male hypogonadal mice, lacking GnRH, are reproductively undeveloped in adulthood and are sexually inactive, rarely showing any mounting behavior and never any intromissions or ejaculations. Perinatal androgen exposure is important for organization of the neural substrate for male sexual behavior in rodents (MacLusky and Naftolin, 1981), as well as a masculine pattern in androgen-sensitive neural and muscular structures such as the spinal nucleus of the bulbocavernosus (Wagner and Clemens, 1989) and associated perineal muscles involved in some copulatory penile reflexes (Sachs, 1982). In light of this, we hypothesized that the absence of GnRH during the neonatal period in the hypogonadal male results in insufficient exposure to androgens in the critical period of organization of the brain. We injected neonatal hypogonadal males with testosterone propionate (TP). In adulthood the mice received TP capsule implants (Livne, Silverman, and Gibson, 1992). Testing with normal ovariectomized estrogen and progesterone-primed female mice began 2 weeks later. After two to four weekly tests, the males were paired with intact normal cycling females. Three of the eight neonatally androgenized hypogonadal males showed complete male sexual behavior, including intromissions and ejaculation, and sired healthy litters. Interestingly, the testicular weights of these fertile animals were about 25% that of normal males, while pituitary gonadotropin levels were less than 10% of normal. In a control group of hypogonadal males that were only given TP implants in adulthood, three of seven also showed high levels of mounts and intromissions, but none ever ejaculated. From these studies, it appears that neonatal androgens in the male mouse may be acting primarily at the level of the spinal nuclei controlling ejaculation. That not all of the animals responded to exogenous TP in adulthood may be due to differential prenatal exposure to androgens. It is reported that males are exposed to higher levels of testosterone when they develop between two males in utero (Vom Saal, 1989). One may speculate that the position of a hypogonadal mouse in utero in regard to heterozygous or normal male siblings may result in differential prenatal androgen priming. We conclude from these studies that those mice with sufficient prenatal exposure to androgens are able to respond to androgen treatment in adulthood with masculine sexual behaviors, while neonatal androgens are necessary for the masculinization of the spinal nucleus of the bulbocavernosus and associated musculature required for the ejaculatory reflex. None of the hypogonadal mice described

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here received any GnRH treatment in the form of grafts or otherwise, indicating that the absence of GnRH does not limit the capacity for the full expression of male sexual behavior in the mouse.

REPLACEMENT OF GnRH BY INJECTION OR MINIPUMP It is widely accepted that the pulsatile secretion of GnRH is necessary for appropriate gonadotropin production and gonadal development. To test this, Charlton et al. (1983) used various regimens of GnRH injections in both male and female 2- to 4-month old hypogonadal mice. In male mice, doses (20 ng – 20 mg) of GnRH were injected once daily for 20 days. Modest testicular growth was seen with all of the doses of GnRH but there was no stimulation of seminal vesicles, which reflect circulating androgens. Pituitary FSH content was dramatically elevated with all treatments, with only slight increases in pituitary LH content. When 60 ng GnRH was administered 12 times/day for 15 days, both testicular and seminal vesicle stimulation was evident. While a bolus of 1 mg/day GnRH was ineffective in stimulating uterine growth in female hypogonadal mice, 60 ng GnRH/2 hr delivered over 15 days induced vaginal opening, cornified vaginal smears, and an 8fold increase in uterine growth to weights of 32 – 48 mg. Treated mice had significantly increased plasma FSH and pituitary content of LH and FSH; plasma LH remained undetectable in all cases. Thus the total amount of GnRH delivered was of less importance than periodic administration, with 720 ng divided into 12 injections yielding more physiological results than a single daily bolus of 1 mg. Similar pulsatile administration of GnRH (60 ng/2 hr) with an infusion pump to hypogonadal female mice (ages 2 to 4 months) for 18 days stimulated uterine weights to about 46 mg (Saade et al., 1989). In these studies, pituitary gonadotropin levels increased to within normal range and plasma LH rose to 1.5 – 2 ng/ml. While these experiments attempted to mimic ‘‘pulsatile’’ patterns of GnRH secretion, we were surprised to see the dramatic ovarian and uterine development that occurred in 2- to 3-month old hypogonadal mice when we used osmotic minipumps tonically secreting 40 ng GnRH per hour over a period of 15 or 30 days (Gibson et al., 1994). After 2 weeks of treatment, ovarian weights increased fivefold and uterine weights increased to those of the normal control mice (90 – 100 mg), considerably greater than that reported in the studies with peri-

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odic administration described above. Although plasma estradiol values were not obtained, vaginal opening and the presence of fully cornified vaginal smears appeared at 5 – 12 days (median: 7 days) in 11 of the 12 treated mice, signifying increased circulating estrogens. Although pituitary stores of LH and FSH remained low in the treated hypogonadal mice, in contrast to the results cited with pulsatile administration, plasma FSH levels were increased to normal levels. FSH secretion is not as tightly linked to patterns of GnRH as is LH (Strobl and Levine, 1988).

REPLACEMENT OF GnRH NEURONS BY NEURONAL GRAFTS When the preoptic area (POA) of normal mouse fetuses is used as a transplant into the anterior hypothalamus or third ventricle of adult hypogonadal mice, a portion of the GnRH neurons within the graft survive and send axonal processes that terminate in the lateral median eminence of the host brain (Silverman, Zimmerman, Gibson, Perlow, Charlton, Kokoris, and Krieger, 1985). Male hypogonadal mice with POA grafts respond with increased pituitary gonadotropin production and testicular and seminal vesicle development (Krieger et al., 1982). Hypogonadal male mice with POA grafts with robust gonadal development, however, do not exhibit normal male sexual behavior (Livne et al., 1992); in fact even mounting behavior is rarely seen in tests with estrogen and progesterone-primed females. However, if hypogonadal males with POA grafts receive TP implants, about 50% will exhibit intromission, similar to the findings in control hypogonadal males. This suggests that the androgen requirement for activation of sexual behavior is greater than that for stimulation of secondary sexual characteristics in the adult animal, since even hypogonadal males with POA grafts that have seminal vesicle weights approaching those of normal males fail to show sexual behavior without supplemental androgens. In adult female hypogonadal mice with POA grafts the first sign of a successful graft is vaginal opening, which occurs at puberty in normal mice (Gibson et al., 1984a). Vaginal opening is estrogen dependent and reflects the activation of the pituitary – gonadal axis by the grafted GnRH cells. However, unlike normal mice, which have 4- to 7-day spontaneous ovulatory cycles, hypogonadal mice with POA grafts generally enter persistent vaginal estrus. While persistent estrus is not seen in normal mice except during aging (Campbell, Ryan,

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and Schwartz, 1976), it is common in several species such as rabbits, cats, or voles. If hypogonadal mice with grafts do not have reproductive cycles as normal mice do, how does their reproductive behavior compare to that of normal female mice? We tested this by comparing hypogonadal females with POA grafts with normal cycling females (Gibson, Moscovitz, Kokoris, and Silverman, 1987b). In this study, groups of mice that had received grafts of fetal POA, or neonatal male or female POA, were included to assess whether the sex of the donor would impact the reproductive development or behavior of the host. The hypogonadal females had all responded to their grafts by entering persistent estrus and were tested on random days. Vaginal cytology was used in the normal cycling females to determine the day of proestrus, when those mice were tested. None of the mice received steroid priming. Two-hour mating tests were conducted in the home cages of reproductively experienced normal male mice. By every measure, the hypogonadal mice with grafts were comparable to the normal female mice. There were no differences in lordotic behavior across the groups. Ovarian and uterine weights of the hypogonadal mice were increased to the normal range, whether donor grafts were of male or female origin. Further, males showed similar latencies and frequencies of mounting behavior, and a similar percentage achieved ejaculation, regardless of the sex of the graft borne by the female. If hypogonadal females with POA grafts in persistent estrus are mated with normal males, reflex ovulation often occurs (Gibson, Krieger, Charlton, Zimmerman, Silverman, and Perlow, 1984b), with an LH surge evident within minutes of mating (Gibson, Moscovitz, Kokoris, and Silverman, 1987a), indicating the ability of the graft to secrete GnRH in response to a sensory stimulus to the host. After reflex ovulation, hypogonadal females with grafts successfully bear litters and nurse them to weaning. This broad range of reproductive function may occur with as few as two immunoreactive GnRH neurons detectable in the POA grafts in the host’s third ventricle (Gibson et al., 1984b). The necessary requirement appears to be innervation of the median eminence of the host by axons of the grafted GnRH neurons. Proven reflex ovulation has not been described in the normal mouse, perhaps because persistent estrus is not seen except during aging. Light-induced persistent estrus rats may ovulate ‘‘reflexively’’ in response to copulation or stressful stimuli (Brown-Grant, Davidson, and Greig, 1973) as well as to contact with soiled cage bedding from males (Johns, Feder, Komisaruk, and Mayer,

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1978). In addition, such rats in persistent estrus may respond to a progesterone challenge with LH release and ovulation (Brown-Grant et al., 1973). When we challenged hypogonadal mice with POA grafts in persistent estrus, approximately 25 – 30% responded to progesterone administration with an LH surge and ovulation (Gibson, Kokoris, and Silverman, 1988), indicating the ability of the grafted GnRH cells (at least in these individuals) to react to alterations in the hormonal milieu with a ‘‘positive feedback’’ response. In the course of the progesterone challenge studies, we first observed that some hypogonadal females with POA grafts displayed spontaneous ovarian cyclicity after weaning their litters (Gibson et al., 1988). Expression of Fos, a product of the immediate-early gene c-fos, is widely used as a marker of neuronal activation (Curran and Morgan, 1995). There is a strong correlation between Fos expression in GnRH neurons and periods of enhanced GnRH neuronal activity, as on the afternoon of proestrus (Lee, Smith, and Hoffman, 1990a,b; Moenter, Karsch, and Lehman, 1993; Doan and Urbanski, 1994) and during a steroid-induced LH surge in ovariectomized rats (Lee et al., 1990b; Moenter et al., 1993) and mice (Wu, Segal, Miller, Gibson, and Silverman, 1992). Increased GnRH mRNA expression is found in those GnRH neurons that express Fos during the proestrous LH surge (Wang, Hoffman, and Smith, 1995), suggesting that Fos expression in GnRH neurons may identify those that are more transcriptionally active. Using double-label immunocytochemical detection of Fos and GnRH, we recently demonstrated (Wu, Silverman, and Gibson, 1996) that when hypogonadal female mice with POA grafts are primed with progesterone prior to pairing with sexually active males, there is robust Fos induction in their grafted GnRH neurons, whether the males only perform intromissions or also ejaculate. An average of approximately 40% of the grafted GnRH neurons expressed Fos, remarkably similar to the levels in normal steroid-primed mice (Wu et al., 1992), rats (Lee et al., 1990b), hamsters (Doan and Urbanski, 1994), and sheep (Moenter et al., 1993). In contrast, there was little or no Fos expression in GnRH neurons in those hypogonadal females with POA grafts that only received progesterone priming or in those that were not primed prior to pairing with males that ejaculated. This evidence of synergism between sensory stimuli and hormonal milieu resulting in enhanced GnRH activation is reminiscent of our findings in normal mice, where both the steroid-induced LH surge and Fos expression in GnRH neurons are prolonged when mating occurs (Wu et al., 1992). Our observations

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are consistent with the hypothesis that the circuitry for sensory-induced or reflex ovulation is present in normal mice. There are reports that copulation affects LH release in many species considered to be spontaneous ovulators, including humans (Jochle, 1975). Ramirez and Beyer have reviewed evidence (Ramirez and Beyer, 1988) that spontaneous ovulation may be a more recent specialization imposed on the basic eutherian pattern of sensory-induced ovulation (Conaway, 1971; Zarrow and Clark, 1968). Pathways involved in transmitting sensory information associated with reproductive behavior to GnRH neurons that stimulate an ovulatory LH surge are not yet defined. Fos expression was also evaluated in regions of the brain activated by reproductive behavior in normal female rats (Rowe and Erskine, 1993; Tetel, Getzinger, and Blaustein, 1993; Rajendren and Moss, 1993). Increased numbers of Fos-positive cells were present in the preoptic area, bed nucleus of the stria terminalis, and medial amygdala when hypogonadal mice with grafts were paired with males that ejaculated or intromitted, regardless of whether the females had been primed with progesterone (Wu et al., 1996). In contrast, there was little or no Fos in these regions in mice that only received progesterone priming, but were not mated. In earlier studies with normal ovariectomized, estrogen- and progesterone-primed female mice (Wu et al., 1992), we also observed (unpublished) that Fos expression was intense in the preoptic area in those females mated with males, but not in those that only received steroid priming. Female mice in both groups had significant Fos expression specifically in GnRH neurons, associated with the LH surge. It appears that Fos activation in GnRH neurons is independent of general activation of the preoptic area in hypogonadal mice.

CONNECTIVITY: PHARMACOLOGY AND ANATOMY Any reproductive behavior seen in the female hypogonadal mice with grafts is only indirectly dependent on the anatomical connectivity of the graft: as long as the GnRH neurons in the graft are successful in innervating the median eminence, with subsequent pituitary and gonadal development, the secretion of ovarian steroid hormones may support normal behavior. Nevertheless, many other aspects of reproductive function do appear to require specific connectivity between the host brain and the grafted GnRH neurons.

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One of the hallmarks of normal GnRH physiology is pulsatile secretion as reflected in measures of plasma levels of LH. Interestingly, the large majority of both male and female hypogonadal mice with POA grafts exhibit LH pulsatility, similar in interval but not amplitude to that seen in normal mice (Gibson, Miller, and Silverman, 1991; Kokoris, Lam, Ferin, Silverman, and Gibson, 1988). In contrast, negative feedback of gonadal steroids on GnRH is rarely if ever seen in the mice with grafts (Gibson and Silverman, 1989). As discussed above, while reflex ovulation commonly occurs in the female hypogonadal with graft, positive feedback or spontaneous cyclicity is infrequent. Understanding the degree to which the grafts receive signals from the host requires both pharmacological and anatomical studies. Glutamate appears to be the major excitatory transmitter in the hypothalamus (Van den Pol and Trombley, 1993). We examined the effect of the glutamate analog, N-methyl-D,L-aspartic acid (NMDA), in adult normal mice and in hypogonadal male and female mice with POA grafts that had supported reproductive development after graft surgery (Saitoh, Silverman, and Gibson, 1991). Sequential blood samples were obtained from awake, freely moving animals before and after challenges with NMDA or vehicle. NMDA caused elevations in plasma LH in normal male mice and in normal and hypogonadal female mice with POA grafts. Male hypogonadal mice were considerably less sensitive to the excitatory amino acid despite GnRH cells surviving in their grafts and innervating the hosts’ median eminence. This study raised the possibility that there may be differences in connectivity in male vs female hypogonadal mice with grafts. We spent an enormous amount of time and effort before finally reporting that normal mice do not respond to challenges of the opioid antagonist, naloxone, with LH release. In contrast to findings in numerous other species, this is true for the mouse regardless of sex, steroid milieu, dose(s) administered, or method of blood sampling. However, we observed in normal male mice that naloxone significantly potentiated LH secretion in response to an NMDA challenge (Miller and Gibson, 1994), indicating that inhibitory opioid afferents to GnRH may be activated by NMDA. This led us to hypothesize that there may be a strong opioid inhibition on GnRH release in hypogonadal males with POA grafts, which rarely respond to NMDA. To test this, male hypogonadal mice were challenged with NMDA in combination with naloxone or saline. Five of 12 males responded occasionally to NMDA with significantly increased LH secretion, but there was no potentiation by opioid blockade. Two of the five respond-

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ers were 16 months old with 12-month-old grafts at the time of NMDA challenge; immunocytochemistry confirmed the presence of GnRH cells and robust innervation of the median eminence in these aging hosts (Miller, Silverman, Rogers, and Gibson, 1995). Since female but not male hypogonadal mice with POA grafts respond to NMDA with significant LH release, we evaluated the role of the steroid milieu after POA implantation. Hypogonadal males with POA grafts were castrated and given an estrogen capsule either at the time of graft implantation or 1 week prior to testing. These mice had significant LH secretory responses to NMDA, in contrast to intact or castrated hypogonadal mice with POA grafts. Significant gonadal development and/or immunocytochemistry for GnRH confirmed the efficacy of all of the POA grafts. GnRH fiber innervation of the host median eminence was present regardless of treatment. GnRH challenge tests indicated that the estradiol effects were primarily central. Hence estrogen treatment altered the responsivity of GnRH neurons to this neuromodulation (Miller et al., 1995). There is a normal distribution of neurons containing estrogen receptors in female hypogonadal mice, as well as substantial numbers of such cells in the POA grafts (Gibson, Silverman, Rosenthal, and Morrell, 1989). The role of endogenous opioids in NMDA-stimulated LH secretion in female hypogonadal mice with POA grafts was also evaluated. As in all our previous work with normal mice, naloxone failed to stimulate LH release in hypogonadal females when administered alone. These findings suggest that tonic opioid inhibition is absent in hypogonadal mice with POA grafts, as it is in normal mice, again in contrast to many other species. However, naloxone significantly potentiated NMDA-induced LH release in female hypogonadal mice with POA grafts. Immunocytochemistry revealed that b-endorphin-ir fibers were present in the grafts of all seven hypogonadal female animals studied, whereas b-endorphin-ir cell bodies were never seen in the POA grafts. In studies with the carbocyanine dye, DiI, we found similar evidence of host innervation of the graft. Small crystals of DiI were applied to the graft or to the host after fixation of the brain, which was subsequently analyzed for retrograde and anterograde movement of the dye. Crystals placed in the graft labeled a small number of axons found in the host median eminence or hypothalamus taking an arching trajectory toward the median eminence, similar to those of GnRH axons. Retrogradely labeled neurons in the host were largely confined to the arcuate nucleus. Immunocytochemical analysis of host and graft tissue with an antibody to b-

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endorphin revealed a large population of positive neurons in the host MBH but none in the graft. However, b-endorphin axons were seen ramifying in the grafts, crossing into the graft from adjacent host tissue (Silverman, Gibson, and Silverman, 1992b). It thus seems evident that NMDA activates both GnRH neurons and host-derived opioidergic afferents to the GnRH neuronal system. Naloxone blocks the inhibitory action of endogenous opioids on GnRH release, permitting an enhanced response to NMDA. Whether this occurs within the graft or at the level of the median eminence is not known.

GRAFTS OF IMMORTALIZED GnRH CELLS The immortalized GT1 cell line, which expresses the GnRH gene and makes releasable peptide (Mellon, Windle, Goldsmith, Padula, Roberts, and Weiner, 1990), is a widely used tool for studying GnRH function. We injected these cells into the preoptic area/anterior hypothalamus of hypogonadal male and female mice (Silverman et al., 1992a). GnRH-immunoreactive cells migrated widely in the central nervous system and exhibited varying degrees of morphological development, including axonal development and occasionally complex dendritic arborization. Cleavage products of the GnRH prohormone were identified. In many cases, however, large tumors formed, presumably due to the SV40 T antigen construct used to generate the cell line. While modest gonadal development occurred in some hosts, it was most evident in those individuals in which tumors or axons were present in the region of the median eminence. Subsequently, nine female hpg mice with GT1 implants were tested with NMDA challenges at 42 – 65 days after implantation (Miller et al., 1993). Three of the mice responded to NMDA challenges with significant increases in circulating LH, while a fourth had measurable LH episodes. While this study provided the first evidence that intrahypothalamic GT1 cells can support LH release in the hypogonadal mouse and that this secretion can be modified by pharmacological agents, we have decided not to continue this work for the following reasons: the very modest stimulation of gonadal development supported by these cells, the formation of tumors, and importantly, the failure of the cells to innervate the median eminence. This failure of targeting was described in the progenitor mice from which the cells were derived (Weiner, Thind, Windle, Mellon, and Goldsmith, 1991). GT1 cells may show

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pulsatile release in vitro (Wetsel, Valenc¸a, Merchenthaler, Liposits, Lo´pez, Weiner, Mellon, and Negro-Vilar, 1992), but we saw little evidence of that in vivo. From our studies described above using continuous GnRH infusion with osmotic minipumps (Gibson et al., 1994), we believe the modest gonadal development seen with these cell implants may well be due to diffusion of GnRH into the portal vessels and thence to the pituitary.

the graft supports this. Defining the specific connectivity underlying the discrete components of reproductive function described here is made more feasible by the use of grafts containing GnRH neurons. This model provides opportunities to dissociate these various capabilities. Elucidating such connectivity is one of the major goals of this work.

ACKNOWLEDGMENTS SUMMARY The discrete knockout of GnRH in the hypogonadal mouse provides an invaluable model to dissect apart the various roles of GnRH in reproductive physiology and behavior. From studies using administration of gonadal steroids, it is clear that GnRH is not essential for the full repertoire of male or female sexual behavior. The use of brain grafts containing GnRH neurons demonstrates that distinct components of reproductive function are dissociable: hosts may be capable of reflex but not spontaneous ovulation; others may show positive but not negative feedback. We have preliminary evidence (Rajendren and Gibson, 1995) that in certain mice grafted GnRH cells may be activated to show Fos expression even when the GnRH neurons in that animal have not innervated the median eminence or stimulated gonadal development. This emphasizes the two distinct aspects of innervation of the grafts: innervation of the graft by the host to transmit sensory and/or steroid signals, and the innervation of the host by the grafted GnRH neurons, necessary for robust stimulation of the reproductive system. A large number of neuromodulators have been implicated in regulating GnRH neuronal activity in various species. There are few such studies in mice, but in our work with both normal and hypogonadal mice we have begun to describe actions of excitatory amino acids and inhibitory endogenous opioids. Studies with the hypogonadal male indicate that sensitivity to the glutamate analog, NMDA, is dependent upon exposure to estrogens (Miller et al., 1995). In contrast to many other species, the GnRH system in mice does not appear to be under tonic endogenous opioid inhibition. However, in both normal and hypogonadal female mice, excitation of the NMDA receptors stimulates b-endorphin neurons (Saitoh et al., 1991); this activation dampens the GnRH response, as shown with the use of opioid blockers. It is thus likely that endogenous opioids play a modulatory role in regulating GnRH function. The finding that b-endorphin fibers of host origin innervate

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This work was supported by NIH Grants NS20335 and HD19077. We are grateful to the many colleagues, students, and associates who have collaborated in these studies over the years, and whose names are listed in the various references here. Special thanks to the anonymous person at the breeding facility associated with Oxford University who first detected the spontaneously mutated and unique hypogonadal mice.

REFERENCES Brown-Grant, K., Davidson, J. M., and Greig, F. (1973). Induced ovulation in albino rats exposed to constant light. J. Endocrinol. 57, 7 – 22. Campbell, C. S., Ryan, K. D., and Schwartz, N. B. (1976). Estrous cycles in the mouse: relative influence of continuous light and the presence of a male. Biol. Reprod. 14, 292 – 299. Cattanach, B. M., Iddon, C. A., Charlton, H. M., Chiappa, S. A., and Fink, G. (1977). Gonadotropin releasing hormone deficiency in a mutant mouse with hypogonadism. Nature 269, 338 – 340. Charlton, H. M., Halpin, D. M. G., Iddon, C., Rosie, R., Levy, G., McDowell, I. F. W., Megson, A., Morris, J. F., Bramwell, A., Speight, A., Ward, B. J., Broadhead, J., Davey-Smith, G., and Fink, G. (1983). The effects of daily administration of single and multiple injections of gonadotropin-releasing hormone on pituitary and gonadal function in the hypogonadal (hpg) mouse. Endocrinology 113, 533 – 544. Conaway, H. (1971). Ecological adaptation and mammalian reproduction. Biol. Reprod. 4, 239 – 247. Curran, T., and Morgan, J. I. (1995). Fos: An immediate-early transcription factor in neurons. J. Neurobiol. 26, 403 – 412. Doan, A., and Urbanski, H. F. (1994). Diurnal expression of Fos in luteinizing hormone-releasing hormone neurons of Syrian hamsters. Biol. Reprod. 50, 301 – 308. Dudley, C. A., Vale, W., Rivier, J., and Moss, R. L. (1981). The effect of LHRH antagonist analogs and an antibody to LHRH on mating behavior in female rats. Peptides 2, 393 – 396. Gibson, M. J., Charlton, H. M., Perlow, M. J., Zimmerman, E. A., Silverman, A. J., and Krieger, D. T. (1984a). Preoptic area brain grafts in hypogonadal (hpg) female mice abolish effects of congenital hypothalamic gonadotropin-releasing hormone (GnRH) deficiency. Endocrinology 114, 1938 – 1940. Gibson, M. J., Kasowski, H., and Dobrjansky, A. (1994). Continuous gonadotropin-releasing hormone infusion stimulates dramatic gonadal development in hypogonadal female mice. Biol. Reprod. 50, 680 – 685. Gibson, M. J., Kokoris, G. J., and Silverman, A. J. (1988). Positive feedback in hypogonadal female mice with preoptic area brain transplants. Neuroendocrinology 48, 112 – 119. Gibson, M. J., Krieger, D. T., Charlton, H. M., Zimmerman, E. A., Sil-

haba

AP: H & B

219

GnRH and Hypogonadal Mice

verman, A. J., and Perlow, M. J. (1984b). Mating and pregnancy can occur in genetically hypogonadal mice with preoptic area brain grafts. Science 225, 949 – 951. Gibson, M. J., Miller, G. M., and Silverman, A. J. (1991). Pulsatile luteinizing hormone secretion in normal female mice and in hypogonadal female mice with preoptic area implants. Endocrinology 128, 965 – 971. Gibson, M. J., Moscovitz, H. C., Kokoris, G. J., and Silverman, A. J. (1987a). Plasma LH rises rapidly following mating in hypogonadal female mice with preoptic area (POA) brain grafts. Brain Res. 424, 133 – 138. Gibson, M. J., Moscovitz, H. C., Kokoris, G. J., and Silverman, A. J. (1987b). Female sexual behavior in hypogonadal mice with GnRHcontaining brain grafts. Horm. Behav. 21, 211 – 222. Gibson, M. J., and Silverman, A. J. (1989). Effects of gonadectomy and treatment with gonadal steroids on luteinizing hormone secretion in hypogonadal male and female mice with preoptic area implants. Endocrinology 125, 1525 – 1532. Gibson, M. J., Silverman, A. J., Rosenthal, M. F., and Morrell, J. I. (1989). Estradiol-concentrating cells in the brains of hypogonadal female mice and in their intraventricular preoptic area implants. Exp. Neurol. 105, 127 – 134. Jochle, W. (1975). Current research in coitus-induced ovulation: A review. J. Reprod. Fertil. 165 – 207. Johns, M. A., Feder, H. H., Komisaruk, B. R., and Mayer, A. D. (1978) Urine-induced reflex ovulation in anovulatory rats may be a vomeronasal effect. Nature 272, 446 – 448. Kokoris, G. J., Lam, N. Y., Ferin, M., Silverman, A. J., and Gibson, M. J. (1988). Transplanted gonadotropin-releasing hormone neurons promote pulsatile luteinizing hormone secretion in congenitally hypogonadal (hpg) male mice. Neuroendocrinology 48, 45 – 52. Krieger, D. T., Perlow, M. J., Gibson, M. J., Davies, T. F., Zimmerman, E. A., Ferin, M., and Charlton, H. M. (1982). Brain grafts reverse hypogonadism of gonadotropin releasing hormone deficiency. Nature 298, 468 – 471. Lee, W.-S., Smith, M. S., and Hoffman, G. E. (1990a). Progesterone enhances the surge of luteinizing hormone by increasing the activation of luteinizing hormone-releasing hormone neurons. Endocrinology 127, 2604 – 2606. Lee, W.-S., Smith, M. S., and Hoffman, G. E. (1990b). Luteinizing hormone-releasing hormone neurons express Fos protein during the proestrous surge of luteinizing hormone. Proc. Natl. Acad. Sci. USA 87, 5163 – 5167. Livne, I., Gibson, M. J., and Silverman, A.-J. (1993). Gonadotropinreleasing hormone (GnRH) neurons in the hypogonadal mouse elaborate normal projections despite their biosynthetic deficiency. Neurosci. Lett. 151, 229 – 233. Livne, I., Silverman, A.-J., and Gibson, M. J. (1992). Reversal of reproductive deficiency in the hpg male mouse by neonatal androgenization. Biol. Reprod. 47, 561 – 567. Luttge, W. G., and Sheets, C. S. (1977). Further studies on the restoration of estrohen-induced sexual receptivity in ovariectomized mice treated with dihydrotestosterone: Effects of progesterone, dihydroprogesterone and LH-RH. Pharmacol. Biochem. Behav. 7, 563 – 566. MacLusky, N. J., and Naftolin, F. (1981). Sexual differentiation of the central nervous system. Science 211, 1294 – 1303. Mason, A. J., Hayflick, J. S., Zoeller, R. T., Young, W. S., III, Phillips, H. S., Nikolics, K., and Seeburg, P. H. (1986). A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the hpg mouse. Science 234, 1366 – 1371. Mellon, P. L., Windle, J. J., Goldsmith, P. C., Padula, C. A., Roberts, J. L., and Weiner, R. I. (1990). Immortalization of hypothalamic

AID

H&B 1387

/

680a$$$$41

06-02-97 08:05:51

GnRH neurons by genetically targeted tumorigenesis. Neuron 5, 1 – 10. Miller, G. M., and Gibson, M. J. (1994). Opioidergic modulation of Nmethyl-D,L-aspartic-acid-stimulated LH release in young adult but not older male mice. Neuroendocrinology 59, 277 – 284. Miller, G. M., Silverman, A.-J., Roberts, J. L., Dong, K. W., and Gibson, M. J. (1993). Functional assessment of intrahypothalamic implants of immortalized gonadotropin-releasing hormone-secreting cells in female hypogonadal mice. Cell Transplant. 2, 251 – 257. Miller, G. M., Silverman, A.-J., Rogers, M.-C., and Gibson, M. J. (1995). Neuromodulation of transplanted gonadotropin-releasing hormone neurons in male and female hypogonadal mice with preoptic area brain grafts. Biol. Reprod. 52, 572 – 583. Moenter, S. M., Karsch, F. J., and Lehman, M. N. (1993). Fos expression during the estradiol-induced gonadotropin-releasing hormone (GnRH) surge of the ewe: Induction in GnRH and other neurons. Endocrinology 133, 896 – 903. Moss, R. L. (1979). Actions of hypothalamic-hypophysiotropic hormones on the brain. Annu. Rev. Physiol. 41, 617 – 631. Moss, R. L., and McCann, S. M. (1973). Induction of mating behaviour in rats by luteinizing hormone-releasing factor. Science 181, 177 – 179. Pfaff, D. W. (1973). Luteinizing hormone-releasing factor potentiates lordosis behaviour in hypophysectomized, ovariectomized female rats. Science 178, 417 – 418. Rajendren, G. V., and Gibson, M. J. (1995). Influence of conspecific males on the GnRH neuronal system in normal and hypogonadal female mice with preoptic area grafts (HPG/POA): An immunocytochemical study. Soc. Neurosci. Abstr. 21: 1893. Rajendren, G., and Moss, R. L. (1993). The role of the medial nucleus of amygdala in the mating-induced enhancement of lordosis in female rats: The interaction with luteinizing hormone-releasing hormone neuronal system. Brain Res. 617, 81 – 86. Ramirez, V. D., and Beyer, C. (1988). The ovarian cycle of the rabbit: Its neuroendocrine control. In E. Knobil and J. D. Neill (Eds.) The Physiology of Reproduction, Vol. 2, pp. 1873 – 1892. Raven Press, New York. Rowe, D. W., and Erskine, M. S. (1993). c-Fos proto-oncogene activity induced by mating in the preoptic area, hypothalamus and amygdala in the female rat: Role of afferent input via the pelvic nerve. Brain Res. 621, 25 – 34. Saade, G., London, D. R., and Clayton, R. N. (1989). The interaction of gonadotropin-releasing hormone and estradiol on luteinizing hormone and prolactin gene expression in female hypogonadal (hpg) mice. Endocrinology 124, 1744 – 1753. Sachs, B. D. (1982). Role of striated penile muscles in penile reflexes, copulation, and induction of pregnancy in the rat. J. Reprod. Fertil. 66, 433 – 443. Saitoh, Y., Silverman, A. J., and Gibson, M. J. (1991). Effects of Nmethyl-D,L-aspartic acid on luteinizing hormone secretion in normal mice and in hypogonadal mice with fetal preoptic area implants. Endocrinology 128, 2432 – 2440. Sakuma, Y., and Pfaff, D. W. (1983). Modulation of the lordosis reflex of female rats by LHRH, its antiserum and analogs in the mesencephalic central gray. Neuroendocrinology 36, 218 – 224. Schmale, H., and Richter, D. (1984). Single base deletion in the vasopressin gene is the cause of diabetes insipidus in Brattleboro rats. Nature 308, 705 – 709. Silverman, A.-J., Roberts, J. L., Dong, K.-W., Miller, G. M., and Gibson, M. J. (1992a). Intrahypothalamic injection of a cell line secreting gonadotropin-releasing hormone results in cellular differentiation

haba

AP: H & B

220

Gibson et al.

and reversal of hypogonadism in mutant mice. Proc. Natl. Acad. Sci. USA 89, 10668 – 10672. Silverman, A. J., Jamandas, J., and Renaud, L. P. (1987). Localization of LHRH neurons that project to the median eminence. J. Neurosci. 7, 2312 – 2319. Silverman, A. J., Zimmerman, E. A., Gibson, M. J., Perlow, M. J., Charlton, H. M., Kokoris, G. J., and Krieger, D. T. (1985). Implantation of normal fetal preoptic area into hypogonadal (hpg) mutant mice: Temporal relationships of the growth of GnRH neurons and the development of the pituitary/testicular axis. Neuroscience 16, 69 – 84. Silverman, R. C., Gibson, M. J., and Silverman, A. J. (1992b). Application of a fluorescent dye to study connectivity between third ventricular preoptic area grafts and host hypothalamus. J. Neurosci. Res. 31, 156 – 165. Strobl, F. J., and Levine, J. E. (1988). Estrogen inhibits luteinizing hormone (LH), but not follicle-stimulating hormone secretion in hypophysectomized pituitary-grafted rats receiving pulsatile LH-releasing hormone infusions. Endocrinology 123, 622 – 630. Tetel, M. J., Getzinger, M. J., and Blaustein, J. D. (1993). Fos stimulation in the rat brain following vaginal-cervical stimulation bymating and manual probing. J. Neuroendocrinol. 5, 397 – 404. Van den Pol, A. N., and Trombley, P. Q. (1993). Glutamate neurons in hypothalamus regulate excitatory transmission. J. Neurosci. 13, 2829 – 2836. Vom Saal, F. S. (1989). Sexual differentiation in litter bearing mammals: Influence of sex of adjacent fetuses in utero. J. Anim. Sci. 67, 1824 – 1840.

AID

H&B 1387

/

680a$$$$41

06-02-97 08:05:51

Wagner, C. K., and Clemens, L. G. (1989). Perinatal modification of a sexually dimorphic motor nucleus in the spinal cord of the B6D2F1 house mouse. Physiol. Behav. 45, 831 – 835. Wang, H. J., Hoffman, G. E., and Smith, M. S. (1995). Increased GnRH mRNA in the GnRH neurons expressing cFos during the proestrous LH surge. Endocrinology 136, 3673 – 3676. Ward, B. J., and Charlton, H. M. (1981). Female sexual behaviour in the GnRH deficient, hypogonadal (hpg) mouse. Physiol. Behav. 27, 1107 – 1109. Weiner, R. I., Thind, K. K., Windle, J. J., Mellon, P. L., and Goldsmith, P. C. (1991). Expression of SV-40 T antigen in GnRH neurons inhibits organization of terminals in the median eminence in transgenic mice. Soc. Neurosci. Abstr. 17, 76.9. Wetsel, W. C., Valenc¸a, M. M., Merchenthaler, I., Liposits, Z., Lo´pez, F. J., Weiner, R. I., Mellon, P. L., and Negro-Vilar, A. (1992). Intrinsic pulsatile secretory activity of immortalized luteinizing hormonereleasing hormone-secreting neurons. Proc. Natl. Acad. Sci. USA 89, 4149 – 4153. Wu, T. J., Segal, A. Z., Miller, G. M., Gibson, M. J., and Silverman, A.-J. (1992). FOS expression in gonadotropin-releasing hormone neurons: Enhancement by steroid treatment and mating. Endocrinology 131, 2045 – 2050. Wu, T. J., Silverman, A. J., and Gibson, M. J. (1996). FOS expression in grafted gonadotropin-releasing hormone neurons in hypogonadal mouse: Mating and steroid induction. J. Neurobiol. 31, 67 – 76. Zarrow, M. X., and Clark, J. H. (1968). Ovulation following vaginal stimulation in a spontaneous ovulator and its implications. J. Endocrinol. 40, 343 – 352.

haba

AP: H & B