Pregnancy outcomes in women with congenital virilizing adrenal hyperplasia.

Although low fertility rates have traditionally been reported among women with classic CAH and especially among women with the salt-wasting variant, more recent data suggest that fertility rates are significantly improved, largely owing to earlier treatment of CAH, improved compliance with therapy, and surgical advances in genital reconstruction. Furthermore, ovulation induction and assisted reproductive techniques are now available to women who remain infertile despite effective adrenal androgen suppression. Although the pregnancy experience in women with classic CAH remains limited, it is apparent that, once pregnant, these women have a high probability of successful outcome. Key issues should be emphasized in the management of CAH during gestation, including the need for assessing adrenal steroid replacement and adrenal androgen suppression, particularly in light of the interplay between maternal hyperandrogenism and the protective effect of placental aromatase activity, which provides a relatively large margin of safety for the female fetus. Maternal hormone levels should be evaluated in the context of laboratory-specific reference ranges for pregnancy. The infant should be examined for ambiguous genitalia and monitored for evidence of adrenal insufficiency. Although an affected female infant with classic CAH has not been reported as a pregnancy outcome of a mother with classic virilizing CAH, these concerns should be discussed during preconception counseling. Patients should also be aware of the importance of medication compliance and careful hormonal monitoring during the entire pregnancy. In most cases, successful gestational management requires the close coordination of care between the obstetrician and endocrinologist.

Estrogen, bone, growth and sex: a sea change in conventional wisdom.

The discovery of a man with a homozygous mutation in the estrogen receptor alpha gene, which results in estrogen-receptor alpha resistance, and of males and females with autosomal recessive mutations in the CYP19 gene encoding aromatase, which leads to a failure to synthesize estrogens, has challenged conventional wisdom about the 'unimportant' role of estrogen in the male. For example, in the male, estrogen (not androgen) derived from direct testicular secretion (approximately 20%) and from extragonadal aromatization of testosterone and androstenedione (approximately 80%), is the critical sex hormone in the pubertal growth spurt, skeletal maturation, accrual of peak bone mass, and the maintenance of bone mass in the adult. Estrogen stimulates chondrogenesis in the epiphyseal growth plate increasing pubertal linear growth. At puberty, estrogen promotes skeletal maturation and the gradual, progressive closure of the epiphyseal growth plate, possibly as a consequence of both estrogen-induced vascular and osteoblastic invasion and the termination of chondrogenesis. In addition, during puberty and into the third decade, estrogen has an anabolic effect on the osteoblast and an apoptotic effect on the osteoclast, increasing bone mineral acquisition in axial and appendicular bone. In the adult, estrogen is important in maintaining the constancy of bone mass through its effects on remodeling and bone turnover. Establishing a role for estrogen does not exclude a direct action of testosterone on bone in the human male (especially on cortical bone), but this action is less characterized than thought in the past and is relatively minor in comparison with the major effect of estrogen in the male.

Estrogen: consequences and implications of human mutations in synthesis and action.

Recent developments have advanced our knowledge of the role of estrogen in the male. Studies of the mutations in CYP19, the gene encoding aromatase, in six females and two males and a mutant estrogen receptor alpha in a man are described. These observations provide illuminating new insights into the critical role of estrogen in the male (as well as female) in the pubertal growth spurt and skeletal maturation, and in the importance of estrogen sufficiency in the accrual and maintenance of bone mass. The weight of evidence supports an effect of androgens on the latter processes, but this effect has not been quantitated. There is a discordance in the estrogen-deficient male between skeletal growth and skeletal maturation and the accrual of bone mass and density. Estrogen synthesis by the testis is limited before puberty, and estrogen deficiency does not affect the age of pubertal onset. Estrogen deficiency in men leads to hypergonadotropism, macroorchidism, and increased testosterone levels. Estrogen lack has a significant effect on carbohydrate and lipid metabolism, and estrogen resistance was associated with evidence of premature coronary atherosclerosis in a man. These observations have highlighted the role of extraglandular estrogen synthesis and intracrine and paracrine actions. In the human, in contrast to nonprimate vertebrates, aromatase deficiency and estrogen resistance (alpha) does not seem to affect gender identity or psychosexual development. The clinical repercussions of mutations in CYP19 on the fetal-placental unit have highlighted the major role of placental aromatase in the protection of the female fetus from androgen excess, thus preventing androgen-induced pseudohermaphrodism and virilization of the mother. These features are compared with the virilization that occurs in utero in the female spotted hyena. The novel features of the aromatase deficiency syndrome in the affected female--in the fetus, during childhood, and at puberty--are discussed, including virilization at puberty and development of polycystic ovaries. The severity of the syndrome correlates with the severity of impairment of aromatase formation in expression systems. Finally, the structural consequences of missense mutations in CYP19 are described in accordance with a model of the structure of human aromatase.

Congenital hypogonadotropic hypogonadism and micropenis: effect of testosterone treatment on adult penile size why sex reversal is not indicated.

Micropenis is commonly due to fetal testosterone deficiency. The clinical management of this form of micropenis has been contentious, with disagreement about the capacity of testosterone treatment to induce a functionally adequate adult penis. As a consequence, some clinicians recommend sex reversal of affected male infants. We studied 8 male subjects with micropenis secondary to congenital pituitary gonadotropin deficiency from infancy or childhood to maturity (ages 18 to 27 years). Four patients were treated with testosterone before 2 years of age (group I) and four between age 6 and 13 years (group II). At presentation, the mean penile length in group I was 1.1 cm (-4 SD; range, 0.5 to 1.5 cm) and in group II it was 2.7 cm (-3.4 SD; range, 1.5 to 3.5 cm). All patients received one or more courses of 3 intramuscular injections of testosterone enanthate (25 or 50 mg) at 4-week intervals in infancy or childhood. At the age of puberty the dose was gradually increased to 200 mg monthly and later to an adult replacement regimen. As adults, both group I and II had attained a mean final penile length of 10.3 cm 2.7 cm with a range of 8 to 14 cm (mean adult stretched penile length for Caucasians is 12.4 2.7 cm). Six of 8 men were sexually active, and all reported normal male gender identity and psychosocial behavior. We conclude that 1 or 2 short courses of testosterone therapy in infancy and childhood augment penile size into the normal range for age in boys with micropenis secondary to fetal testosterone deficiency; replacement therapy at the age of puberty results in an adult size penis within 2 SD of the mean. We found no clinical, psychologic, or physiologic indications to support conversion of affected male infants to girls. Further, the results of this study do not support the notion, derived from data in the rat, that testosterone treatment in infancy or childhood impairs penile growth in adolescence and compromises adult penile length.

Normal female infants born of mothers with classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Women with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, especially those patients with the salt-losing form, have decreased fertility rates. Pregnancy experience in this population is limited. We report the pregnancy outcomes and serial measurements of maternal serum steroid levels in four women with classic 21-hydroxylase deficiency, three of whom were female pseudohermaphrodites with the salt-losing form. These glucocorticoid-treated women gave birth to four healthy female newborns with normal female external genitalia, none of whom were affected with 21-hydroxylase deficiency. In three women, circulating androgen levels increased during gestation, but remained within the normal range for pregnancy during glucocorticoid therapy. In the fourth patient, androgen levels were strikingly elevated during gestation despite increasing the dose of oral prednisone from 5 to 15 mg/day (two divided doses). Notwithstanding the high maternal serum concentration of androgens, however, placental aromatase activity was sufficient to prevent masculinization of the external genitalia of the female fetus and quite likely the fetal brain, consistent with the idea that placental aromatization of androgens to estrogens is the principal mechanism that protects the female fetus from the masculinizing effects of maternal hyperandrogenism. These four patients highlight key issues in the management of pregnancy in women with 21-hydroxylase deficiency, particularly the use of endocrine monitoring to assess adrenal androgen suppression in the mother, especially when the fetus is female. Recommendations for the management of pregnancy and delivery in these patients are discussed.

Acute N-methyl-D,L-aspartate administration stimulates the luteinizing hormone releasing hormone pulse generator in the ovine fetus.

To assess whether fetal luteinizing hormone releasing hormone (LH-RH) neurosecretory neurons have the capacity to respond to an exogenous stimulus, a synthetic excitatory amino acid analogue, N-methyl-D-L-aspartate (NMDA; 15 mg/kg), was given rapidly intravenously to 8 chronically catheterized fetuses (130-142 days of gestation; term 147 +/- 3 days). All 8 fetuses exhibited a rise in plasma ovine luteinizing hormone (oLH) and ovine follicle-stimulating hormone (oFSH) within 5 min. The mean maximal increments of oLH (2.25 +/- 0.36 ng/ml) and oFSH (1.21 +/- 0.32 ng/ml) were significantly greater than in 6 normal saline-injected controls (oLH p < 0.0002; oFSH p < 0.03). The secretion of ovine prolactin (oPRL) and ovine growth hormone (oGH) was unaffected. LH-RH (5 microg) evoked a greater oLH response (p < 0.0009) and a greater oFSH response (p < 0.03) than NMDA (n = 6). Desensitization of the fetal gonadotrope by a potent LH-RH agonist, D-Trp6Pro9NEt-LH-RH (10 microg/day i.v. x 4 days), abolished the fetal oLH and the oFSH response to NMDA (n = 5). Moreover, D, L-2-amino-5-phosphonovalerate, a specific competitive antagonist for the NMDA receptor, completely inhibited the fetal oLH and oFSH response to NMDA, whereas D-L-2-amino-5-phosphonovalerate alone did not affect the plasma oLH or oFSH levels, the gonadotropin response to LH-RH, or the release of oGH or oPRL (n = 3). In primary ovine fetal pituitary cell cultures, NMDA (10(-10) to 10(-6) M) had no effect on oLH, oFSH, oGH, or oPRL secretion, whereas LH-RH stimulated oLH (10(-8) M; p < 0.0004) and oFSH (10(-8) M; p < 0. 0001) release, evidence that NMDA did not have a direct pituitary effect. The results suggest that NMDA induces oLH and oFSH secretion by stimulation of the fetal LH-RH pulse generator and is mediated by central NMDA receptors. Fetal LH and FSH secretion and the response to LH-RH decrease in late gestation in the ovine and human fetus. The relative importance of sex steroid dependent and sex steroid independent central nervous system inhibition in this developmental change is unclear. It appears that central neural inhibition in addition to sex steroid negative feedback contributes to the decrease in fetal gonadotropin concentrations in late gestation. NMDA did not affect fetal oGH or oPRL secretion.

The growth hormone cascade: progress and long-term results of growth hormone treatment in growth hormone deficiency.

The growth hormone (GH) cascade and the remarkable advances over the past four decades in our knowledge of its components are considered. It is now over 40 years since human pituitary GH (pit-hGH) was purified and the first GH-deficient patient, a 17-year-old male, was successfully treated with pit-hGH. However, the shortage of pit-hGH limited its use and the dose, the biopotency of preparations varied, strict criteria of GH deficiency (GHD) were used for patient selection including peak plasma immunoreactive GH levels after provocative stimuli of <3.5-5 ng/ml, treatment was not infrequently interrupted, the mean age for initiating treatment was often late in childhood (12-13 years) and the growth deficiency severe (height -4 to -6 SDS), and finally pit-hGH therapy was often discontinued when girls attained a height of 5' and boys 5'5". Nonetheless, the effects of pit-hGH were dramatic; the final height SDS increased in isolated GHD to about -2 SDS in boys and -2.5 to -3.0 SDS in girls, and in multiple pituitary hormone deficiencies to between -1 and -2 SDS. Between 1962 and 1985 when the Creutzfeldt-Jakob disease crisis struck, the number of GH-deficient patients treated with pit-hGH increased from about 150 to over 3,000. The advent of biosynthetic GH (rhGH) and its availability to treat large numbers of idiopathic GH-deficient children (the minimum prevalence rate of which in the USA and UK is between 1 in 3,400 and 4,000) dramatically changed this picture in 1985. It is estimated that more than 60,000 patients have been or are now on treatment. With rhGH treatment the attained mean adult height SDS is now about -1.0, and in our experience with the treatment of patients under 4 years of age, final height may exceed the target height. It is now recognized that (a) the replacement dose of rhGH ranges from 0.175 to 0.35 mg/kg/week and should be individualized; (b) dividing this dose into 6 or 7 daily subcutaneous injections is more effective than giving the same total dose in three weekly portions, and (c) final height correlates significantly with pretreatment chronologic age, height SDS and predicted adult height, duration of therapy, birth length, in some studies height SDS and age at start of puberty, weight, and serum GHBP (an indicator of GH receptor mass). Early recognition of GHD is essential for an optimal height outcome. rhGH treatment should not be delayed in children with documented GHD; the greater the height deficit, the lower the probability that target height will be reached. GHD needs to be detected earlier in children with organic hypopituitarism whether due to a developmental defect, neoplasm, radiation, head trauma, or a CNS infection. Early rhGH therapy in neonatal hypopituitarism has resulted in excellent growth responses. As the height prognosis in isolated GHD is not as good (especially in girls) as in GHD associated with gonadotropin deficiency, the use of LHRH agonists to delay puberty or potent aromatase inhibitors to delay skeletal maturation should be considered in selected patients with isolated GHD. When the growth response to rhGH is less than predicted, one must consider: (a) poor compliance; (b) improper preparation of rhGH for administration or faulty injection techniques; (c) the timing of administration; (d) the dose of glucocorticoid in the ACTH-deficient patient; (e) occult hypothyroidism; (f) inadequate nutrition; (g) a chronic illness; (h) neutralizing antibodies to rhGH, and (i) the wrong diagnosis. The major cause of mortality (unrelated to Creutzfeldt-Jakob disease or a CNS neoplasm) is adrenal crisis and hypoglycemia in children with both GH and ACTH deficiency. Major adverse effects of rhGH treatment in children are uncommon and include idiopathic intracranial hypertension, slipped capital femoral epiphysis, and acute pancreatitis. The rhGH is not an added risk for leukemia in the US and Europe in the absence of coexisting risk factors, nor is there a higher risk of recurrence of b

Long-term outcome in children and adolescents after transsphenoidal surgery for Cushing's disease.

Cushing's disease refers specifically to an ACTH-producing pituitary adenoma that stimulates excess cortisol production. Transsphenoidal surgery is the treatment of choice in children and adolescents, but disparate cure rates have been reported, ranging from 50-98%. The discrepancies in cure rate are due primarily to the technical success of the surgery and the length and method of follow-up. We studied 42 consecutive children and adolescents (age, < or = 18 yr) who underwent transsphenoidal exploration for the primary treatment of Cushing's disease at University of California-San Francisco from 1974-1993. Only 7 patients had persistent disease, defined as evidence of Cushing's disease within 6 months of surgery, yielding an initial remission rate of 83%. We comprehensively evaluated 26 of the 35 patients who experienced an initial remission, including testing of the ACTH-adrenocortical axis. The mean duration of follow-up is 7.2 yr (range, 1.5-13.6 yr). Seven experienced a relapse of Cushing's disease, yielding a net remission rate of 73%. Relapses occurred an average of 4.2 yr postoperatively (range, 0.75-6.2 yr). Five patients experienced relapse within 5 yr of surgery, whereas 2 relapsed more than 5 yr postoperatively. Repeat transsphenoidal surgery was performed in 8 patients with persistent or recurrent disease, and 6 of these remain in remission. Low serum or urinary cortisol measurements within the first post-operative week predicted remission of Cushing's disease, but were not necessarily predictive of long-term cure. Hypercortisolism had significant effects on bone metabolism, as reflected by both diminished bone density in the majority of patients examined and decreased growth rate. Both parameters improved after surgical care, although they did not fully normalize. We conclude that transsphenoidal surgery is a safe and effective treatment for pediatric Cushing's disease, but long-term surveillance is necessary to detect possible recurrences.

Idiopathic hypothalamic diabetes insipidus, pituitary stalk thickening, and the occult intracranial germinoma in children and adolescents.

We report nine consecutive children and adolescents [five females and four males; aged 2 yr 8 months (m) to 18 yr 1 m] studied over the last 5 yr with idiopathic central diabetes insipidus. In addition to vasopressin deficiency, anterior pituitary hormone deficiencies were detected, either on evaluation at presentation or during follow-up studies over the following 3 yr. Four patients had an increased concentration of plasma PRL. One patient had multiple pituitary hormone deficiencies at diagnosis, and two others developed the same by 21 m of follow-up. Brain magnestic resonance imaging scans, performed at presentation, were originally interpreted as normal in four of nine patients, except for absence of the bright posterior pituitary signal; after retrospective review, two of nine were considered normal. All of the brain magnetic resonance imaging (MRI) scans showed positive findings by 14 m of follow-up. The first abnormal finding in all patients was isolated pituitary stalk thickening. Evaluation of cerebrospinal fluid (CSF) for hCG was positive in three of eight evaluated patients; the three positive CSF values were found at presentation and 3 and 9 m after presentation. All eight patients assessed were negative for CSF alpha-fetoprotein and cytology, and no patient had serum tumor markers. Transsphenoidal biopsy of the lesion in seven of nine patients showed a germinoma in six patients and inflammatory cells in one. The six patients with documented germinoma comprise 31% of the intracranial germinomas diagnosed in this age group at the University of California-San Francisco during the last 5 yr. The patient with mononuclear inflammatory cells on biopsy along with one other patient have had spontaneous resolution of their stalk thickening. So-called "idiopathic" central diabetes insipidus warrants close follow-up to determine the etiology, especially if anterior pituitary hormone deficiencies are detected. Normal brain MRI scans or scans that show isolated pituitary stalk thickening merit follow-up with serial contrast enhanced brain MRI for the early detection of an evolving occult hypothalamic-stalk lesion. CSF evaluation is recommended at presentation because elevated CSF hCG may precede MRI abnormalities.

The effect of prepubertal androgen exposure on adult penile length.

PURPOSE: Recent studies in the rat suggest that early exposure to exogenous testosterone accelerates the loss of androgen receptors and compromises eventual penile length. To determine whether this is true in men we measured adult penile length of patients treated in childhood for sexual precocity. MATERIALS AND METHODS: We examined 21 men with sexual precocity due to true precocious puberty (12) or congenital adrenal hyperplasia (9) who had been followed at our institution since childhood. Penile lengths were compared with data from normal men. RESULTS: Mean stretched penile length plus or minus standard deviation was 12.7 +/- 2.6 cm. in all patients, 12.1 +/- 2.6 cm. in those with true precocious puberty and 13.6 +/- 1.6 cm. in those with congenital adrenal hyperplasia. These lengths were not significantly different from those of normal men (12.4 +/- 2.7 cm.). CONCLUSIONS: In contrast to findings in rats, exposure to endogenous testosterone during gestation and/or childhood does not reduce adult penile length in men. Thus, the use of testosterone to treat childhood genitourinary anomalies would likely not compromise mature penile size.

Molecular genetics and pathophysiology of 17 beta-hydroxysteroid dehydrogenase 3 deficiency.

Autosomal recessive mutations in the 17 beta-hydroxysteroid dehydrogenase 3 gene impair the formation of testosterone in the fetal testis and give rise to genetic males with female external genitalia. Such individuals are usually raised as females, but virilize at the time of expected puberty as the result of increases in serum testosterone. Here we describe mutations in 12 additional subjects/families with this disorder. The 14 mutations characterized to date include 10 missense mutations, 3 splice junction abnormalities, and 1 small deletion that results in a frame shift. Three of these mutations have occurred in more than 1 family. Complementary DNAs incorporating 9 of the 10 missense mutations have been constructed and expressed in reporter cells; 8 of the 9 missense mutations cause almost complete loss of enzymatic activity. In 2 subjects with loss of function, missense mutations testosterone levels in testicular venous blood were very low. Considered together, these findings strongly suggest that the common mechanism for testosterone formation in postpubertal subjects with this disorder is the conversion of circulating androstenedione to testosterone by one or more of the unaffected 17 beta-hydroxysteroid dehydrogenase isoenzymes.

Aromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens.

The aromatase enzyme complex catalyzes the conversion of androgens to estrogens in a wide variety of tissues, including the ovary, testis, placenta, brain, and adipose tissue. Only a single human gene encoding aromatase P450 (CYP19) has been isolated; tissue-specific regulation is controlled in part by alternative promoters in a tissue-specific manner. We report a novel mutation in the CYP19 gene in a sister and brother. The 28-yr-old XX proband, followed since infancy, exhibited the cardinal features of the aromatase deficiency syndrome as recently defined. She had nonadrenal female pseudohermaphrodism at birth and underwent repair of the external genitalia, including a clitorectomy. At the age of puberty, she developed progressive signs of virilization, pubertal failure with no signs of estrogen action, hypergonadotropic hypogonadism, polycystic ovaries on pelvic sonography, and tall stature. The basal concentrations of plasma testosterone, androstenedione, and 17-hydroxyprogesterone were elevated, whereas plasma estradiol was low. Cyst fluid from the polycystic ovaries had a strikingly abnormal ratio of androstenedione and testosterone to estradiol and estrone. Hormone replacement therapy led to breast development, menses, resolution of ovarian cysts, and suppression of the elevated FSH and LH values. Her adult height is 177.6 cm (+2.5 SD). Her only sibling, an XY male, was studied at 24 yr of age. During both pregnancies, the mother exhibited signs of progressive virilization that regressed postpartum. The height of the brother was 204 cm (+3.7 SD) with eunuchoid skeletal proportions, and the weight was 135.1 kg (+2.1 SD). He was sexually fully mature and had macroorchidism. The plasma concentrations of testosterone (2015 ng/dL), 5 alpha-dihydrotestosterone (125 ng/dL), and androstenedione (335 ng/dL) were elevated; estradiol and estrone levels were less than 7 pg/mL. Plasma FSH and LH concentrations were more than 3 times the mean value. Plasma PRL was low; serum insulin-like growth factor I and GH-binding protein were normal. The bone age was 14 yr at a chronological age of 24 3/12 yr. Striking osteopenia was noted at the wrist. Bone mineral densitometric indexes of the lumbar spine (cancellous bone) and distal radius (cortical bone) were consistent with osteoporosis; the distal radius was -4.7 SD below the mean value for age- and sex-matched normal men; indexes of bone turnover were increased. Hyperinsulinemia, increased serum total and low density lipoprotein cholesterol, and triglycerides and decreased high density lipoprotein cholesterol were detected.(ABSTRACT TRUNCATED AT 400 WORDS)

Long-term effect of gonadotropin-releasing hormone agonist therapy on final and near-final height in 26 children with true precocious puberty treated at a median age of less than 5 years.

We report a long term study on the effectiveness of chronic GnRH agonist treatment on final or near-final height in 26 patients (20 females and six males) with true precocious puberty (TPP). This study differs from other treatment studies in that the median age at onset of therapy was 4.7 yr for females and 6.2 yr for males, the youngest cohort of treated patients reported to date. We compared patients treated with GnRH agonists who attained final or near-final height with a historical control group of untreated children with TPP (n = 116) matched for mean age of pubertal onset, etiology of TPP (idiopathic or neurogenic), rate of progression, and sex ratio. The current mean height of GnRH agonist-treated females who began therapy at more than 5 yr of age (157.6 +/- 6.6 cm) is already significantly greater than the mean final height of untreated females (152.7 +/- 8.6 cm). The current mean predicted height of the treated females is 164.6 +/- 9.7 cm. The current mean height of females whose treatment was started before 5 yr of age is greater (164.1 +/- 7.7 cm) than that of females whose treatment began after 5 yr of age (157.6 +/- 6.6 cm). The final height of untreated children whose age of sexual precocity was less than 5 yr at diagnosis is significantly less than that of treated patients who were less than 5 yr when they developed TPP (P = 0.0006). The current mean height of GnRH agonist-treated males is 166.3 +/- 12.2 cm, and the current mean predicted height is 170.8 +/- 11.3 cm. This is in sharp contrast to the mean final height of untreated males (155.6 +/- 7.7 cm). The current predicted height correlates negatively with the age at initiation of treatment and the initial bone age and positively with height SD for bone age in the agonist-treated children. The current mean height deviation from target height is significantly less in the 20 treated females (-1 SD) than in 93 untreated females (-2.4 SD; P = 0.006). The mean final height deviation from target height in 23 untreated males (-3.7 SD) is significantly greater than the current height deviation from target height in 6 treated males (-1.7 SD; P = 0.03). The salutary effects of long term GnRH agonist therapy on stature are more clear-cut in the younger treated children. Young untreated children may have the worst outcome with respect to final height.(ABSTRACT TRUNCATED AT 400 WORDS)

Nitric oxide synthesized by gonadotropin-releasing hormone neurons is a mediator of N-methyl-D-aspartate (NMDA)-induced GnRH secretion.

N-Methyl-D-aspartate (NMDA) directly stimulates gonadotropin-releasing hormone (GnRH) neurons to secrete GnRH. It is not known if this stimulatory effect of NMDA is mediated by NO. Northern blot analysis of the immortalized hypothalamic GnRH neuronal cells (GT1-1) mRNA with a neuronal NOS cDNA revealed this clonal cell line expressed neuronal NOS transcripts as a single 10.5-kb band. Immunoblot analysis of GT1-1 proteins with anti-neuronal NOS serum showed that the GT1-1 cells contain neuronal NOS. GT1-1 cells were used to study the effects of NO and NMDA on GnRH release. L-Arginine (10(-2) M), a precursor of NO enhances basal GnRH secretion. Both oxyhemoglobin (Hb)(10(-6)-10(-4) M), a NO scavenger and N omega-nitro-L-arginine (NNA)(10(-3),10(-2) M), a NOS inhibitor and inactivator block basal as well as NMDA-induced GnRH release. Sodium nitroprusside (SNP) (10(-4), 10(-3) M), a NO donor stimulates GnRH release, an effect inhibited by Hb. Incubation of GT1-1 cells in Ca(2+)-free medium abolished the stimulatory effect of NMDA on GnRH release. In contrast, incubation in medium with increasing concentrations of Ca2+ enhances basal GnRH release as well as augments NMDA-mediated GnRH release. The results demonstrate that L-arginine-NO pathway is functional in the GT1-1 cells and an increase in intracellular Ca2+ [Ca2+]i following NMDA receptor activation activates NOS to generate NO. We conclude that endogenous NO mediates, at least in part, basal as well as NMDA-stimulated GnRH release and may play a role as an intercellular messenger in synchronizing pulsatile GnRH release.