Ovarian cancer stem-like side-population cells are tumourigenic and chemoresistant.

BACKGROUND: Ovarian cancer is the most lethal gynaecological malignancy. Although ovarian cancer patients often respond initially to chemotherapy, they usually develop chemoresistance. We hypothesised that a small portion of ovarian cancer cells have stem-like cell properties that contribute to tumourigenesis and drug resistance. METHODS: Flow cytometry and Hoechst 33342 efflux isolated side-population (SP) cells from ascites derived from ovarian cancer patients and from mice inoculated with human ovarian cancer cell lines. The SP cells were examined for stem cell markers OCT4, NANOG, STELLAR, and ABCG2/BCRP1 by immunocytochemistry and RT-PCR. The SP cells and non-SP cells were studied for tumourigenesis and chemoresistance in vitro and in vivo. RESULTS: The SP cells expressed ABCG2/BCRP1, OCT4, STELLAR, and NANOG, detected by immunocytochemistry and RT-PCR. ABCG2/BCRP1 expression was higher in SP than in non-SP cells. Xenogeneic mice inoculated with SP cells yielded more tumours than did mice inoculated with non-SP cells. In parallel, SP cell culture resulted in extensive cell proliferation, which was markedly more than in non-SP cells. SP cells resisted chemotherapy compared with non-SP cells, both in vivo and in vitro. CONCLUSION: Ovarian cancer SP cells are tumourigenic and chemoresistant. ABCG2/BCRP1 has an important role in chemoresistance, which has implications for new therapeutic approaches.

Biologically active chorionic gonadotropin: synthesis by the human fetus.

The kidney, and to a slight extent the liver, of human fetuses were found to synthesize and secrete the alpha subunit common to glycoprotein hormones. Fetal lung and muscle did not synthesize this protein. Since fetal kidney and liver were previously found to synthesize beta chorionic gonadotropin, their ability to synthesize bioactive chorionic gonadotropin was also determined. The newly synthesized hormone bound to mouse Leydig cells and elicited a biological response: namely, the synthesis of testosterone. These results suggest that the human fetus may participate in metabolic homeostasis during its development.

Developmental and functional biology of the primate fetal adrenal cortex.

The unique characteristics of the primate (particularly human) fetal adrenal were first realized in the early 1900s when its morphology was examined in detail and compared with that of other species. The unusual architecture of the human fetal adrenal cortex, with its unique and disproportionately enlarged fetal zone, its compact definitive zone, and its dramatic remodeling soon after birth captured the interest of developmental anatomists. Many detailed anatomical studies describing the morphology of the developing human fetal adrenal were reported between 1920 and 1960, and these morphological descriptions have not changed significantly. More recently, it has become clear that fetal adrenal cortical growth involves cellular hypertrophy, hyperplasia, apoptosis, and migration and is best described by the migration theory, i.e. cells proliferate in the periphery, migrate centripetally, differentiate during their migration to form the functional cortical zones, and then likely undergo apoptosis in the center of the cortex. Consistent with this model, cells of intermediate phenotype, arranged in columnar cords typical of migration, have been identified between the definitive and fetal zones. This cortical area has been referred to as the transitional zone and, based on the expression of steroidogenic enzymes, we consider it to be a functionally distinct cortical zone. Elegant experiments during the 1950s and 1960s demonstrated the central role of the primate fetal adrenal cortex in establishing the estrogenic milieu of pregnancy. Those findings were among the first indications of the function and physiological role of the human fetal adrenal cortex and led Diczfalusy and co-workers to propose the concept of the feto-placental unit, in which DHEA-S produced by the fetal adrenal cortex is used by the placenta for estrogen synthesis. Tissue and cell culture techniques, together with improved steroid assays, revealed that the fetal zone is the primary source of DHEA-S, and that its steroidogenic activity is regulated by ACTH. In recent years, function of the human and rhesus monkey fetal adrenal cortical zones has been reexamined by assessing the localization and ontogeny of steroidogenic enzyme expression. The primate fetal adrenal cortex is composed of three functionally distinct zones: 1) the fetal zone, which throughout gestation does not express 3 beta HSD but does express P450scc and P450c17 required for DHEA-S synthesis; 2) the transitional zone, which early in gestation is functionally identical to the fetal zone but late in gestation (after 25-30 weeks) expresses 3 beta HSD, P450scc, and P450c17, and therefore is the likely site of glucocorticoid synthesis, and 3) the definitive zone, which lacks P450c17 throughout gestation but late in gestation (after 22-24 weeks) expresses 3 beta HSD and P450scc, and therefore is the likely site of mineralocorticoid synthesis. Indirect evidence, based on effects of P450c21 deficiency and maternal estriol concentrations, indicate that the fetal adrenal cortex produces cortisol and DHEA-S early in gestation (6-12 weeks). However, controversy exists as to whether cortisol is produced de novo or derived from the metabolism of progesterone, as data regarding the expression of 3 beta HSD in the fetal adrenal cortex early in gestation are conflicting. During the 1960s, Liggins and colleagues demonstrated that in the sheep, cortisol secreted by the fetal adrenal cortex late in gestation regulates maturation of the fetus and initiates the cascade of events leading to parturition. Those pioneering discoveries provided insight into the mechanism underlying the timing of parturition and therefore were of particular interest to obstetricians and perinatologists confronted with the problems of preterm labor. However, although cortisol emanating from the fetal adrenal cortex promotes fetal maturation in primates as it does in sheep, its role in the regulation of primate parturition, unlike that in sheep

Development and regulation of growth and differentiated function in human and subhuman primate fetal gonads.

We have attempted to summarize the research on primate fetal gonadal development that has occurred over the past three decades. Many similarities exist between fetal gonadal development in human and subhuman primates; therefore, comparisons and analogies between these species can be made. Fetal gonadal development is a complex process dependent on timely maturation and differentiation of several cell types with different functions. Adequate development is important for normal sexual development and intact adult fertility potential as well as for intrauterine priming of neural centers in the central nervous system. While the fetal primate testis is active in steroidogenesis, the fetal ovary seems to be quiescent throughout most of gestation, although some ovarian steroidogenic enzymes have been demonstrated. Growth and development of both gonads are controlled during late gestation at least in part by pituitary hormones, while earlier in gestation other yet undefined regulators (placental, intragonadal) likely also are active. The main goal of this review was to demonstrate that gonadal growth and differentiation, both in males and females, is regulated by endocrine factors as well as by intragonadal, autocrine/paracrine agents. Although many parts of the puzzle are still missing it is probable that, similar to fetal development of other endocrine tissues and to events in postnatal gonads, these local regulators have important functions. Currently, primate fetal gonadal research is lacking in at least two key aspects: 1) the definition of paracrine and autocrine nonsteroidal factors that are involved in the regulation of gonadal growth and differentiation in vitro; and 2) in vivo studies in subhuman primates that might better help to clarify the biological roles of the multiple extra- and intragonadal hormones and their complex interactions. To date, the regulation of gonadal steroidogenesis has been investigated more thoroughly than the regulation of gonadal growth. Most of our knowledge stems from observations of gonadal development in anencephalics or subhuman primates after pituitary ablation. Because of the constraints of small organ size and limitation of material, studies of fetal primate gonadal development have been limited. Given such limitations, new molecular biological techniques, including polymerase chain reaction and in situ hybridization, may provide the means of addressing these questions. Further, because of these limitations, sensitive cell separation techniques need to be developed to achieve enriched primary gonadal cell cultures from individual gonads.

Hypophysiotropic and neuromodulatory regulation of adrenocorticotropin in the human fetal pituitary gland.

Synthetic human corticotropin-releasing factor (hCRF) stimulated ACTH secretion by human fetal pituitaries in superfusion and dispersed human fetal pituitary cells cultured on an extracellular matrix in static incubation from 14 to 23 wk gestational age. The action of hCRF in vitro was potentiated by arginine vasopressin (AVP) at all ages studied. 8-Br-cAMP induced a response similar to hCRF. The AVP effect on ACTH was synergistic with both CRF and 8-Br-cAMP. hCRF-mediated secretion of ACTH was noncompetitively inhibited by 24-h pretreatment, or by 3-h concomitant treatment, with dexamethasone. Neither oxytocin, catecholamines, prostaglandins, nor indomethacin exerted significant effects on ACTH secretion, either alone or in combination with hCRF or AVP during the gestational ages studied. These results support a physiologic role for CRF in the regulation of secretion by corticotropic cells as early as 14 wk gestation, by which time corticotropes and ability to secrete ACTH have been demonstrated.

Activin and inhibin in the human adrenal gland. Regulation and differential effects in fetal and adult cells.

Recent experimental data have revealed that activins and inhibins exert pivotal effects on development. As part of our studies on growth and differentiation of the human fetal adrenal gland, we examined the subunit localization, as well as the mitogenic and steroidogenic actions of activin and inhibin in human fetal and adult adrenals. All three activin and inhibin subunit proteins (alpha, beta A, and beta B) were detected in the fetal and adult adrenal cortex. Immunoreactive activin-A dimer was demonstrated in midgestation fetal and neonatal adrenals. ACTH1-24-stimulated fetal adrenal cell expression of alpha and beta A subunit messenger RNA. In addition, ACTH elicited a rise in levels of immunoreactive alpha subunit secreted by fetal and adult adrenal cells. Human recombinant activin-A inhibited mitogenesis and enhanced ACTH-stimulated cortisol secretion by cultured fetal zone cells, but not definitive zone or adult adrenal cells. Recombinant inhibin-A had no apparent mitogenic or steroidogenic effects. Thus, activin selectively suppressed fetal zone proliferation and enhanced the ACTH-induced shift in the cortisol/dehydroepiandrosterone sulfate ratio of fetal zone steroid production. These data indicate that activin-A may be an autocrine or paracrine factor regulated by ACTH, involved in modulating growth and differentiated function of the human fetal adrenal gland.

Fetal tissue can synthesize a placental hormone. Evidence for chorionic gonadotropin beta-subunit synthesis by human fetal kidney.

Metabolically active tissues from second trimester human fetuses were examined for their ability to synthesize the placental hormones chorionic gonadotropin and chorionic somatomammotropin. During short-term incubation studies both placenta and fetal kidney were found to synthesize and secrete the beta-subunit of chorionic gonadotropin, whereas its synthesis was not observed in fetal liver, lung or muscle. In addition, chorionic somatomammotropin synthesis and secretion was demonstrated with placental tissue but could not be detected in any of the fetal tissues examined. These observations constitute the first evidence that the genome of a fetal tissue directs the synthesis of what is considered a placental hormone.

Activin-A as an intraovarian modulator: actions, localization, and regulation of the intact dimer in human ovarian cells.

The actions, localization, and regulation of activin in the human ovary are unknown. Therefore, the aims of this study were (a) to define the effects of recombinant activin-A and its structural homologue, inhibin-A, on mitogenesis and steroidogenesis (progesterone secretion and aromatase activity) in human preovulatory follicular cells; (b) to localize the activin-A dimer in the human ovary by immunohistochemistry; and (c) to examine regulation of intracellular activin-A production in cultured human follicular cells. In addition to stimulating mitogenic activity, activin-A causes a dose- and time-dependent inhibition of basal and gonadotropin-stimulated progesterone secretion and aromatase activity in human luteinizing follicular cells on day 2 and day 4 of culture. Inhibin-A exerts no effects on mitogenesis, basal or gonadotropin-stimulated progesterone secretion and aromatase activity, and does not alter effects observed with activin-A alone. Immunostaining for dimeric activin-A occurs in granulosa and cumulus cells of human ovarian follicles and in granulosa-lutein cells of the human corpus luteum. cAMP, and to a lesser degree human chorionic gonadotropin and follicle-stimulating hormone, but not inhibin-A, activin-A, or phorbol 12-myristate 13-acetate, increased the immunostaining for activin-A in cultured granulosa cells. These results indicate that activin-A may function as an autocrine or paracrine regulator of follicular function in the human ovary.

Lysophosphatidic acid induction of vascular endothelial growth factor expression in human ovarian cancer cells.

BACKGROUND: Lysophosphatidic acid (LPA) stimulates ovarian tumor growth at concentrations present in ascitic fluid. Vascular endothelial growth factor (VEGF) stimulates angiogenesis and plays a pivotal role in the formation of ovarian cancer-associated ascites. We examined whether LPA promotes ovarian tumor growth by increasing angiogenesis via VEGF. METHODS: VEGF expression was examined in a simian virus 40 T-antigen-immortalized ovarian surface epithelial cell line (IOSE-29) and in ovarian cancer cell lines (OVCAR-3, SKOV-3, and CAOV-3) treated with LPA. VEGF promoter activity was measured in OVCAR-3 cells after transfection or cotransfection with c-Fos and c-Jun, components of AP1 transcription factor, potential binding sites for which are present in the VEGF promoter. The expression of the LPA receptors Edg2 and Edg4 was also assessed. All statistical tests were two-sided. RESULTS: LPA treatment increased steady-state VEGF messenger RNA (mRNA) levels in OVCAR-3 cells in a time- and dose-dependent fashion and stimulated VEGF promoter activity without prolonging mRNA half-life in these cells, but LPA had little effect on IOSE-29 cells. Forced overexpression of c-Jun and c-Fos in OVCAR-3 cells stimulated VEGF promoter activity fourfold. LPA also elevated VEGF protein levels by 1.5-fold in SKOV-3 cells (P =.0148), 1.9-fold in CAOV-3 cells (P<.001), and threefold in OVCAR-3 cells (P<.0001). Both Edg2 and Edg4 were detected in ovarian cancer cells; however, only Edg2 was present in normal ovarian surface epithelial cells and IOSE-29 cells. CONCLUSIONS: LPA stimulates ovarian tumor growth, at least in part, via induction of VEGF expression through transcriptional activation. However, this LPA response is not evident in normal ovarian surface epithelial cells. Our data suggest that Edg4, but not Edg2, plays a role in LPA stimulation of ovarian tumor growth.

Distinctive expression and functions of the type 4 endothelial differentiation gene-encoded G protein-coupled receptor for lysophosphatidic acid in ovarian cancer.

Endothelial differentiation gene (edg)-encoded G protein-coupled receptors (Edg Rs)-1, -3, and -5 bind sphingosine 1-phosphate (S1P), and Edg-2 and -4 bind lysophosphatidic acid (LPA). Edg Rs transduce signals from LPA and S1P that stimulate ras- and rho-dependent cellular proliferation, enhance cellular survival, and suppress apoptosis. That high levels of LPA in plasma and ascitic fluid of patients with ovarian cancer correlate with widespread invasion suggested the importance of investigating expression and functions of Edg Rs in ovarian cancer cells (OCCs) as compared with nonmalignant ovarian surface epithelial cells (OSEs). Analyses of Edg Rs by semiquantitative reverse transcription-PCR, a radioactively quantified variant of PCR, and Western blots developed with monoclonal antibodies showed prominent expression of Edg-4 R in primary cultures and established lines of OCCs but none in OSEs. In contrast, levels of Edg-2, -3, and -5 were higher in OSEs than OCCs. LPA stimulated proliferation and signaled a serum response element-luciferase reporter of immediate-early gene activation in OCCs but not OSEs, whereas S1P evoked similar responses in both OSEs and OCCs. Pharmacological inhibitors of Edg R signaling suppressed OCC responses to LPA. A combination of monoclonal anti-Edg-4 R antibody and phorbol myristate acetate, which were inactive separately, evoked proliferative and serum response element-luciferase responses of OCCs but not OSEs. Thus the Edg-4 R may represent a distinctive marker of OCC that transduces growth-promoting signals from the high local concentrations of LPA characteristic of aggressive ovarian cancer.

In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002).

Phosphatidylinositol 3-kinase (PI3-K) induces mitogenesis, cell growth, and cell transformation. Amplification of the gene encoding the P110alpha subunit likely is an important event in ovarian cancer progression, and PI3-K inhibitors are possible therapeutic agents for this disease. We evaluated effects of LY294002, a potent inhibitor of PI3-K, on growth of ovarian carcinoma in vivo and in vitro, and on ascites formation in vivo. Athymic mice were inoculated i.p. with the ovarian cancer cell line OVCAR-3. Seven days after inoculation, mice were treated with or without LY294002 (100 mg/kg of body weight) for 3 weeks. Body weight and abdominal circumference were measured twice weekly. At the end of the experiment, mice were sacrificed, ascites volume was measured, and tumors were excised. Mean tumor burden in the LY294002-treated group was reduced by approximately 65% versus controls. Virtually no ascites developed in the treatment group; mean volume of ascites in controls was 3.3 +/- 0.38 ml. OVCAR-3 cells also were cultured in vitro without and with LY294002 (1, 5, and 10 microM) for 24 h. The number of cells in 1, 5, and 10 microM LY294002-treated wells was reduced by 27, 56, and 75%, respectively, versus controls. In vivo and in vitro morphological studies demonstrated that LY294002 induced marked nuclear pyknosis and diminished cytoplasmic volume in the tumor cells, confirmed as apoptosis. Thus, LY294002 significantly inhibits growth and ascites formation of ovarian carcinoma in vivo and markedly inhibits ovarian cancer cell proliferation in vitro, suggesting an important role of PI3-K inhibitors as a potentially useful treatment for women with ovarian carcinoma.

Functional maturation of the primate fetal adrenal in vivo: 3. Specific zonal localization and developmental regulation of CYP21A2 (P450c21) and CYP11B1/CYP11B2 (P450c11/aldosterone synthase) lead to integrated concept of zonal and temporal steroid biosynthesis.

Previous studies in the primate fetal adrenal gland have indicated that the gland is comprised of three functional zones: 1) the inner fetal zone (FZ), which has the enzymes necessary for dehydroepiandrosterone sulfate (DHEAS) production beginning early in gestation; 2) the transitional zone (TZ), which possesses enzymes necessary for cortisol production; and 3) the outer, definitive zone (DZ), which appears to function as a reservoir of progenitor cells that may populate the remainder of the gland and does not acquire a steroidogenic phenotype with the capacity to produce mineralocorticoids until near term. The enzymes CYP21A2 (P450 21 hydroxylase, or P450c21), CYP11B1 (11beta hydroxylase or P450c11) and CYP11B2 (aldosterone synthase) are necessary for glucocorticoid and mineralocorticoid synthesis but have not been localized previously in an ontogenic manner in the primate fetal adrenal gland. Therefore, we used immunocytochemistry (ICC) to assess specific zonal localization and developmental regulation of CYP21A2 and CYP11B1/CYP11B2 in the human (13-24 weeks' gestation) and rhesus monkey (109 d-term) fetal adrenal gland. In the fetal rhesus, ICC was performed with and without metyrapone administration to the fetus to assess the effects of endogenously increased fetal ACTH. In the human fetal adrenal, CYP21A2 immunoreactivity (IR) was present in only a few isolated cells in the DZ but was detectable in almost all cells in the TZ and FZ. In the fetal rhesus, CYP21A2-IR was present in cells throughout the DZ and TZ and, to a lesser degree, in the FZ. Staining intensity increased with advancing gestational age and was up-regulated in the DZ and TZ, but not the FZ, of the metyrapone-treated fetuses. In the human fetal adrenal gland, CYP11B1/CYP11B2-IR was absent in the DZ but present in the TZ and FZ. In the fetal rhesus monkey adrenal, CYP11B1/CYP11B2-IR was present in all cells of the TZ and FZ but was absent from the DZ until near term. After metyrapone, CYP11B1/CYP11B2-IR was induced in the DZ and was up-regulated in the TZ and FZ. Taken together, these data indicate that in the primate fetal adrenal gland, the FZ has the capacity to synthesize DHEA and DHEAS beginning early in development, the TZ has the capacity to synthesize cortisol after midgestation, and the DZ has the capacity to synthesize mineralocorticoids, but not until near term. The spatial localization of steroid metabolizing enzymes and steroid products in the human and rhesus monkey fetal adrenal suggests analogies of the three functional zones of the fetus (DZ, TZ, and FZ) to their adult counterparts (zona glomerulosa, zona fasciculata, and zona reticularis) and their steroid products (mineralocorticoids, glucocorticoids and androgens, respectively), although the reason for the presence of CYP11B1/CYP11B2- and CYP21A2-IR in the FZ remains to be elucidated.

Cortisol-cortisone interrelationship in the late gestation rhesus monkey fetus in utero.

The metabolic interrelationship between cortisol (F) and cortisone (E) was studied in four long term catheterized rhesus monkey fetuses in utero during the last third of gestation. The MCR of E (50.8 +/- 5.4 liters/day) was greater than that of F (22.4 +/- 2.1, P less than 0.005) as was the plasma concentration (187.9 +/- 5.0 vs 86.1 +/- 2.5 ng/ml, P less than 0.001). The production rate of E (9.6 +/- 1.4 mg/day) was several-fold greater than for F (1.9 +/- 0.2, P less than 0.005). Of all fetal F, 79.5 +/- 7.0% was metabolized to E, and 43.4 +/- 3.9% originated from E within the fetal circulation. A significant mass of F was infused in these experiments because of the low specific activity of [14C]F. Nevertheless, the fetus was able to maintain F concentrations in the normal range. The MCR of F was similar to that which we previously found using trace amounts of [3H]F. This indicates that the fetus regulates the amount of F in the fetal compartment, probably by decreasing fetal; adrenal secretion rate. We conclude that F in the primate fetus is extensively oxidized to E. We conclude also that E is produced and metabolized much more extensively than is F. Reduction of E back to F could be an important source of fetal F, and increasing activity of this pathway, if present, could contribute to the increase in fetal F levels observed in late gestation in the primate.

Sequential patterns of circulating LH and FSH in female sheep during the early postnatal period: effect of gonadectomy.

To determine the basis for the marked daily variability in circulating luteinizing hormone (LH) in the female lamb shortly after birth and to determine whether circulating follicle-stimulating hormone (FSH) exhibits similar variability during this period of life, serum levels of both gonadotropins were monitored frequently (20-min intervals) over a 3- or 6-hr period each week for the first 9 weeks of life. Similar measurements of circulating LH and FSH were made in lambs ovariectomized at 2 weeks of age to assess whether the ovary influences the secretion of these gonadotropins. Pulsatile release of LH, but not FSH, was observed in both groups of lambs. Although mean concentrations of circulating FSH in intact female lambs were similar to basal levels of the cyclic adult, mean concentrations of serum LH were much higher. Because of the discontinuous release of LH in intact lambs after 4-5 weeks of age, patterns of LH were often indistinguishable from those of the long-term ovariectomized adult. At 9 weeks of age the frequency of episodic LH release in intact lambs ranged from 0.33-1.1 pulses/hr. Castration at 2 weeks of age produced a concomitant, but delayed, rise in mean serum LH and FSH beginning 4-5 weeks later. By 9 weeks of age serum LH, but not FSH, had attained levels comparable to those of the castrated adult, and the mean frequency of LH release (about hourly) was similar for both groups. The results indicate that by 9 weeks of age the ovary of the lamb influences the secretion of LH and FSH. Because in the 9-week-old intact female lamb episodic release of LH occurs producing high (castrate) levels of this gonadotropin while circulating concentrations of FSH remain stable and within basal levels of the adult cycling female, it is suggested that acute concomitant release of LH and FSH does not occur at this age and that separate negative feedback loops for the control of LH and FSH secretion may exist during early postnatal life.

Prolactin concentrations in the monkey fetus during the last third of gestation.

PRL concentrations were measured in cord plasma obtained at hysterotomy from 26 rhesus monkey fetuses between 111--170 days gestational age (GA). Mean PRL concentrations increased significantly from 23.7 +/- 10.1 (X +/- SE) ng/ml at 121--130 days GA to 126.9 +/- 16.9 ng/ml at 161--170 days GA. A similar significant increase in PRL with age also was observed in samples obtained from 16 fetuses chronically catheterized in utero between 130--155 days GA. Mean PRL levels were 34 +/- 3.2 ng/ml at 131--140 days GA and rose to 82 +/- 9.7 at 150--155 days GA. No difference in PRL concentrations was found between cord blood samples and fetal peripheral blood samples at the ages studied. Maternal PRL levels did not change in samples obtained from chronically catheterized, chair-restrained mothers between 130--155 days GA. A tendency toward an increase in maternal PRL with advancing gestational age was observed in samples collected after hysterotomy. These data indicate that the fetal rhesus monkey demonstrates an increase in plasma PRL similar to that in the human, suggesting a possible physiological role for this hormone in the primate fetus late in gestation.

Sequential patterns of circulating luteinizing hormone and follicle-stimulating hormone in female sheep from early postnatal life through the first estrous cycles.

Patterns of circulating LH and FSH were examined during several 6-h periods in developing female sheep from 3 weeks of age through the first estrous cycles (30--40 weeks of age) and early pregnancy. With the onset of pulsatile LH secretion, beginning 11 weeks after birth, circulating LH increased to levels 2- to 5-fold greater than those observed in the adult. Thereafter, high and variable levels of circulating LH persisted through young adulthood when ovarian cyclicity including ovulation became manifest. Although prior to the initial estrus changes in frequency of LH release (number of LH discharges/6-h period) appeared to occur randomly from week to week, after first estrus, changes in frequency became predictable. During the early (day 1) and late (day 15) luteal phases of the first estrous cycles, when circulating progesterone was low (less than 0.6 ng/ml), the frequency of LH release was increased (5--7 discharges/6 h) while during the mid-luteal phase (day 7--12), when circulating progesterone was high (2--4 ng/ml), the frequency was diminished (0--2 discharges/6 h). Massive and sustained discharges of LH which resembled preovulatory surges were observed only shortly before first estrus. In contrast to the pulsatile release pattern of LH, concentrations of circulating FSH within the 6-h periods were relatively constant and within the range found in the adult. These findings lead to the following conclusions: a) changes in negative feedback control are not directly responsible for the onset of ovarian cyclicity in the sheep as indicated by the lack of differences in mean concentrations of circulating LH and FSH before and after first ovulation; b) the onset of the surge mode of gonadotropin secretion occurs only shortly before first ovulation; and c) progesterone may play an inhibitory role in regulating tonic LH secretion during the first estrous cycles.

Isolation and characterization of three forms of joining peptide from adult human pituitaries: lack of adrenal androgen-stimulating activity.

Three structural variants of the joining peptide (JP) fragment of POMC have been purified from human pituitaries. Ion exchange and reverse phase tissue extraction procedures were combined with reverse phase HPLC to achieve complete purification of each form of JP. Fragments resulting from tryptic hydrolysis of each form were characterized by amino acid analysis and fast atom bombardment mass spectrometry. The predominant form of human JP, accounting for about 50% of the total purified, was found to be conjugated to glutathione through the lone cysteine residue at position 9. The other two variants were identified as human JP with a free cysteine residue and human JP dimer and accounted for 35% and 15%, respectively, of the total purified. Recently, human JP-(1-18) has been suggested as having adrenal androgen-stimulating activity. None of the three JP variants or their respective 1-20 amino-terminal fragments resulting from tryptic hydrolysis showed any ability to promote the secretion of dehydroepiandrosterone sulfate by cultured human fetal adrenal cells. Similarly, no potentiation of the stimulatory effects of ACTH-(1-39) was observed. The three variants of human JP as well as JP purified from rat, porcine, and bovine pituitaries were tested for their ability to stimulate androgenic steroids from dispersed fetal rabbit adrenal cells. None showed any significant biological activity either in stimulating steroid secretion or in potentiating the action of ACTH-(1-39).

Functional maturation of the primate fetal adrenal in vivo: I. Role of insulin-like growth factors (IGFs), IGF-I receptor, and IGF binding proteins in growth regulation.

The rapid growth of the primate fetal adrenal from midgestation until term is regulated by ACTH secreted by the fetal pituitary. Previous studies suggest that the trophic actions of ACTH are mediated by insulin-like growth factor II (IGF-II) synthesized by fetal adrenal cortical cells. To characterize further the role of IGF-II in the regulation of fetal adrenal growth, we investigated the expression of the messenger RNAs (mRNAs) encoding IGF-I, IGF-II, IGF-I receptor (IGF-IR) and IGF binding protein (IGFBP) 1-6 in the fetal rhesus monkey adrenal in vivo from 109 days of gestation until term (165 +/- 5 days) using in situ hybridization. To assess the role of ACTH in the regulation of expression of the IGF system in vivo, we administered metyrapone (3-7 days) to late gestation fetal rhesus monkeys (n = 4) in utero to increase fetal pituitary ACTH secretion. IGF-II mRNA was abundant in the definitive, transitional and fetal zones of the adrenal cortex from 109 days until term. IGF-IR mRNA was expressed in the definitive, transitional and fetal zones and decreased to nondetectable levels at term. IGFBP-2 and IGFBP-6 mRNAs were expressed in the definitive, transitional, and fetal zones, whereas IGFBP-1, -3, -4, and -5 were not detected in adrenal cells. The effects of increasing ACTH secretion on the growth of the specific zones of the adrenal were determined using morphometric techniques. Metyrapone treatment approximately doubled adrenal weight, which was due to an increase in the area of the definitive, transitional, and fetal zones with decreased cell density of the definitive, transitional, and fetal zones compared with controls and not due to a change in total cell number. Therefore, the increase in adrenal weight after metyrapone treatment was due to hypertrophy of the three cortical zones; there was no effect on adrenal medullary growth. The relative abundance of the mRNAs encoding IGF-II and the IGF-IR was increased after metyrapone treatment, whereas the localization and relative abundance of IGFBP 1-6 mRNAs were not altered by metyrapone treatment. We conclude that the ontogenetic increase in adrenal growth may be regulated, at least in part, by locally synthesized IGF-II, and the cessation of adrenal growth that occurs at term may be mediated by the decrease in the IGF-IR. The adrenal cortical expression of IGFBP-2 and IGFBP-6 suggests that these IGFBPs may modulate the IGF-IGF-IR interaction. Metyrapone treatment, which likely increased fetal pituitary ACTH secretion, causes a coordinated increase in expression of IGF-II and IGF-IR in fetal adrenal cortical cells, which may be an important mechanism of regulation of fetal adrenal cortical growth.

Functional maturation of the primate fetal adrenal in vivo. II. Ontogeny of corticosteroid synthesis is dependent upon specific zonal expression of 3 beta-hydroxysteroid dehydrogenase/isomerase.

Cortisol, produced by the primate fetal adrenal, regulates the maturation of organ systems necessary for extrauterine life. During most of primate pregnancy, however, the fetal adrenal lacks the enzyme 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD), which is essential for cortisol synthesis. Therefore, we used immunohistochemistry and in situ hybridization techniques to investigate the developmental expression of 3 beta HSD in the fetal rhesus monkey adrenal from 109 days' gestation until term (165 +/- 5 days) and assessed the role of ACTH in the induction of its expression and localization. We also examined whether ACTH regulates the expression of two other steroidogenic enzymes, cytochrome P450 cholesterol side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase, 17/20-lyase (P450c17), in the fetal rhesus monkey adrenal. To stimulate ACTH secretion from the fetal pituitary in vivo, we administered metyrapone to late gestation fetal rhesus monkeys for 3-7 days. Adrenals were collected from untreated fetuses at 109-125 days (n = 5), 130-148 days (n = 7), 155-172 days (n = 4), and after metyrapone treatment at 135-137 days (n = 4). The cortical width and total amount of 3 beta HSD staining were measured using an image analysis system. 3 beta HSD was localized primarily in the definitive zone cells of the adrenal from fetuses between 109-148 days, whereas at term (155-172 days), 3 beta HSD was localized in both definitive and transitional zone cells. The cortical width and total amount of 3 beta HSD staining in the adrenal increased significantly (P < 0.05) between 148 days (137 +/- 14 microns and 3,689 +/- 522 grains) and 155 days (315 +/- 61 microns and 7,321 +/- 2,008 grains). Interestingly, in metyrapone-treated fetuses at 135-137 days, 3 beta HSD messenger RNA (mRNA) and protein were localized extensively in both the definitive and transitional zones, a pattern seen only in term fetal adrenals in untreated animals. In addition, metyrapone treatment significantly (P < 0.05) increased cortical width (386 +/- 95 microns) and total 3 beta HSD immunostaining (29,063 +/- 13,692 grains) compared with age-matched controls. In contrast to 3 beta HSD, P450scc mRNA was detected in the definitive, transitional, and fetal zones, and its expression was not altered after metyrapone treatment. P450c17 mRNA was detected in the transitional and fetal zones, and the relative abundance was greater in the transitional zone. The relative abundance of P450c17 mRNA was increased in the fetal zone after metyrapone treatment. In summary, at term or after metyrapone treatment, expression of 3 beta HSD is induced in the transitional zone of the fetal rhesus monkey adrenal gland, an indication of functional maturation of the primate adrenal cortex. These data suggest that the ontogenetic increase in fetal pituitary ACTH secretion plays an important role in the induction of 3 beta HSD expression in the transitional zone.

Stimulation of testosterone production in vivo and in vitro in the male rhesus monkey fetus in late gestation.

To assess intrauterine fetal testicular function, the carotid or femoral vessels of rhesus monkey fetuses, 129-145 days gestational age, were catheterized following hysterotomy of the mother. The fetus was returned to the uterus, the catheters were exteriorized through the mother's vagina and the pregnancy was allowed to continue. In this chronic preparation, basal levels of testosterone (measured with an RIA with 65% cross-reactivity with 5 alpha-dihydrotestosterone) in male fetal serum were 0.85 +/- 0.29 (SD) ng/ml. Administration of a 10 or 100 IU intra-arterial bolus of hCG into the fetal circulation stimulated in increase in fetal serum testosterone levels of 70 and 630%, respectively. Other fetuses were challenged with bolus infusions of 10 and 50 micrograms of synthetic gonadotropin releasing hormone (GnRH). The lower dose caused an increase in serum testosterone concentrations in only one of four fetuses, while the higher dose resulted in a positive response in all three experiments performed. With this dose, the mean increase in circulating testosterone concentration after 1 h was 105%. In vitro, specific binding of iodinated hCG was demonstrated in testicular homogenates from rhesus fetuses near term and hCG stimulated testosterone biosynthesis in testicular minces. Maximal stimulation was achieved at hCG concentrations between 5 and 50 ng/ml. The data indicate that the testes of fetal rhesus monkeys during late gestation are capable of androgen biosynthesis and can bind and respond to gonadotropin stimulation. Furthermore, the pituitary-gonadal axis in the fetal male monkey is capable of responding to GnRH stimulation at this stage of gestation.