Expression of DAX-1, the Gene Responsible for X-Linked Adrenal Hypoplasia Congenita and Hypogonadotropic Hypogonadism, in the Hypothalamic-Pituitary-Adrenal/Gonadal Axis
WEIWEN GUO, THOMAS P. BURRIS, AND EDWARD R. B. MCCABE Department of Pediatrics, UCLA School of Medicine, Los Angeles, California 90024
Received June 29, 1995
DAX-1, an orphan member of the nuclear hormone receptor superfamily, is responsible for X-linked adrenal hypoplasia congenita (AHC) and the fre- quently associated hypogonadotropic hypogo- nadism (HH). The entire DAX-1 genomic region has been sequenced and a putative steroidogenic fac- tor-1 response element has been identified in the promoter region of the gene. The purpose of these investigations was to determine if DAX-1 was ex- pressed in the central nervous system, particularly the hypothalamus and pituitary, in order to better understand the relationship of mutations in this gene to HH associated with AHC. We used Northern blot analysis and reverse transcription PCR to dem- onstrate that DAX-1 was expressed in the hypothal- amus and the pituitary, and to confirm its expres- sion in adrenal cortex and gonads. The expression of DAX-1 in these tissues indicates the involvement of DAX-1 in the development of the reproductive system at multiple levels within the hypothalamic- pituitary-adrenal/gonadal axis. We also observed the expression of DAX-1 in a human adrenocortical carcinoma cell line, NCI-H295, that has features characteristic of the fetal adrenal cortex. Therefore, NCI-H295 cells will be a useful cellular model for investigating the involvement of DAX-1 in the reg- ulation of steroidogenesis. 1995 Academic Press, Inc.
Adrenal hypoplasia congenita (AHC) causes adre- nal insufficiency in infants and children, occurs with a frequency of one per 12,500 births (1), and was first described by Sikl (2). Two distinct forms have been identified, based on the histological appearance of the adrenal cortex. The miniature adult form, also called the anencephalic or secondary form, is char- acterized by small adrenal glands with a well- defined, but generally thinner than normal, perma- nent zone and a greatly reduced fetal zone (3). The miniature adult form of adrenal hypoplasia is asso- ciated with congenital abnormalities in pituitary or
hypothalamic function. Studies have shown this form of AHC can be either sporadic or an autosomal recessive disorder (4, 5). The primary or X-linked cytomegalic form of AHC (MIM 300200) (6) is char- acterized by an absence of the permanent zone of the adrenal cortex and by structural disorganization of the fetal cortex (7). The cytomegalic cells are larger than normal fetal adrenal cells and have nuclear inclusions due to cytoplasmic invaginations (8). The disorder, which is lethal if untreated, results in adrenal insufficiency early in infancy, with low se- rum concentration of glucocorticoids, mineralocorti- coids, and androgens, and failure to respond to ACTH stimulation.
Hypogonadotropic hypogonadism (HH) is com- monly associated with the X-linked form of the AHC (9, 10). Low serum concentrations of LH, FSH, and testosterone are usually observed and the response of serum gonadotropin concentrations to a single GnRH bolus is generally low (11), although response to pulsatile GnRH has been reported (12). HH often is noted in boys with X-linked AHC at the expected time of pubertal maturation (9, 10, 13). It is not clear if this form of HH is of pituitary or hypothalamic origin (14). Furthermore, abnormalities of the geni- tourinary system appear to occur with increased fre- quency in boys with AHC, and these include: cryptor- chidism, hypospadias, small external genitalia, ure- teral reflux, and urethral stenosis (8, 15-17). The HH of AHC should not be confused with the HH of Kallmann syndrome (MIM 308700), a disorder that maps to Xp22 (6). Patients with Kallmann syndrome also have anosmia, and this disorder appears to re- sult from defective migration of GnRH neurons from the olfactory placode into the arcuate nucleus of the hypothalamus during fetal development (18-20).
The gene responsible for X-linked AHC was mapped to the Xp21 region, distal to the glycerol kinase (GK) gene (21, 22) and adjacent to the dosage
sensitive sex reversal (DSS) locus, a region that is duplicated in some 46 XY patients with an intact SRY gene and ambiguous or female genitalia (23, 24). The gene responsible for AHC, termed DAX-1, for dosage sensitive sex reversal locus, adrenal hy- poplasia congenita at chromosome X, number 1, has been identified and belongs to the nuclear hormone receptor superfamily (25-27). Intragenic mutations have been identified in the DAX-1 gene among pa- tients with AHC associated with HH, indicating that this gene is responsible for both of these clinical fea- tures. The genomic structure of DAX-1 has been elu- cidated and a putative steroidogenic factor 1 (SF-1) response element has been observed in the 5’ flank- ing region of the gene (28). SF-1 is a nuclear receptor that regulates the expression of many steroidogenic enzymes and is essential for development of the adrenal, the gonads, and the ventromedial nucleus of the hypothalamus (29-32).
The purpose of the investigations reported here was to determine if DAX-1 was expressed in the ner- vous system, particularly in the hypothalamus and pituitary, in order to better understand the patho- genesis of the HH that is frequently associated with X-linked AHC. Our results showed that DAX-1 was not only expressed in the adrenal cortex and gonads, as has been reported previously (25, 27), but also in the brain. Further study demonstrated the expres- sion of DAX-1 in the hypothalamus and pituitary gland. In the course of these investigations, we also observed DAX-1 expression in a human adrenocor- tical carcinoma cell line NCI-H295 that has previ- ously been shown to exhibit features of the fetal cor- tex. These results broaden our concept of DAX-1 be- yond involvement in the adrenal gland and gonads, and give insight into the mechanism responsible for HH, while also providing a possible cellular model for investigating the regulatory role of DAX-1 in the fetal adrenal cortex.
MATERIALS AND METHODS
Northern Blotting
A human multiple tissue Northern blot was pur- chased from Clontech (cat. 7759-1, lot 52715; Clon- tech, Palo Alto, CA). The Northern blot contained 2 ug of poly (A)+ RNA per lane from various human tissues. The 1.6-kb SacI genomic DNA fragment, which included the first exon of the DAX-1 gene, was used as the hybridization probe. The Northern blot was prehybridized in 5x SSPE, 10x Denhardt’s, 2% SDS, and 8-10 µg/ml sheared human placen-
tal DNA, for 3 h. The probe was labeled with [a-32P]dCTP using the random hexamer primer la- beling method (33), and was preassociated with 8-10 µg/ml sheared human placental DNA in 5x SSPE, 10x Denhardt’s, and 2% SDS for 3 h. The hybridization was carried out at 65℃ for 18-20 h. Following hybridization, the blot was washed in 2x SSC and 0.05% SDS at room temperature for up to 20 min. If required because of excess residual radio- activity, a second wash in 2x SSC and 0.1% SDS was performed for up to 15 min at 65°C.
Cell Lines and cDNA Libraries
A human adrenocortical carcinoma cell line, NCI- H295 (34), was obtained from the American Type Culture Collection (ATCC; Rockville, MD). The fol- lowing human cDNA libraries were purchased from Clontech: fetal adrenal (dgt11HL1118b); hypothala- mus (Agt11 HL1172B); kidney (Agt11 HL1071b); liver (Agt11 HL1115b); and testis (Agt11 HL1161a). Human poly (A)+ RNA, prepared from total adult brain (6516-1), fetal brain (6525-1), pituitary (6584- 1), and placenta (6518-1), was purchased from Clon- tech and was used for RT-PCR preparation of cDNA.
RNA Isolation
Total RNA was isolated from 1 × 108 NCI-H295 cells by the guanidinium thiocyanate method (35) using Promega RNA Agents RNA isolation kit (Promega, Madison, WI).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
The DAX-1 transcript was detected by reverse transcription of mRNA from tissues and from cul- tured cells, with amplification of the resultant cDNA by PCR (RT-PCR) amplification. For reverse tran- scription of the RNA (100 ng each), the reaction mix- tures contained 2 ul of random hexamer (300 ng/ul), 2 ul of oligo(dT) (300 ng/ul), 2 ul of 20 mM DTT, 1.6 ul of 25 mM dNTP, 4 ul of 10x buffer (750 mM KCI, 30 mM MgCl2, and 500 mM Tris-HCI, pH 8.3, 0.5 p.l of RNAsin (Promega, Madison, WI), 1 ul of M-MLV (GIBCO BRL, Gaithersburg, MD), and water to bring the total volume to 40 ul. The reaction was carried out at 70°℃ for 5 min, then 37℃ for 90 min. For amplification of a 251-bp fragment of the DAX-1 cDNA after RT, the primer pair was selected, such that primer 2937 (5’-AAGGAGTACGCCTACCT- CAA-3’) was located in the first exon of the DAX-1 gene and 2851 (5’-TCCATGCTGACTGTGCCGAT- 3’) was located in the second exon; therefore, any
contamination from genomic DNA or unprocessed RNA would have been detectable by its larger size. Amplification conditions were as follows: 5 min at 95℃ without Taq polymerase, then 0.5 min dena- turation at 94℃, 0.5 min annealing at 57°C, and 0.5 min extension at 72℃ for 32 cycles using Taq poly- merase (Boehringer-Mannheim) and amplification buffer (Perkin-Elmer).
Southern Blotting of cDNA Gel
The RT-PCR products were separated electropho- retically on a 2% agarose gel containing ethidium bromide (10 µg/100 ml) in 1x TBE. The gel was transferred to Biodyne B membrane (GIBCO BRL) and was prehybridized in 5x SSPE, 10x Den- hardt’s, 2% SDS, and 8-10 ug/ml sheared human placental DNA for 3 h. The membrane was hybrid- ized with a probe consisting of the full-length cDNA of DAX-1. The probe was labeled with [a-32P]dCTP, using the random hexamer primer labeling method (33) and was preassociated with 8-10 ug/ml sheared human placental DNA in 5x SSPE, 10x Denhardt’s, and 2% SDS for 3 h. The hybridization was carried out at 65℃ for 18-20 h. Following hybridization, the blot was washed in 2x SSC and 0.05% SDS at room temperature for up to 20 min. A second wash in 2x SSC and 0.1% SDS was performed for up to 15 min at 65°℃ (36).
RESULTS
Expression of DAX-1 in Human Tissues
A Northern blot containing RNA from a variety of human tissues was hybridized with the SacI restric- tion fragment containing the first exon of the DAX-1 gene (Fig. 1). The results show that the gene is strongly expressed in testis and weakly expressed in ovary. Two different RNA species were observed, one approximately 1.9 kb in length and the other about 6 kb in size. The 1.9-kb band was consistent with the size of the cDNA. The 6-kb band, but not the 1.9-kb band, hybridized with a probe containing only intronic sequence from the DAX-1 gene (data not shown), indicating that the 6-kb band repre- sented unprocessed RNA.
Expression of DAX-1 in Human Hypothalamus, Pituitary Gland, and an Adrenocortical Carcinomal Cell Line
RT-PCR amplified a 251-bp DAX-1 sequence from mRNA and cDNA libraries. Despite the observation of unprocessed RNA in the Northern blot, only the
Spleen
Thymus
Prostate
Testis
Ovary
Small Int.
Colon
Blood
9.5 Kb
7.5 Kb
4.4 Kb
2.4 Kb
-
*
1.35 Kb -
smaller amplification product from processed mRNA was observed, presumably due to reduced efficiency of amplification of the longer sequence containing the intron. RT-PCR amplified the 251-bp DAX-1 product from mRNA extracted from total adult brain, total fetal brain, pituitary gland, and the hu- man adrenocortical carcinoma cell line, NCI-H295, as well as from cDNA libraries of the hypothalamus, fetal adrenal gland, and testis (Fig. 2A). The DAX-1 product was negative in mRNA from placental tis- sue and cDNA libraries from liver and kidney. In order to increase the sensitivity of this analysis a Southern blot was prepared from the gel (Fig. 2B). Placenta, liver, and kidney remained negative for DAX-1.
DISCUSSION
Recently, studies have shown DAX-1 is the gene responsible for X-linked AHC and is an orphan member of the nuclear hormone receptor superfam- ily, based on the presence of a conserved ligand bind- ing domain in its carboxy terminal portion. Since DAX-1 appears to contain a novel zinc finger domain in its amino terminal portion, it very likely serves as a transcription factor that is required for adrenal development and, particularly, for development of the fetal adrenal cortex (25-27). Intragenic muta- tions in the DAX-1 gene were found in AHC patients who also exhibited HH, indicating that DAX-1 is re- sponsible for HH. Furthermore, as a putative tran- scription factor expressed in steroidogenic tissues,
3
Placenta
Fetal Brain
Pituitary
Hypothalamus
A
Marker
NCI-H295
Fetal Adrenal
Hypothalamus
Brain
Testis
Liver
Kidney
Marker
Marker
NCI-H295
Placenta
Fetal Brain
Pituitary
Fetal Adrenal
Brain
Testis
Liver
Kidney
Marker
NC
NC
369bp
369bp
246bp
246bp
the DAX-1 protein product is a candidate gene for the sex reversal locus since DAX-1 is contained within the DSS region (24). In this study we provide evidence that DAX-1 is not only expressed in mRNA from adrenal and gonadal tissues, but also from adult and fetal brain. More specifically, DAX-1 is expressed in the hypothalamic-pituitary axis. These studies, together with the clinical features of pa- tients with DAX-1 mutations, directly link DAX-1 to the normal development of the adrenal cortex and gonadal tissue.
We also demonstrated that DAX-1 is expressed in a human adrenocortical carcinoma cell line, NCI- H295, that is a steroidogenic model for the human fetal cortex, and expresses most of the enzymes as- sociated with adrenal steroidogenesis (37). The genes encoding the steroidogenic enzymes in this cell line respond to stimulation by second messenger pathways in a manner similar to the human adrenal cortex (37). Investigation of this cell line will help us to understand the regulation of DAX-1 expression and the role of DAX-1 in the fetal adrenal. In addi- tion, DAX-1 may be a useful marker for tumor cell transformation in the specific tissues of the hypotha- lamic-pituitary-adrenal/gonadal axis.
The development of the mammalian reproductive system requires elaborate interactions within the hypothalamic-pituitary-adrenal/gonadal axis. The gonadal steroids, essential reproductive hormones, are produced by testes and ovaries under the regu- lation of the tropic hormones LH and FSH (38). The
release of LH and FSH by the gonadotropes of the anterior pituitary gland is stimulated by GnRH, which is secreted by specific neurons located within the hypothalamus (39, 40). A number of neuronal centers deliver the modulatory inputs to GnRH neu- rons that transduce the elaborate effects of behav- ioral and environmental stimuli on the reproductive axis (40, 41). All of these complex interactions are modulated at both the hypothalamic and the pitu- itary levels through negative and positive feedback regulation (42). The association of HH with AHC, along with our data showing that DAX-1 is ex- pressed in hypothalamus and pituitary gland, sug- gest that DAX-1 may be involved with the normal development of GnRH neurons and gonadotropes or the regulation of GnRH and gonadotropin expres- sion. Since pulsatile administration of GnRH to males with HH associated with AHC can result in testosterone secretion (12), the action of DAX-1 would appear to be proximal to GnRH in the hypo- thalamus and/or pituitary.
Interestingly, the expression pattern of DAX-1 is very similar to that for SF-1. SF-1 is an orphan nu- clear receptor that is an essential regulator of ste- roid hydroxylase gene expression (29, 30) and is ex- pressed at multiple levels of the reproductive system from hypothalamus and pituitary to the adrenal cor- tex and gonads (31, 43). SF-1 has been demonstrated to be essential for development of the adrenal cortex, gonads, and the ventromedial nucleus of the hypo- thalamus (31, 32). We have shown previously that
the promoter region of the DAX-1 gene contains a putative SF-1 response element (28) and now we show that DAX-1 is expressed in hypothalamic- pituitary-adrenal/gonadal axis. Collectively, these results strongly suggest that DAX-1 may be regu- lated by SF-1 or may act in concert with SF-1 as a coregulator at various levels in gonadal and adrenal development.
ACKNOWLEDGMENTS
This work was supported by a grant from the National Institute of Child Health and Human Development (RO1 HD22563). Dr. Burris is supported by the American Cancer Society (PF-4074). Dr. McCabe is an Academic Associate for the Corning-Nichols Laboratories. Walter Miller, M.D., Department of Pediatrics, University of California, San Francisco, kindly provided initial samples of NCI-H295 poly (A)+ RNA.
REFERENCES
1. Kelch RP, Virdis R, Rapaport R, Greig F, Levine LS, New MI. Congenital adrenal hypoplasia. Pediatr Adolesc Endo- crinol 13:156-161, 1984.
2. . Sikl H. Addison’s disease due to congenital hypoplasia of the adrenals in an infant aged 33 days. J Pathol Bacteriol 60:323-324, 1948.
3. . Laverty CRA, Fortune DW, Beischer NA. Congenital idio- pathic adrenal hypoplasia. Obstet Gynecol N.Y. 41:655-664, 1973.
4. Favara BE, Franciosi RA, Miles V. Idopathic adrenal hy- poplasia in children. Amer J Clin Pathol 57:287-296, 1972.
5. Ohlbaum P, Hehunstre PP, Bouchet JL, Deminiere C. In- suffisance surrenale chronique et hyalinose segmentaire et focale familiale: unenouvelle association. Pediatrie 41:86, 1986.
. McKusick VA. Mendelian inheritance in man. 10th ed. Bal- timore: The Johns Hopkins Univ. Press, 1992, pp. 1841- 1843.
7. Seltzer WK, Firminger H, Klein J, Pike A, Fennessey P, McCabe ERB. Adrenal dysfunction in glycerol kinase defi- ciency. Biochem Med 33:189-199, 1985.
8. Marsden HB, Zakkhour HD. Cytomegalic adrenal hypopla- sia with pituitary cytomegaly. Virchows Arch Abt A Path Anat Histol 378:105-110, 1978.
9. Martin MM, Martin ALA. The syndrome of congenital he- reditary adrenal hypoplasia and hypogonadotropic hypogo- nadism. Int J Adol Med Hlth 1:119-137, 1985.
10. Prader A, Zachman M, Illig R. Luteinizing hormone defi- ciency in hereditary congenital adrenal hypoplasia. J Pedi- atr 86:421-422, 1975.
11. Dunkel L, Perheentupa J, Virtanen M, Maenpaa J. GnRH and hCG tests are both necessary in differential diagnosis of male delayed puberty. Am J Dis Child 139:494-498, 1985.
12. Partsch C-J, Sippel WG. Hypothalamic hypogonadism in congenital adrenal hypoplasia. Horm Metabol Res 21:623- 625, 1989.
13. Virdis R, Levine LS, Levy D, Pang S. Congenital adrenal
hypoplasis: Two new cases. J Endocrinol Invest 6:51-54, 1983.
14. Kletter GB, Gorski JL, Kelch RP. Congenital adrenal hy- poplasia and isolated gonadotropin deficiency. Trends Endo- crinol Metabol 2:123-128, 1991.
15. Hensleigh PA, Moore WV, Wilson K, Tulchinsky D. Congen- ital X-linked adrenal hypoplasia. Obstet Gynecol 52:228- 232, 1978.
16. Zachmann M, Illig R, Prader A. Gonadotropin deficiency and crytorchidism in three prepubertal brothers with congenital adrenal hypoplasia. J Pediatr 92:255-256, 1980.
17. Wise JE, Matalon R, Morgan AM, McCabe ERB. Phenotypic features of patients with congenital adrenal hypoplasia and glycerol kinase deficiency. Am J Dis Child 141:744-747, 1987.
18. Schwanzel-Fukuda M, Jorgensen KL, Bergen HT, Weesner GD, Pfaff DW. Biology of normal luteinizing hormone- releasing hormone neurons during and after their migration from olfactory placode. Endocrinol Rev 13:623-634, 1992.
19. Franco B, Guiol S, Pragliola A, Incerti B, Bardoni B, Ton- lorenzi R, Carrozzo R, Maestrini E, Pieretti M, Taillon- Miller P, Brown CJ, Willard HF, Lawrence C, Persico MG, Camerion G, Ballabio A. A gene deleted in Kallmann’s syn- drome shares homology with neural cell adhesion and ax- onal path-finding molecules. Nature 353:529-536, 1991.
20. Legouis R, Hardelin J-P, Levillers J, Claverie J-M, Compain S, Wunderlw V, Millassear P, Le Paslier D, Cohen D, Cate- rina D, Bougueleret L, Delemarre-Van de Wall H, Luttalla G, Weissenbach J, Petit C. The candidate gene for the X-linked Kallmann syndrome encodes a protein related to adhesion molecules. Cell 67:423-435, 1991.
21. Worley KC, Ellison KA, Zhang Y-H, Wang D-F, Mason J, Roth EJ, Adams V, Fogt DD, Zhu XM, Tobin JA, Chinault AC, Zoghbi H, McCabe ERB. Yeast artificial chromosome cloning in the glycerol kinase and adrenal hypoplasia con- genita region of Xp21. Genomics 16:407-416, 1993.
22. Walker AP, Muscatelli F, Monaco AP. Isolation of the hu- man Xp21 glycerol kinase gene by positional cloning. Hum Mol Genet 2:107-114, 1993.
23. Arn P, Chen H, Tuck Muller CM, Mankinen C, Wachtel G, Li S, Shen CC, Wachtel SS. A sex reversing locus in Cp21.2 to p22.11. Hum Genet 93:389-393, 1994.
24. Bardoni B, Zanaria E, Guioli S, Floridia G, Worley K, Tonini G, Ferrante E, Chiumello G, McCabe ERB, Fraccaro M, Zuf- fardi O, Camerino G. X-linked sex reversal due to a dosage sensitive gene which interferes testis formation. Nature Genet 7:497-501, 1994.
25. Zanaria E, Muscatelli F, Bardoni B, Strom TM, Guioli S, Guo W, Lalli E, Moser C, Walker AP, McCabe ERB, Meit- inger T, Monaco AP, Sassone-Corsi P, Camerino G. An un- usual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita. Na- ture 372:635-641, 1994.
26. Muscatelli F, Strom TM, Walker AP, Zanaria E, Recan D, Meindl A, Bardoni B, Guioli S, Zehetner G, Rabl W, Schwarz HP, Kaplan JC, Camerino G, Meitinger T, Monaco AP. Mu- tation in the DAX-1 gene gives rise to both X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Nature 372:672-676, 1994.
27. Guo W, Mason J, Stone CG, Morgan SA, Muda S, Baldini A,
Lindsay EA, Biesecker LG, Copelland K, Horlick MNB, Pet- tigrew A, Zanaria E, McCabe ERB. Diagnosis of X-linked adrenal hypoplasia congenita by mutation analysis of the DAX-1 gene. JAMA 274:324-330, 1995.
28. Guo W, Mason J, Burris TP, McCabe ERB. X-linked adrenal hypoplasia congenita: Characterization of the genomic re- gion. Pediatr Res 37:90A, 1995.
29. Lala DS, Rice DA, Parker KL. Steroidogenic factor I, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor I. Mol Endocrinol 6:1249- 1258, 1992.
30. Ikeda Y, Shen W-H, Ingraham HA, Parker KL. Development expression of mouse steroidogenic factor 1, an essential reg- ulator of the steroid hydroxylases. Mol Endocrinol 8:652- 654, 1994.
31. Luo X, Ikeda Y, Parker KL. A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77:481-490, 1994.
32. Ikeda Y, Luo X, Abbud R, Hilson JH, Parker KL. The nu- clear receptor steroidogenic factor 1 is essential for the for- mation of the ventromedial hypothalamic nucleus. Mol En- docrinol 9:478-486, 1995.
33. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific ac- tivity. Anal Biochem 132:6-13, 1993.
34. Gazder AF, Oie HK, Shackleton CH, Chen TR, Triche TJ, Myers CE, Chrousos GP, Brennan MF, Stein CA, La-Rocca RV. Establishment and characterization of a human adre- nocortical carcinoma cell line that expresses multiple path-
ways of steroid biosynthesis. Cancer Res 50:5488-5496, 1990.
35. Chomczynski P, Sacchi N. Single step method of RNA iso- lation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159, 1987.
36. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning --- A Laboratory Manual. 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989.
37. Staels BS, Hum DW, Miller WL. Regulation of steroidogen- esis in NCI-H295 cells: A cellular model of the human fetal adrenal. Mol Endocrinol 7:423-433, 1993.
38. Pierce JD, Parsons RF. Glycoprotein hormones: Structure and function. Annu Rev Biochem 50:465-495, 1981.
39. Waldhauser F, Weissenbacher G, Frisch H. Pulsatile secre- tion of gonadotropins in early infancy. Eur J Pediatr 137:71-74, 1981.
40. Fink G. Gonadotropin secretion and its control. In The Phys- iology of Reproduction. (Knobil E, Neill JD, Eds.) New York: Raven Press, 1988, pp. 1349-1377.
41. Schwanzel-Fukuda M, Pfaff DW. Origin of luteining hor- mone-releasing hormone neurons. Nature 338:161, 1989.
42. Gharib SD, Wierman ME, Shupnik MA, Chin WW. Molecu- lar biology of the pituitary gonadotropins. Endocrinol Rev 11:177-199, 1990.
43. Ingraham HA, Lala DS, Ikeda Y, Lou X, Shen W-H, Nachti- gal MW, Abbud R, Nilson JH, Parker KL. The nuclear re- ceptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 8:2302-2312, 1994.