Alternative (backdoor) androgen production and masculinization in the human fetus.

Masculinization of the external genitalia in humans is dependent on formation of 5α-dihydrotestosterone (DHT) through both the canonical androgenic pathway and an alternative (backdoor) pathway. The fetal testes are essential for canonical androgen production, but little is known about the synthesis of backdoor androgens, despite their known critical role in masculinization. In this study, we have measured plasma and tissue levels of endogenous steroids in second trimester human fetuses using multidimensional and high-resolution mass spectrometry. Results show that androsterone is the principal backdoor androgen in the male fetal circulation and that DHT is undetectable (<1 ng/mL), while in female fetuses, there are significantly lower levels of androsterone and testosterone. In the male, intermediates in the backdoor pathway are found primarily in the placenta and fetal liver, with significant androsterone levels also in the fetal adrenal. Backdoor intermediates, including androsterone, are only present at very low levels in the fetal testes. This is consistent with transcript levels of enzymes involved in the alternate pathway (steroid 5α-reductase type 1 [SRD5A1], aldo-keto reductase type 1C2 [AKR1C2], aldo-keto reductase type 1C4 [AKR1C4], cytochrome P450 17A1 [CYP17A1]), as measured by quantitative PCR (qPCR). These data identify androsterone as the predominant backdoor androgen in the human fetus and show that circulating levels are sex dependent, but also that there is little de novo synthesis in the testis. Instead, the data indicate that placental progesterone acts as substrate for synthesis of backdoor androgens, which occurs across several tissues. Masculinization of the human fetus depends, therefore, on testosterone and androsterone synthesis by both the fetal testes and nongonadal tissues, leading to DHT formation at the genital tubercle. Our findings also provide a solid basis to explain why placental insufficiency is associated with disorders of sex development in humans.

Persistent organic pollutants in pregnant women potentially affect child development and thyroid hormone status.

BACKGROUND: Potentially harmful effects of persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT) on prenatal development and the endocrine system have been controversially discussed. METHODS: Working with a German cohort of 324 pregnant women, we assessed POP levels and used robust linear regression models to determine potential associations between maternal POP concentrations and pre- and postnatal development in the children, as well as the thyroid hormone status of the mother and child. RESULTS: Maternal p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) and most measured PCBs positively correlated with postnatal weight gain. We detected no correlation between newborn birth weight and head circumference, respectively, and maternal PCB and p,p'-DDE serum levels, while body length at birth was negatively associated with the maternal serum concentration of PCB 183. Maternal p,p'-DDE and nearly all PCB serum levels showed a negative correlation with maternal free triiodothyronine (FT3). p,p'-DDE and PCB 74 and 118 were negatively associated with maternal thyroid-stimulating hormone levels. In addition, we identified significant associations between maternal POP levels and thyroid hormone parameters of the child. CONCLUSIONS: These results indicate that POP exposure likely affects different aspects of pre- and postnatal development and impacts the thyroid hormone status of both mother and child. IMPACT: Pregnant women in a German cohort display a substantial accumulation of POPs. Body mass index and age influence maternal serum POP levels. Maternal POP levels show correlations with the child's length at birth and weight gain, and FT3 levels in the mother and child. Our data provide additional evidence for the potentially harmful influence of POPs. Our data indicate that POPs influence pre- and postnatal development.

Self-organising human gonads generated by a Matrigel-based gradient system.

BACKGROUND: Advances in three-dimensional culture technologies have led to progression in systems used to model the gonadal microenvironment in vitro. Despite demonstrating basic functionality, tissue organisation is often limited. We have previously detailed a three-dimensional culture model termed the three-layer gradient system to generate rat testicular organoids in vitro. Here we extend the model to human first-trimester embryonic gonadal tissue. RESULTS: Testicular cell suspensions reorganised into testis-like organoids with distinct seminiferous-like cords situated within an interstitial environment after 7 days. In contrast, tissue reorganisation failed to occur when mesonephros, which promotes testicular development in vivo, was included in the tissue digest. Organoids generated from dissociated female gonad cell suspensions formed loosely organised cords after 7 days. In addition to displaying testis-specific architecture, testis-like organoids demonstrated evidence of somatic cell differentiation. Within the 3-LGS, we observed the onset of AMH expression in the cytoplasm of SOX9-positive Sertoli cells within reorganised testicular cords. Leydig cell differentiation and onset of steroidogenic capacity was also revealed in the 3-LGS through the expression of key steroidogenic enzymes StAR and CYP17A1 within the interstitial compartment. While the 3-LGS generates a somatic cell environment capable of supporting germ cell survival in ovarian organoids germ cell loss was observed in testicular organoids. CONCLUSION: The 3-LGS can be used to generate organised whole gonadal organoids within 7 days. The 3-LGS brings a new opportunity to explore gonadal organogenesis and contributes to the development of more complex in vitro models in the field of developmental and regenerative medicine.

The relationship between pubertal timing and under-nutrition in rural Pakistan.

INTRODUCTION: Sexual development in females and males are routinely measured according to the Tanner Stages. Sparse data exist on the timing of pubertal milestones in Pakistan. To fill this gap, the age of attainment of pubertal milestones and their relationship with nutritional status was explored among children and adolescents living in the rural district of Matiari, Pakistan. METHODS: Anthropometry, nutrition biomarkers and Tanner Stage were assessed among girls aged 9.0-14.9 years (n = 723) and boys aged 10.0-15.9 years (n = 662) who were free from known disease in the rural District of Matiari, Pakistan. Median age was calculated for all Tanner Stages and menarche. Multivariable linear regressions were undertaken to determine covariates associated with the timing (age) of pubertal milestones. RESULTS: Among participants living in this rural community, the median age of puberty onset for girls was 11.9 years (95%CI:10.9; 12.5) and boys was 12.3 years (95%CI:11.5; 12.9). Age at first menarche was 12.9 years (95%CI:12.1; 13.3). Undernutrition was widespread among adolescents in this community. Thirty-seven percent of females and 27.0% of males were stunted while 20.5% of females and 31.3% of males were thin. Only 8% (n = 58) of females and 12% (n = 78) of males were free from any nutrient deficiency with most adolescents having two or three nutrient deficiencies. CONCLUSIONS: Undernutrition (stunting or thinness) was associated with relatively older ages for early puberty stages but not puberty completion. This may decrease the duration of the pubertal growth spurt and curtail potential catch-up growth that may occur during puberty. Efforts to decrease nutrient deficiencies, stunting and thinness beyond childhood should be made in rural Pakistan.

Later puberty onset among chronically undernourished adolescents living in a Karachi slum, Pakistan.

AIM: To assess factors associated with the timing of puberty onset (Tanner Stage Breast 2/Genital 2) among adolescents living in an urban slum in Karachi, Pakistan. METHODS: Girls enrolled at 8-10 years (n = 1009) and boys 9-11 years (n = 863) were followed every 6 months from 2006 to 2010. Parametric survival analysis for interval-censored data was used to estimate median age at puberty onset and assess the association between exposures and timing of puberty onset. RESULTS: The overall median age at Tanner Stage Breast 2 (AAB2) was 10.1 years (95% CI: 10.1; 10.5), and the median age at Tanner Stage Genital 2 (AAG2) was 10.1 years (95% CI: 10.1; 10.6). Stunting delayed AAB2 1 year and AAG2 about 6 months when compared to non-stunted peers. In the multivariable model, after adjusting for age at enrolment, stunting, thinness and vitamin A deficiency (VAD) were significantly associated with delayed AAB2, while stunting and anaemia were significantly associated with delayed AAG2. CONCLUSION: Among adolescents living in the Karachi slum, stunting and highly prevalent anaemia delayed AAG2, while stunting, thinness and VAD delayed AAG2. Parental and household factors were not significantly associated with the timing of puberty onset.

Impact of uteroplacental insufficiency on ovarian follicular pool in the rat.

BACKGROUND: A low oxygen supply to the fetus causes intrauterine growth restriction and can affect gonadal development of the offspring, having a potential impact on fertility. We investigated histology and gene expression in the postnatal rat ovary after fetal hypoxia induced by uterine artery ligation. METHODS: Sprague-Dawley rats underwent uterine artery ligation at day 19 of gestation. Offspring were sacrificed at 5, 20 and 40 days post-partum. Follicles were counted and classified in hematoxylin-eosin stained sections. Gene expression of 90 genes was analyzed by TaqMan® Low Density Array. RESULTS: A significantly lower number of total and primordial follicles was detected in 20 days post-partum intrauterine growth restricted animals. Follicle density was not different at 40 days post-partum, suggesting that compensatory mechanisms occurred during the pre-pubertal window. Uterine artery ligation modified the expression of 24 genes involved in different cellular functions, among which proliferation, apoptosis and metabolism. CONCLUSION: Ovarian follicle pool was affected by fetal hypoxia in early life, but this effect did not persist in puberty. Genes involved in cellular processes were affected at all ages, potentially implying long-term genetic alterations. Further analyses are needed to elucidate later effects of fetal hypoxia on ovarian function and fertility.

Telomere length regulators are activated in young men after pediatric kidney transplantation compared to healthy controls and survivors of childhood cancer-A cross-sectional study.

Chronic diseases are known to cause premature aging and frailty. Data about telomere length and telomere length-regulating proteins after pediatric KTx are scarce. Leukocyte telomere length and gene expression level of eight telomere-binding proteins were analyzed in 20 KTx recipients, eight childhood NBL survivors, and nine healthy controls. The influence of key clinical parameters on telomere length and on regulators of telomere length was evaluated. The telomere length in the KTx recipients tended to be shorter (0.53 AU) than in the healthy controls (0.64 AU) but longer than in the NBL survivors (0.38 AU). There was no significant difference in telomere length between the NBL survivors and the KTx recipients (P = .110). The gene expression level of telomere length-preserving protein RPA1 was significantly higher in the KTx recipients than among the NBL survivors or healthy controls, while the expression of TRF2 and the tumor suppressor gene p16 was significantly higher in the KTX recipients when compared to the controls. TRF2 and TIN2 correlated significantly with hsCRP; additionally, TRF2 showed significant correlation with plasma creatinine and eGFR. KTx recipients have near to normal telomere length, but they have significantly higher gene expression levels of telomere regulatory proteins compared with healthy controls, suggesting activation of mechanisms preserving telomere length among KTx recipients. Our results suggest that declined graft function and consequent inflammatory response may have influence on telomerase activity.

Child Health Systems in Sweden.

On a national level, several factors are responsible for Sweden's leading position in achieving the excellent health of children because Sweden has experienced a long history of peace and success in establishing a parliamentary democracy throughout the 20th century. Among the different sectors of society, Sweden has been able to focus on prevention and health promotion. The Swedish health care system is publicly financed based on local taxation. Pediatricians working in secondary and tertiary care are employed by the public sector, whereas family physicians are employed by both the private and public sectors. The pediatric departments at county and university levels provide a high quality of inpatient care for neonates and children. The county hospital pediatric departments typically include one neonatal ward and one ward for older children. Subspecialization exists even at the county level, and there is close cooperation between the county level and subspecialist units at the university level. Within the primary care sector, most children receive care from family physicians. The majority of family physicians have completed 3 months of pediatrics in their basic training program. In the more densely populated areas there are also pediatric ambulatory care centers working mostly with referrals from the family physicians. Preventive care is carried out at midwife-led maternity health centers, nurse-led Child Health Centers, and nurse-led school health care settings and reach almost everyone (99%). All health care for children and adolescents is free of charge up to 18 years of age.

Leydig cell steroidogenesis unexpectedly escapes mitochondrial dysfunction in prematurely aging mice.

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in tissues during aging in animals and humans and are the basis for mitochondrial diseases. Testosterone synthesis occurs in the mitochondria of Leydig cells. Mitochondrial dysfunction (as induced here experimentally in mtDNA mutator mice that carry a proofreading-deficient form of mtDNA polymerase γ, leading to mitochondrial dysfunction in all cells types so far studied) would therefore be expected to lead to low testosterone levels. Although mtDNA mutator mice showed a dramatic reduction in testicle weight (only 15% remaining) and similar decreases in number of spermatozoa, testosterone levels in mtDNA mutator mice were unexpectedly fully unchanged. Leydig cell did not escape mitochondrial damage (only 20% of complex I and complex IV remaining) and did show high levels of reactive oxygen species (ROS) production (>5-fold increased), and permeabilized cells demonstrated absence of normal mitochondrial function. Nevertheless, within intact cells, mitochondrial membrane potential remained high, and testosterone production was maintained. This implies development of a compensatory mechanism. A rescuing mechanism involving electrons from the pentose phosphate pathway transferred via a 3-fold up-regulated cytochrome b5 to cytochrome c, allowing for mitochondrial energization, is suggested. Thus, the Leydig cells escape mitochondrial dysfunction via a unique rescue pathway. Such a pathway, bypassing respiratory chain dysfunction, may be of relevance with regard to mitochondrial disease therapy and to managing ageing in general.

Bortezomib treatment causes long-term testicular dysfunction in young male mice.

BACKGROUND: With increased long-term survivors of childhood cancer patients, therapy-associated infertility has become one of the most common late side-effects and significantly affects their life-quality. Therefore, evaluation of anti-cancer agents on male reproduction and infertility prevention are urgently demanding. The proteasome inhibitor bortezomib has been launched in clinical trials for childhood cancers, however, its potential side effects on reproduction have so far been neither investigated experimentally nor reported in treated children. Thus the present study is designed to explore the impact of bortezomib on male reproductive function and to gain insights into how bortezomib exerts its adverse effects on man gonad, thereby providing pediatric oncologists relevant information. METHODS: 35 day-old male mice were treated with one 11-day cycle of bortezomib and then sacrificed 2 days, 45 days, or 6 months later. A mating study was performed in the group followed for 6 months, and their pups were analyzed on postnatal day 50. Serum follicle-stimulating hormone (FSH) and testicular testosterone levels were measured. Testicular morphology was evaluated by light- and electron microscopy, and the underlying mechanisms and pathways of testis damage were investigated. RESULTS: Testicular damage was visible already 2 days after stopping bortezomib and increased in severity by day 45. Then 80% of seminiferous tubules exhibited hypospermatogenesis with arrest at the levels of spermatogonia, spermatocytes and round spermatids. Germ cells were specifically targeted by bortezomib as evidenced by increased apoptosis mediated through activation of p53 and caspases. Even six months after the bortezomib treatment, testis weight, sperm concentration and seminiferous tubule length remained at a decreased level, indicating that spermatogenesis and tubular outgrowth could not fully recover. Combined with persistently increased serum levels of FSH in these mice, our results demonstrate that bortezomib can have long-term effects on testicular function, although fertility of bortezomib-exposed males remained and their offspring looked healthy. CONCLUSION: Bortezomib treatment causes long-term gonadal dysfunction in male mice. Careful monitoring of gonadal function in male childhood cancer patients treated with bortezomib is thus strongly recommended.

Growth charts and long-term sequelae in extreme preterm infants--from full-term age to 10 years.

AIM: To describe growth pattern from full-term age to 10 years in infants born before 26 weeks of gestation. METHOD: This retrospective longitudinal cohort contained 123 children from Karolinska Hospital, Stockholm, during 1990-2002. Length/height (Ht), weight (Wt) and head circumference (HC) were recorded monthly during the first year, every 3 months until 2 years and yearly thereafter, but HC at 15 months and at median age of 8.1/9.7 years (range 2-14) in boys/girls. RESULTS: For boys/girls at birth, the mean Z-score for Ht was -0.2/-0.2, for Wt 0.0/-0.2 and for HC 0.0/-0.3. At term, the mean Z-score for Ht was -3.8/-3.1, for Wt -3.0/-2.5 and for HC -1.7/-1.2. At 1 year, the mean Z-score for Ht was-1.3/-1.3, for Wt -1.9/-1.7 and for HC -1.2/-1.0. At 2 years, the mean Z-score for Ht was -1.3/-1.1, for Wt -1.6/-1.2 and at 10 years for Ht -0.7/-0.4; that was on average -0.3 below mid-parental height; for Wt -0.2/-0.2. Long-term sequelae were found in 48% of the boys and 34% of the girls. CONCLUSION: By 10 years of age, the attained mean Ht was in accordance with their genetic potential and almost half of these children had significant long-term sequelae.

Identification of an isoform of colony-stimulating factor 1 receptor mRNA in the rat testis.

Because alternative RNA splicing regulation in the testis is prevalent, we explored testes of Sprague-Dawley rats for existence of alternatively spliced colony-stimulating factor 1 receptor (CSF-1R) mRNA. Using RT-PCR and sequencing, we identified a variant of CSF-1R mRNA that was 284 bp shorter than the full-length CSF-1R transcript. This variant was present in the testis (late fetal stage to adult) and in other organs of rats (7 and 60 days old). The deletion of 284 bp disrupted the open reading frame, resulting in a noncoding mRNA product. When testicular macrophages were stimulated with CSF-1R ligand and lipopolysaccharide, proportionally increased expression of both short isoform and full-length CSF-1R mRNA was observed. Thus, the identified isoform of CSF-1R mRNA may interfere with the expression of full-length CSF-1R mRNA, thereby affecting the biological activity of the ligand/receptor signaling axis in Sprague-Dawley rats.

Science and policy on endocrine disrupters must not be mixed: a reply to a "common sense" intervention by toxicology journal editors.

The "common sense" intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU.

Medium- and time-related effects on hypothermic storage of rat testicular cells.

OBJECTIVE: Testicular samples obtained for fertility preservation often need to be transported between clinics. This study aimed to mimic this short-term hypothermic storage (4-8 °C) and explore the impact of these conditions and the transport medium composition on prepubertal rat testicular tissue samples. METHODS: Testicular tissue samples obtained from seven days post-partum rats were transferred to six compositionally different basal culture media and a balanced salt solution, which had been kept at 4-8 °C prior to transfer. The samples were preserved for either 12 or 24 hours in these hypothermic conditions. The potential effects of the short-term storage were evaluated by assessing the morphology, measuring the testosterone levels by radioimmunoassay and analysing 96 genes with TaqMan Low-Density Arrays. Summarizing results: Levels of gene expression related to energy, apoptosis and angiogenesis pathways were altered after hypothermic storage for 12 and especially 24 hours. We observed only minor differences in gene expression profiles for germ and testicular somatic cells, and no differences in tissue morphology and testosterone production levels. CONCLUSIONS: Short-term hypothermic storage of testicular tissue with a maximum duration of 24 hours does not affect the overall expression profile of testicular cell-specific genes; however, in a minor way, it affects the expression of specific cellular genes.

Proinsulin C-peptide interferes with insulin fibril formation.

Insulin aggregation can prevent rapid insulin uptake and cause localized amyloidosis in the treatment of type-1 diabetes. In this study, we investigated the effect of C-peptide, the 31-residue peptide cleaved from proinsulin, on insulin fibrillation at optimal conditions for fibrillation. This is at low pH and high concentration, when the fibrils formed are regular and extended. We report that C-peptide then modulates the insulin aggregation lag time and profoundly changes the fibril appearance, to rounded clumps of short fibrils, which, however, still are Thioflavine T-positive. Electrospray ionization mass spectrometry also indicates that C-peptide interacts with aggregating insulin and is incorporated into the aggregates. Hydrogen/deuterium exchange mass spectrometry further reveals reduced backbone accessibility in insulin aggregates formed in the presence of C-peptide. Combined, these effects are similar to those of C-peptide on islet amyloid polypeptide fibrillation and suggest that C-peptide has a general ability to interact with amyloidogenic proteins from pancreatic ß-cell granules. Considering the concentrations, these peptide interactions should be relevant also during physiological secretion, and even so at special sites post-secretory or under insulin treatment conditions in vivo.

De Novo and Depot-Specific Androgen Production in Human Adipose Tissue: A Source of Hyperandrogenism in Women with Obesity.

INTRODUCTION: Obesity in women is often associated with hyperandrogenism, but the role of adipose tissue (AT) in androgen synthesis remains unclear. Therefore, we studied whether AT could be a source of androgens promoting hyperandrogenism. METHODS: Subcutaneous and visceral (visc) AT was collected from lean and obese women. Androgen levels were evaluated in serum, AT, and cell-culture supernatant. Gene and protein expression of steroidogenic enzymes were determined. RESULTS: Obese subjects had elevated serum androgen levels, which reduced after weight loss. Androgens were measurable in AT and in cell-culture supernatants of adipocytes. Steroids were higher in AT from obese women, with the highest difference for testosterone in visc AT (+7.9-fold, p = 0.032). Steroidogenic enzymes were expressed in human AT with depot-specific differences. Obese women showed a significantly higher expression of genes of the backdoor pathway and of CYP19 in visc AT. CONCLUSION: The whole steroidogenic machinery of the classical and backdoor pathways of steroidogenesis, and the capacity for androgen biosynthesis, were found in both AT depots and cultured adipocytes. Therefore, we hypothesize that AT is a de novo site of androgen production and the backdoor pathway of steroidogenesis might be a new pathomechanism for hyperandrogenism in women with obesity.

Timing of Puberty, Pubertal Growth, and Adult Height in Short Children Born Small for Gestational Age Treated With Growth Hormone.

CONTEXT: Growth hormone (GH) is used to treat short children born small for gestational age (SGA); however, the effects of treatment on pubertal timing and adult height are rarely studied. OBJECTIVE: To evaluate adult height and peak height velocity in short GH-treated SGA children. METHODS: Prospective longitudinal multicenter study. Participants were short children born SGA treated with GH therapy (n = 102). Adult height was reported in 47 children. A reference cohort of Danish children was used. Main outcome measures were adult height, peak height velocity, age at peak height, and pubertal onset. Pubertal onset was converted to SD score (SDS) using Danish reference data. RESULTS: Gain in height SDS from start of treatment until adult height was significant in both girls (0.94 [0.75; 1.53] SDS, P = .02) and boys (1.57 [1.13; 2.15] SDS, P < .001). No difference in adult height between GH dosage groups was observed. Peak height velocity was lower than a reference cohort for girls (6.5 [5.9; 7.6] cm/year vs 7.9 [7.4; 8.5] cm/year, P < .001) and boys (9.5 [8.4; 10.7] cm/year vs 10.1 [9.7; 10.7] cm/year, P = .002), but no difference in age at peak height velocity was seen. Puberty onset was earlier in SGA boys than a reference cohort (1.06 [-0.03; 1.96] SDS vs 0 SDS, P = .002) but not in girls (0.38 [-0.19; 1.05] SDS vs 0 SDS, P = .18). CONCLUSION: GH treatment improved adult height. Peak height velocity was reduced, but age at peak height velocity did not differ compared with the reference cohort. SGA boys had an earlier pubertal onset compared with the reference cohort.

From cohorts to molecules: Adverse impacts of endocrine disrupting mixtures.

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio, as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.

Excessive ovarian production of nerve growth factor elicits granulosa cell apoptosis by setting in motion a tumor necrosis factor α/stathmin-mediated death signaling pathway.

Excessive nerve growth factor (NGF) production by the ovary, achieved via a transgenic approach, results in arrested antral follicle growth, reduced ovulatory capacity, and a predisposition to cyst formation in response to mildly elevated LH levels. Two salient features in these mutant mice (termed 17NF) are an elevated production of 17α-hydroxyprogesterone (17-OHP(4)), testosterone, and estradiol (E(2)) in response to gonadotropins, and an increased frequency of granulosa cell (GC) apoptosis. In this study, we show that the increase in steroidal response is associated with enhanced expression of Cyp17a1, Hsd17b, and Cyp19a1, which encode the enzymes catalyzing the synthesis of 17-OHP(4), testosterone, and E(2) respectively. Using a proteomic approach, we identified stathmin (STMN1), as a protein that is overproduced in 17NF ovaries. In its phosphorylated state, STMN1 mediates a cell death signal initiated by tumor necrosis factor α (TNF). STMN1 is expressed in GCs and excessive NGF increases its abundance as well as that of its forms phosphorylated at serine (Ser) 16, 25, and 38. TNF synthesis is also increased in 17NF ovaries, and this change is abolished by blocking neurotrophic tyrosine kinase receptors. Inhibiting TNF actions in vivo by administering a soluble TNF receptor prevented the increase in total and phosphorylated STMN1 production, as well as GC apoptosis in NGF-overproducing ovaries. These results indicate that an excess of NGF in the ovary promotes steroidogenesis by enhancing the expression of enzyme genes involved in 17-OHP(4), testosterone, and E(2) synthesis, and causes GC apoptosis by activating a TNF/ STMN1-mediated cell death pathway.

The prenylflavonoid phytoestrogens 8-prenylnaringenin and isoxanthohumol diferentially suppress steroidogenesis in rat Leydig cells in ontogenesis.

8-Prenylnaringenin and isoxanthohumol are prenylflavonoids found in the hop plant, Humulus lupulus (Cannabaceae), which is traditionally used to add bitterness and flavor to beer. Flavonoids have previously been reported to exert endocrine disrupting actions. Therefore, we investigated the effects of 8-prenylnaringenin and isoxanthohumol on steroidogenesis activated by human chorionic gonadotropin (hCG) in primary cultures of rat Leydig cells at different stages of their development. The present study is the first to demonstrate that the prenylflavonoids 8-prenylnaringenin and isoxanthohumol exert complex maturation-dependent effects on Leydig cell steroidogenesis. Those compounds inhibited hCG-stimulated androgen production by Leydig cells at all stages of their development, a process that was associated with the reduced ability of the cells to produce cAMP. However, these same compounds up-regulated hCG-activated StAR expression in progenitor (PLC) and immature (ILC) but not adult types of Leydig cells (ALC). Further, 8-prenylnaringenin and isoxanthohumol were not able to suppress androgen production activated by an exogenous analog of cAMP, (Bu)2 cAMP, in ALC and ILC but synergistically stimulated steroidogenesis in PLC. Our data suggest that 8-prenylnaringenin and isoxanthohumol affect cAMP-dependent cellular processes up-stream transport of cholesterol into mitochondria.