The biochemical basis for increased testosterone production in theca cells propagated from patients with polycystic ovary syndrome.

Ovarian theca cells propagated from patients with polycystic ovary syndrome (PCOS) convert steroid precursors into T more efficiently than normal theca cells. To identify the basis for increased T production by PCOS theca cells, we examined type I-V 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isoform expression in long-term cultures of theca and granulosa cells isolated from normal and PCOS ovaries. RT-PCR analysis demonstrated that theca cells express type V 17 beta HSD a member of the aldo-keto reductase (AKR) superfamily (17 beta HSDV, AKR1C3), whereas expression of type I, II, and IV 17 beta HSD, which are members of the short-chain dehydrogenase/reductase superfamily, was limited to granulosa cells. Type III 17 beta HSD, the testicular isoform, was not detected in either granulosa or theca cells. Northern and real-time PCR analyses demonstrated that 17 beta HSDV transcripts were not significantly increased in PCOS theca cells compared with normal theca cells. RT-PCR analysis revealed that theca cells also express another AKR, 20 alpha HSD (AKR1C1). Both basal and forskolin-stimulated 20 alpha HSD mRNA levels were increased in PCOS theca cells compared with normal theca cells. However, 17 beta HSD enzyme activity per theca cell was not significantly increased in PCOS, suggesting that neither AKR1C3 nor AKR1C1 contributes to the formation of T in this condition. In contrast, 17 alpha-hydroxylase/C17,20 lyase and 3 beta HSD enzyme activities were elevated in PCOS theca cells, driving increased production of T precursors. These findings indicate that 1) increased T production in PCOS theca cells does not result from dysregulation of "androgenic" 17 beta HSD activity or altered expression of AKRs that may express 17 beta HSD activity; and 2) increased synthesis of T precursors is the primary factor driving enhanced T secretion in PCOS.

Pediatric implications in bioterrorism part II: postexposure diagnosis and treatment.

Bioterrorism is an old tactic used in warfare. The use of fatal diseases or man-made poisons to subdue others has not been limited to wartime. Trauma personnel should be aware of commonly used agents, the signs and symptoms of their exposure, and recommended treatment. Children can be victims of terrorists' acts and need special attention because of their unique response to exposure, varying dosages of medications, and lack of active immunity.

Limited cooperation between peroxisome proliferator-activated receptors and retinoid X receptor agonists in sebocyte growth and development.

We have found that sebaceous epithelial cell (sebocyte) differentiation is induced by cognate ligand-agonists of either peroxisome proliferator-activated receptors (PPARs) or retinoid X receptors (RXRs). In this study, we tested the hypothesis that PPAR-RXR cooperation is used in sebocytes as was reported to occur in gene transfection systems and liposarcoma cells through PPAR-RXR heterodimerization. PPAR agonists at maximally effective concentrations were tested in combination with a specific RXR agonist (the rexinoid CD2809) at doses ranging from submaximal to maximal in a primary rat preputial cell monolayer culture system. We evaluated ligand-agonists of PPARalpha (WY-14643 = WY), PPARgamma (troglitazone = TRO), and PPARdelta,alpha (carbaprostacyclin = cPGI2). Cell differentiation was determined by analysis of lipid staining and proliferation by cell counting. The RXR agonist induced a more diffuse and granular pattern of lipid staining throughout colonies than did PPAR agonists. The PPAR ligands WY, TRO, and cPGI2 induced 37, 35, and 59% lipid-forming colonies (LFCs), respectively (P < 0.05 vs controls, which averaged 19%). Low-dose rexinoid (10(-8) M) alone exerted no significant effect but amplified the effect of cPGI2 (P < 0.05). Middose rexinoid (10(-7) M), which alone induced about 40% LFCs, had an additive effect on differentiation with WY, TRO, and cPGI2 (71, 48, and 83% LFCs respectively, P < 0.05 vs each agonist alone). Proliferation was enhanced significantly by either rexinoid or cPGI2, but there was no change in growth when the two were added together. The greater effectiveness of cPGI2 than the other PPAR agonists may be explained by the predominance of PPARdelta gene expression in cultured sebocytes, as demonstrated by RNase protection assay. These studies demonstrate that a submaximal dose of RXR agonist augmented the stimulation of sebocyte differentiation by PPAR agonists, as expected from PPAR-RXR heterodimerization. However, the evidence for PPAR-RXR cooperativity is limited. The pattern of lipid staining is compatible with an independent effect of rexinoid on sebocyte differentiation. Furthermore, since there is no enhancement of the growth-promoting effects of RXR agonist and cPGI2 when they are combined, this effect also does not appear to be mediated by PPAR-RXR interaction.

Polycystic ovary syndrome and insulin-resistant hyperinsulinemia.

In a broad sense, polycystic ovary syndrome (PCOS) may be considered to be synonymous with chronic unexplained hyperandrogenemia, which accounts for approximately 95% of hyperandrogenism in women. PCOS comprises a mosaic of classic and nonclassic forms, which may ultimately prove to have distinct genetic determinants. The hyperandrogenism appears to arise from generalized abnormal regulation (dysregulation) of steroidogenesis. This dysregulation seems to result from imbalance among the various extrinsic and intrinsic factors involved in the modulation of trophic hormone action. Hyperinsulinemia seems to be an important extrinsic factor in many cases of PCOS; it results from resistance to the effects of insulin on glucose metabolism. The elevation in insulin levels may precipitate hyperandrogenemia in genetically vulnerable individuals by unmasking latent abnormalities in the regulation of steroidogenesis. One of these may be a polycystic ovary gene that is expressed in the male as pattern baldness. Insulin also seems to be one of many factors that interact with androgen to regulate pilosebaceous unit development. Treatment of PCOS with antidiabetic insulin-lowering agents may improve ovarian function and androgen levels; it remains to be determined whether it will benefit the pilosebaceous unit manifestations of the disorder.

Diagnosis of the polycystic ovary syndrome in adolescence: comparison of adolescent and adult hyperandrogenism.

We performed gonadotropin releasing hormone agonist (GnRHag) tests on 23 consecutive hyperandrogenic girls 9.9-17.5 years of age who were referred to our pediatric endocrinology clinic with symptoms suggestive of PCOS. They were compared to contemporaneously studied groups of adult normal and hyperandrogenic women. We found that hyperandrogenic adolescents had clinical and endocrine features similar to those of hyperandrogenic adults. However, there were some noteworthy unique features of adolescent hyperandrogenism, such as presentation in mid-childhood with premature pubarche and the occasional diagnosis before the age of 10 years. Some differences between adolescents and adults were statistically significant, for example, pelvic ultrasonography was not as helpful in the diagnosis of FOH as it is in adults. Nevertheless, a number of questions about the development of the ovarian dysfunction remain to be answered. For example, we are unable to diagnose ovarian dysfunction before puberty or in early puberty, and the relationship of "physiologic adolescent anovulation" to PCOS remains to be defined.

Role of hormones in pilosebaceous unit development.

Androgens are required for sexual hair and sebaceous gland development. However, pilosebaceous unit (PSU) growth and differentiation require the interaction of androgen with numerous other biological factors. The pattern of PSU responsiveness to androgen is determined in the embryo. Hair follicle growth involves close reciprocal epithelial-stromal interactions that recapitulate ontogeny; these interactions are necessary for optimal hair growth in culture. Peroxisome proliferator-activated receptors (PPARs) and retinoids have recently been found to specifically affect sebaceous cell growth and differentiation. Many other hormones such as GH, insulin-like growth factors, insulin, glucocorticoids, estrogen, and thyroid hormone play important roles in PSU growth and development. The biological and endocrinological basis of PSU development and the hormonal treatment of the PSU disorders hirsutism, acne vulgaris, and pattern alopecia are reviewed. Improved understanding of the multiplicity of factors involved in normal PSU growth and differentiation will be necessary to provide optimal treatment approaches for these disorders.

Expression of 17 beta-hydroxysteroid dehydrogenase type 5 in human ovary: a pilot study.

OBJECTIVE: Conversion of androstenedione to testosterone, the most potent androgen secreted by the ovary, is carried out by androgenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity. The molecular basis for this is unclear. We tested the hypothesis that type 5 17 beta-HSD (17 beta-HSD5) is responsible for testosterone formation from androstenedione in the human ovary. METHODS: We used primers specific for each type of 17 beta-HSD to identify quantitatively and directly sequence the polymerase chain reaction products of a human ovary library. RESULTS: 17 beta-HSD1, 17 beta-HSD4, and 17 beta-HSD5 were detected in the library lysate, but not 17 beta-HSD2 or 17 beta-HSD3. 17 beta-HSD5 was the predominant androgenic form of 17 beta-HSD expressed in human ovary. CONCLUSION: These data suggest that 17 beta-HSD5 may play a major role in testosterone biosynthesis by the human ovary. Further investigation of the regulation of 17 beta-HSD5 gene expression is warranted with regard to ovarian testosterone secretion in normal and abnormal states of ovarian function, such as polycystic ovary syndrome.

The role of specific retinoid receptors in sebocyte growth and differentiation in culture.

Retinoic acid derivatives (retinoids) exert their pleiotropic effects on cell development through specific nuclear receptors, the retinoic acid receptors and retinoid X receptors. Despite recent progress in understanding the cellular and molecular mechanisms of retinoid activity, it is unknown which of the retinoid receptor pathways are involved in the specific processes of sebocyte growth and development. In this study, we investigated the roles of specific retinoid receptors in sebocyte growth and differentiation, by testing the effects of selective retinoic acid receptor and retinoid X receptor ligands at concentrations between 10-10 M and 10-6 M in a primary rat preputial cell monolayer culture system. Cell growth was determined by number of cells and colonies, and cell differentiation by analysis of lipid-forming colonies. All-trans retinoic acid and selective retinoic acid receptor agonists (CD271 = adapalene, an RAR-beta,gamma agonist; CD2043 = retinoic acid receptor pan-agonist; and CD336 = Am580, an RAR-alpha agonist) caused significant decreases in numbers of cells, colonies, and lipid-forming colonies, but with an exception at high doses of all-trans retinoic acid (10-6 M), with which only a small number of colonies grew but they became twice as differentiated as controls (42.2 +/- 4.0% vs 22.6 +/- 2.7%, mean +/- SEM, lipid-forming colonies, p < 0.01). Furthermore, the RAR-beta,gamma antagonist CD2665 antagonized the suppressive effects of all-trans retinoic acid, adapalene, and CD2043 on both cell growth and differentiation. In contrast, the retinoid X receptor agonist CD2809 increased cell growth slightly and lipid-forming colonies dramatically in a clear dose-related manner to a maximum of 73.7% +/- 6.7% at 10-6 M (p < 0. 001). Our data suggest that retinoic acid receptors and retinoid X receptors differ in their roles in sebocyte growth and differentiation: (i) retinoic acid receptors, especially the beta and/or gamma subtypes, mediate both the antiproliferative and antidifferentiative effects of retinoids; (ii) retinoid X receptors mediate prominent differentiative and weak proliferative effects; (iii) the antiproliferative and antidifferentiative effects of all-trans retinoic acid are probably mediated by retinoic acid receptors, whereas its differentiative effect at high dose may be mediated by retinoid X receptors via all-trans retinoic acid metabolism to 9-cis retinoic acid, the natural ligand of retinoid X receptors.

Peroxisome proliferator-activated receptors and skin development.

PPARs are nuclear hormone receptors. PPAR subtypes (alpha, gamma, delta, the latter a xPPARbeta homologue) were initially investigated in skin because of their known role in regulating lipid metabolism. Studies adding specific PPAR ligand activators to cultured skin or skin cells are compatible with the concepts that PPARalpha activation mediates early lipogenic steps common to the function of both skin epidermal cells (keratinocytes) and sebaceous cells (sebocytes), PPARgamma activation plays a unique role in stimulating sebocyte lipogenesis, and PPARdelta activation may contribute to lipid biosynthesis in both sebocytes and keratinocytes under certain circumstances. Epidermal keratinocytes appear to express small amounts of PPARalpha and PPARdelta mRNA and a trace of PPARgamma mRNA which is up-regulated with differentiation. Sebocytes express all subtypes; PPARgamma gene expression excedes that in epidermis. The emerging data on PPAR protein expression suggests that epidermis normally expresses predominantly PPARalpha, while sebocytes express more PPARgamma than PPARalpha. These expression patterns may change during hyperplasia, differentiation and inflammation. Gene disruption studies in mice are compatible with a contribution of PPARalpha to skin barrier function, suggest that PPARgamma is necessary for sebocyte differentiation, and indicate that PPARdelta can ameliorate inflammatory responses in skin. PPARs appear to play a role in keratinocyte synthesis of the lipids that they export to the intercellular space to form the skin permeability barrier. They also appear to be important for sebocyte formation of the intracellular fused lipid droplets that constitute the holocrine secretion of the sebaceous gland. In addition, they may play roles in keratinocyte growth and differentiation and the inhibition of skin inflammation by diverse mechanisms not necessarily related to fat metabolism.

Intrauterine growth retardation associated with maternal uniparental disomy for chromosome 6 unmasked by congenital adrenal hyperplasia.

We report the first case of maternal uniparental disomy for chromosome 6 (UPD6mat) ascertained through congenital adrenal hyperplasia (CAH), which arose because of reduction to homozygosity of an autosomal recessive mutation. This case suggests that UPD6mat is associated with intrauterine growth retardation (IUGR). A case of paternal UPD (involving only the short arm of chromosome 6) ascertained as CAH has previously been reported, but was not stated to have IUGR. Our patient was born with IUGR followed by extraordinarily good catch-up growth. She had a history of a marked lag in motor development. She presented at 2.65 y of age with pubarche of 3 mo duration, clitoral enlargement, and an advanced bone age. Simple virilizing CAH was diagnosed by elevations of plasma 17-hydroxyprogesterone and testosterone. Mutation analysis showed that the CAH was due to homozygosity for the 1172N exon 4 mutation. When parental DNA was examined, the mother was found to be heterozygous for the uncommon exon 4 mutation, while the father had no detectable mutations. DNA microsatellite analysis was subsequently performed on the patient and parents using polymorphic markers spanning the entire chromosome 6. Seven markers were informative for inheritance of a single maternal allele and absence of paternal alleles in the proband. Analysis of microsatellite markers from other chromosomes confirmed biparental inheritance at these loci. This combination of findings is diagnostic of UPD6mat. The only other reported case of UPD6mat was discovered serendipitously when genotyped for renal transplantation; this patient had a history of IUGR. Since both cases of UPD6mat had IUGR, the phenotype appears to include IUGR as well as the potential to unmask an autosomal recessive trait.

Growth hormone and insulin-like growth factors have different effects on sebaceous cell growth and differentiation.

Several observations suggest that GH stimulates sebaceous gland growth and development. Therefore, we studied the effects of GH and insulin-like growth factors (IGFs), alone and with androgen, on sebaceous epithelial cell (sebocyte) growth and differentiation in vitro. The rat preputial cell culture model system was used to judge differentiation (induction of lipid-forming colonies, LFCs) and DNA synthesis. GH increased sebocyte differentiation. At a dose of 10(-8) M in the presence of micromolar insulin, GH was 3.8 times more potent than IGF-I (38.1+/-4.2%, SEM, vs. 10+/-1.5% LFCs) and 6 times more potent than IGF-II (6+/-0.5% LFCs). IGF-I 10(-8) M alone stimulated a similar amount of differentiation as insulin 10(-6) M, although it was less effective than insulin in augmenting the effect of GH on differentiation. GH had no effect on sebocyte uptake of 3H-thymidine at doses up to 10(-6) M. On the other hand, IGF-I was the most potent stimulus of DNA synthesis (168% of control; P < 0.001 vs. all others). IGF-II 10(-8) M stimulated 3H-thymidine incorporation similarly to insulin 10(-6) M. In the presence of insulin, dihydrotestosterone (DHT) 10(-6) M induced 31.4+/-1.7% LFCs, and there was a tendency of DHT and GH to interact in promoting differentiation. When insulin was omitted from the system, differentiation was decreased overall, but GH +/- DHT slightly improved differentiation. The IGFs had no effect on the response to DHT. DHT decreased DNA synthesis by 40%, an effect unaltered by GH or IGFs. These results suggest that GH and IGFs have different functions in sebaceous cell growth and differentiation: GH stimulated differentiation beyond that found with IGFs or insulin, yet had no effect on DNA synthesis, a parameter stimulated most potently by IGF-I. While GH augmented the effect of DHT on differentiation, the IGFs had no effect on the response of DHT. These data indicate that GH may in part act directly on sebocytes rather than indirectly through IGF production. These data are consistent with the concept that increases in GH and IGF production contribute in complementary ways to the increase in sebum production during puberty and in acromegaly.

Ovarian and adrenal function in polycystic ovary syndrome.

Androgens are secreted by both the ovaries and adrenal glands in response to their respective trophic hormones LH and ACTH. Androgens in women are not specifically under negative feedback control by these pituitary hormones because they are by-products of estradiol and cortisol secretion. Rather, androgen secretion seems to be regulated mostly by intraglandular mechanisms. Functional ovarian hyperandrogenism is found in about 70% of patients with PCOS. It is characterized by excessive secretion of 17-hydroxyprogesterone in response to GnRH agonist or hCG stimulation. Failure of dexamethasone to suppress plasma free testosterone normally in the presence of normal adrenocortical suppression is also typical. Functional adrenal hyperandrogenism is found in about half of patients with PCOS. It is most often characterized by moderately increased secretion of the 17-ketosteroid DHEA in response to ACTH. The most likely cause of the excessive androgen secretion by both glands seems to be abnormal regulation (dysregulation) of the 17-hydroxylase and 17,20-lyase activities of P-450c17, the rate-limiting step in androgen biosynthesis. There are also subtle generalized disturbances of steroid metabolism, including tendencies toward excessive estrogen and cortisol secretion. The cause of dysregulation of steroidogenesis is unknown. The hyperinsulinemia that is compensatory for resistance to the glucose-metabolic effect of insulin seems to have a role in many cases. In most cases, intrinsic intraovarian or intra-adrenal autocrine or paracrine regulatory mechanisms are most likely malfunctioning.

Prevalence of impaired glucose tolerance and diabetes in women with polycystic ovary syndrome.

OBJECTIVE: NIDDM occurs commonly among women with polycystic ovary syndrome (PCOS). The prevalence and natural history of its precursor, impaired glucose tolerance (IGT), is less well known. The objective of this study was to characterize the prevalence and incidence of glucose intolerance in a large cohort of women with well-characterized PCOS. RESEARCH DESIGN AND METHODS: A total of 122 women with clinical and hormonal evidence of PCOS were recruited from the Medicine, Endocrinology, Gynecology, and Pediatrics Clinics at the University of Chicago. All women had a standard oral glucose tolerance test (OGTT) with measurement of glucose and insulin levels. A subset of 25 women were subsequently restudied with the aim of characterizing the natural history of glucose tolerance in PCOS. RESULTS: Glucose tolerance was abnormal in 55 (45%) of the 122 women: 43 (35%) had IGT and 12 (10%) had NIDDM at the time of initial study. The women with NIDDM differed from those with normal glucose tolerance in that they had a 2.6-fold higher prevalence of first-degree relatives with NIDDM (83 vs. 31%, P < 0.01 by chi 2) and were significantly more obese (BMI 41.0 +/- 2.4 vs. 33.4 +/- 1.1 kg/m2, P < 0.01). For the entire cohort of 122 women, there was a significant correlation between fasting and 2-h glucose concentrations (r = 0.76, P < 0.0001); among the subset with IGT, the fasting glucose concentration was poorly predictive of the 2-h level (r = 0.25, NS). After a mean follow-up of 2.4 +/- 0.3 years (range 0.5-6.3), 25 women had a second OGTT. The glucose concentration at 2 h during the second glucose tolerance test was significantly higher than the 2-h concentration during the first study (161 +/- 9 vs. 139 +/- 6 mg/dl, P < 0.02). CONCLUSIONS: The prevalence of IGT and NIDDM in women with PCOS is substantially higher than expected when compared with age- and weight-matched populations of women without PCOS. The conversion from IGT to NIDDM is accelerated in PCOS. The fasting glucose concentration does not reliably predict the glucose concentration at 2 h after an oral glucose challenge, particularly among those with IGT, the subgroup at highest risk for subsequent development of NIDDM. We conclude that women with PCOS should periodically have an OGTT and must be closely monitored for deterioration in glucose tolerance.

Rat preputial sebocyte differentiation involves peroxisome proliferator-activated receptors.

The hallmark of sebaceous epithelial cell (sebocyte) differentiation is the accumulation of fused neutral fat droplets. Very little sebocyte differentiation occurs, however, in primary or organ culture, even upon incubating with androgens, which are required for maturation in vivo. We hypothesized that sebocyte cell culture systems lack activators of the peroxisome proliferator-activated receptors that are involved in adipocyte differentiation. We here report that activation of peroxisome proliferator-activated receptor gamma and alpha by their respective specific ligands, a thiazolidinedione and a fibrate, induced lipid droplet formation in sebocytes but not epidermal cells. Linoleic acid and carbaprostacyclin, both peroxisome proliferator-activated receptor delta and alpha ligand-activators, were more effective but less specific, stimulating lipid formation in both types of cells. Either was more effective than the combination of peroxisome proliferator-activated receptor gamma and alpha activation, suggesting that peroxisome proliferator-activated receptor delta is involved in this lipid formation. Linoleic acid 0.1 mM stimulated significantly more advanced sebocyte maturation than any other treatment, including carbaprostacyclin, which suggests a distinct role of long chain fatty acids in sebocyte differentiation. Peroxisome proliferator-activated receptor gammal mRNA was demonstrated in sebocytes, but not in epidermal cells; it was more strongly expressed in freshly dispersed than in cultured sebocytes. In contrast, peroxisome proliferator-activated receptor delta mRNA was expressed to a similarly high extent before and after culture in both sebocytes and epidermal cells. These findings are compatible with the concepts that peroxisome proliferator-activated receptor gamma1 gene expression plays a unique role in the differentiation of sebocytes, while peroxisome proliferator-activated receptor delta activation and long chain fatty acids finalize sebocyte maturation and are capable of stimulating epidermal lipid formation. These findings have implications for the development of new modalities of treatment for acne vulgaris.

Optimizing estrogen replacement treatment in Turner syndrome.

Estrogen has a biphasic effect on growth, stimulatory at low doses but inhibitory at higher doses. Therefore, designing optimal sex hormone replacement treatment in girls with Turner syndrome (TS) who are being treated with growth hormone (GH) involves considering the dose and form of the estrogen as well as the route and timing of its administration. We report here a preliminary analysis of a study to test the concept that an optimal estrogen replacement regimen should consist of estradiol administered in a low dose by a systemic route. The study population consisted of 9 girls with TS who had been treated with GH for 6 or more months. When the girls were 12 to 15 years old, we added depot estradiol at a monthly intramuscular dose of 0.2 mg and increased the dose at 6-month intervals to 0.4, 0.6, and, in 7 of the girls, 0.8 mg. We compared the results in these subjects with those in a matched group of 37 patients with TS in whom routine estrogen treatment had been started at similar ages and who were treated with a similar course of GH therapy. The gain in height at 2 years was 2.6 cm greater in those who were treated with depot estradiol than in those who were treated with routine estrogen. The bone age in the patients who were treated with depot estradiol increased in proportion to their chronologic age, suggesting that this difference indicates an increase in their predicted adult height. We conclude that using very low doses of systemic estradiol to induce puberty before the age of 15 years in girls with TS who are treated with GH, instead of using routine estrogen therapy, can result in increased final heights.

Mechanisms of androgen induction of sebocyte differentiation.

It has been difficult to induce the expected sebocyte differentiation in vitro with dihydrotestosterone (DHT). We reasoned that our culture system lacks differentiating factors, and peroxisome proliferator-activated receptors (PPARs) were the prime candidates. We tested PPAR activators informative about diverse PPAR subtypes, with and without DHT (10(-6) M): BRL-49653 (10(-6) M, PPAR-gamma), WY-14643 (10(-6) M, PPAR-alpha), and linoleic acid (LIN, 10(-4) M, PPAR-delta). Treatments were added in serum-free medium to cultures of rat preputial sebocytes. Control, DHT, BRL and BRL + DHT treatments caused 11, 25, 66 and 80%, respectively, of preputial cell colonies to differentiate into lipid-forming colonies (LFCs) (p < 0.001). WY induced 20% and LIN over 95% LFC formation. PPAR-gamma mRNA was identified in preputial sebocytes by the RNase protection assay. These data suggest that differentiation of sebocytes is transduced by PPARs and have implications for the development of new treatments for acne.