Gonadotrophin stimulation for in vitro fertilization significantly alters the hormone milieu in follicular fluid: a comparative study between natural cycle IVF and conventional IVF.

STUDY QUESTION Is the steroid hormone profile in follicular fluid (FF) at the time of oocyte retrieval different in naturally matured follicles, as in natural cycle IVF (NC-IVF), compared with follicles stimulated with conventional gonadotrophin stimulated IVF (cIVF)? SUMMARY ANSWER Anti-Mullerian hormone (AMH), testosterone (T) and estradiol (E2) concentrations are ∼3-fold higher, androstenedione (A2) is ∼1.5-fold higher and luteinizing hormone (LH) is ∼14-fold higher in NC-IVF than in cIVF follicles, suggesting an alteration of the follicular metabolism in conventional gonadotrophin stimulated IVF. WHAT IS KNOWN ALREADY In conventional IVF, the implantation rate of unselected embryos appears to be lower than in NC-IVF, which is possibly due to negative effects of the stimulation regimen on follicular metabolism. In NC-IVF, the intrafollicular concentration of AMH has been shown to be positively correlated with the oocyte fertilization and implantation rates. Furthermore, androgen treatment seems to improve the ovarian response in low responders. STUDY DESIGN, SIZE, DURATION This cross-sectional study involving 36 NC-IVF and 40 cIVF cycles was performed from 2011 to 2013. Within this population, 13 women each underwent 1 NC-IVF and 1 cIVF cycle. cIVF was performed by controlled ovarian stimulation with HMG and GnRH antagonists. PARTICIPANTS/MATERIALS, SETTING, METHODS Follicular fluid was collected from the leading follicles. AMH, T, A2, dehydroepiandrosterone (DHEA), E2, FSH, LH and progesterone (P) were determined by immunoassays in 76 women. Aromatase activity in follicular fluid cells was analysed by a tritiated water release assay in 33 different women. For statistical analysis, the non-parametric Mann-Whitney U or Wilcoxon tests were used. MAIN RESULTS AND ROLE OF CHANCE In follicular fluid from NC-IVF and from cIVF, median levels were 32.8 and 10.7 pmol/l for AMH (P < 0.0001), 47.2 and 18.8 µmol/l for T (P < 0.0001), 290 and 206 nmol/l for A2 (P = 0.0035), 6.7 and 5.6 pg/ml for DHEA (n.s.), 3292 and 1225 nmol/l for E2 (P < 0.0001), 4.9 and 7.2 mU/ml for FSH (P < 0.05), 14.4 and 0.9 mU/ml for LH (P < 0.0001) and 62 940 and 54 710 nmol/l for P (n.s.), respectively. Significant differences in follicular fluid concentrations for AMH, E2 and LH were also found in the 13 patients who underwent both NC-IVF and cIVF when they were analysed separately in pairs. Hormone analysis in serum excluded any relevant impact of AMH, T, A2, and E2 serum concentration on the follicular fluid hormone concentrations. Median serum concentrations were 29.4 and 0.9 mU/ml for LH (P < 0.0001) and 2.7 and 23.5 nmol/l for P (P < 0.0001) after NC-IVF and c-IVF, respectively. Positive correlations were seen for FF-AMH with FF-T (r = 0.35, P = 0.0002), FF-T with FF-LH (r = 0.48, P < 0.0001) and FF-E2 with FF-T (r = 0.75, P < 0.0001). The analysis of aromatase activity was not different in NC-IVF and cIVF follicular cells. LIMITATION, REASONS FOR CAUTION Any association between the hormone concentrations and the implantation potential of the oocytes could not be investigated as the oocytes in cIVF were not treated individually in the IVF laboratory. Since both c-IVF and NC-IVF follicles were stimulated by hCG before retrieval, the endocrine milieu in the natural cycle does not represent the pure physiological situation. WIDER IMPLICATIONS OF THE FINDINGS The endocrine follicular milieu and the concentration of putative markers of oocyte quality, such as AMH, are significantly different in gonadotrophin-stimulated conventional IVF compared with natural cycle IVF. This could be a cause for the suggested lower oocyte quality in cIVF compared with naturally matured oocytes. The reasons for the reduced AMH concentration might be low serum and follicular fluid LH concentrations due to LH suppression, leading initially to low follicular androgen concentrations and then to low follicular AMH production. STUDY FUNDING/COMPETING INTERESTS Funding for this study was obtained from public universities (for salaries) and private industry (for consumables). Additionally, the study was supported by an unrestricted grant from MSD Merck Sharp & Dohme GmbH and IBSA Institut Biochimique SA. The authors are clinically involved in low-dose monofollicular stimulation and IVF therapies, using gonadotrophins from all gonadotrophin distributors on the Swiss market, including Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH. Otherwise, the authors have no competing interests. TRIAL REGISTRATION NUMBER Not applicable.

The 17, 20-lyase activity of cytochrome p450c17 from human fetal testis favors the delta5 steroidogenic pathway.

Cytochrome P450c17 catalyzes both 17alpha-hydroxylation and 17,20-lyase conversion of 21-carbon steroids to 19-carbon precursors of sex steroids. P450c17 can mediate testosterone biosynthesis via the conversion of pregnenolone to dehydroepiandrosterone (the delta(5) pathway) or via conversion of progesterone to androstenedione (the delta(4) pathway). In many species, the 17, 20-lyase activity of P450c17 for one pathway dominates, reflecting the preferred steroidogenic pathway of that species. All studies of recombinant human P450c17 and of human adrenal microsomes have found high 17, 20-lyase activity only in the delta(5) pathway. Because the 17, 20-lyase activities in both the delta(4) and delta(5) pathways for testicular P450c17 have not been directly compared, however, it is not known if the delta(5) pathway dominates in the human testis. To resolve this issue, we assayed the conversion of 17alpha-hydroxypregnenolone to dehydroepiandrosterone (delta(5) 17, 20-lyase activity) and of 17alpha-hydroxyprogesterone to androstenedione (delta(4) 17, 20-lyase activity) by human fetal testicular microsomes. We obtained apparent Michaelis constant (K(m)) and maximum velocity (V(max)) values of 1.0 microM and 0.73 pmol.min(-1). microg(-1) for delta(5) 17, 20-lyase activity and of 3.5 microM and 0.23 pmol.min(-1). microg(-1) for delta(4) 17, 20-lyase activity. Catalytic efficiencies, expressed as the ratio V(max)/K(m), were 0.73 and 0.066 for the delta(5) and delta(4) reactions, respectively, indicating 11-fold higher preference for the delta(5) pathway. We conclude that the majority of testosterone biosynthesis in the human testis proceeds through the conversion of pregnenolone to dehydroepiandrosterone via the delta(5) pathway.

Aromatase deficiency caused by a novel P450arom gene mutation: impact of absent estrogen production on serum gonadotropin concentration in a boy.

We identified a new point mutation in the CYP19 gene responsible for aromatase (P450arom) deficiency in a 46,XY male infant with unremarkable clinical findings at birth. This boy is homozygote for a 1-bp (C) deletion in exon 5 of the aromatase gene causing a frame-shift mutation. The frame-shift results in a prematurely terminated protein that is inactive due to the absence of the functional regions of the enzyme. Aromatase deficiency was suspected prenatally because of the severe virilization of the mother during the early pregnancy, and the diagnosis was confirmed shortly after birth. Four weeks after birth, the baby boy showed extremely low levels of serum estrogens, but had a normal level of serum free testosterone; in comparison with the high serum concentration of androstenedione at birth, a striking decrease occurred by 4 weeks postnatally. We previously reported elevated basal and stimulated FSH levels in a female infant with aromatase deficiency in the first year of life. In contrast, in the male infant, basal FSH and peak FSH levels after standard GnRH stimulation tests were normal. This finding suggests that the contribution of estrogen to the hypothalamic-pituitary gonadotropin-gonadal feedback mechanism is different in boys and girls during infancy and early childhood. In normal girls, serum estradiol concentrations strongly correlate with circulating inhibin levels, and thus, low inhibin levels may contribute to the striking elevation of FSH in young girls with aromatase deficiency. In contrast, estradiol levels are physiologically about a 7-fold lower in boys than in girls, and serum inhibin levels remain elevated even though levels of FSH, LH, and testosterone are decreased.

Mounting behaviour in finishing pigs: Stable individual differences are not due to dominance or stage of sexual development

Every year around 100 million male piglets are castrated in the EU, usually without anaesthesia or post-operative analgesia. This surgical intervention is painful and stressful. Several main players within the pig industry have voluntarily agreed to end the practice of surgical pig castration in the EU by 2018. One alternative to castration is entire male pig production. However, entire males behave differently than castrates, for example, by performing more mounting behaviour, which is suggested to be a welfare problem. The aim of our study was to develop a comprehensive ethogram of different types of mounting and to investigate properties, causes and consequences of mounting behaviour in finishing pigs. The study included 80 entire male and 80 female pigs from two farrowing batches born six weeks apart. Mixed sex and single-sex housing of pigs are both common in pig farming, so to ensure our study was representative, the 160 pigs were assigned to social groups of 20 in three treatments: entire male pigs only (MM, 2 groups, n = 40), entire females only (FF, 2 groups, n = 40) and entire males and females mixed together (MF, 4 groups, n = 80). Measurements took place during the final six weeks before slaughter (between 63.5 and 105.5 kg). Observations of mounting behaviour on 12 days per batch suggested that: (i) males mounted more than females, (ii) within sex, there was no effect of treatment on the amount of mounting (although the statistical power of the study to detect these effects was low), and (iii) there were individual differences in mounting that were stable over time (within sex). Classification of mounting into different categories revealed that sexual mounting was most common overall and in males but only rare in females. Compared to other types of mounting (e.g. caused by crowding or during a fight), sexual mounts lasted longer and provoked more screaming by the recipient. There were no relationships between mounting behaviour on the one hand and dominance rank in food competition tests, the circulating levels of sex hormones (oestradiol, testosterone and progesterone) at the end of the study, the health scores (lameness and scratches) or weight gain on the other hand. The stable individual differences of mounting over time suggest that mounting behaviour is a trait of the individual rather than the appearance of random outbreaks. However, these differences in mounting cannot be explained by dominance behaviour or by differences in sex hormone concentrations that could indicate the onset of puberty. Mounting behaviour and in particular sexual mounting provoked high pitched screaming of the recipients indicating that mounting is a welfare problem. For the welfare assessment of entire male pig production the performance of mounting behaviour should be considered. (C) 2013 Elsevier B.V. All rights reserved.

Significance of Increased Tissue Transglutaminase in Hormone Refractory Prostate Cancer

The progression of hormone responsive to hormone refractory prostate cancer poses a major clinical challenge in the successful treatment of prostate cancer. The hormone refractory prostate cancer cells exhibit resistance not only to castrate levels of testosterone, but also to other therapeutic modalities and hence become lethal. Currently, there is no effective treatment available for managing this cancer. These observations underscore the urgency to investigate mechanism(s) that contribute to the progression of hormone-responsive to hormone-refractory prostate cancer and to target them for improved clinical outcomes. Tissue transglutaminase (TG2) is a multifunctional pro-inflammatory protein involved in diverse physiological processes such as inflammation, tissue repair, and wound healing. Its expression is also implicated in pathological conditions such as cancer and fibrosis. Interestingly, we found that the androgen-independent prostate cancer cell lines, which lacked androgen receptor (AR) expression, contained high basal levels of tissue transglutaminase. Inversely, the cell lines that expressed androgen receptor lacked transglutaminase expression. This attracted our attention to investigate the possible role this protein may play in the progression of prostate cancer, especially in view of recent observations that its expression is linked with increased invasion, metastasis, and drug resistance in multiple cancer cell types. The results we obtained were rather surprising and revealed that stable expression of tissue transglutaminase in androgen-sensitive LNCaP prostate cancer cells rendered these cells independent of androgen for growth and survival by silencing the AR expression. The AR silencing in TG2 expressing cells (TG2-infected LNCaP and PC-3 cells) was due to TG2-induced activation of the inflammatory nuclear transcription factor-kB (NF-kB). Thus, TG2 induced NF-kB was found to directly bind to the AR promoter. Importantly, TG2 protein was specifically recruited to the AR promoter in complex with the p65 subunit of NF-kB. Moreover, TG2 expressing LNCaP and PC-3 cells exhibited epithelial-to-mesenchymal transition, as evidenced by gain of mesenchymal (such as fibronectin, vimentin, etc.) and loss of epithelial markers (such as E-cadherin, b-catenin). Taken together, these results suggested a new function for TG2 and revealed a novel mechanism that is responsible for the progression of prostate cancer to the aggressive hormone-refractory phenotype.

An examination of the characteristics, concentration and distribution of androgen receptor in rat testis during sexual maturation

Previously reported androgen receptor concentrations in rat testis and testicular cell types have varied widely. In the studies reported here a nuclear exchange assay was established in rat testis in which exchange after 86 hours at 4$\sp\circ$C was greater than 85% complete and receptor was stable. Receptor concentration per DNA measured by exchange declined between 15 and 25 days of age in the rat testis, then increased 4-fold during sexual maturation. Proliferation of germ cells which had low receptor concentration appeared to account for the early decline in testicular receptor concentration, whereas increase in receptor number per Sertoli cell between 25 and 35 days of age contributed to the later increase. Increase in Leydig cell number during maturation appeared to account for the remainder of the increase due to the high receptor concentration in these cells. Detailed studies showed that other possible explanations for changes in receptor number (e.g. shifts in receptor concentration between the cytosol and nuclear subcellular compartments or changes in the affinity of the receptor for its ligands) were not likely.^ Androgen receptor dynamics in testicular cells showed rapid, specific uptake of ($\sp3$H) -testosterone that was easily blocked by unlabeled testosterone (RA of 7 nM in both cell types), and medroxyprogesterone acetate (RA of 28 and 16 nM in Sertoli and peritubular cells, respectively), but not as well by the anti-androgens cyproterone acetate (RA of 116 and 68 nM) and hydroxyflutamide (RA of 300 and 180 nM). The affinity of the receptor for the ligand dimethylnortestosterone was similar in the two cell types (K$\rm\sb{d}$ values of 0.78 and 0.71 nM for Sertoli and peritubular cells) and was virtually identical with the affinity of the whole testis receptor (0.89 nM). Medroxyprogesterone acetate and testosterone significantly increased nuclear androgen receptor concentration relative to untreated controls in Sertoli and peritubular cells, whereas hydroxyflutamide and cyproterone acetate did not. Despite the different embryological origins of peritubular and Sertoli cells, their responses to both androgens and anti-androgens were similar. In addition, these studies suggest that peritubular cells are as likely as Sertoli cells to be primary androgen targets. ^

Synthesis and properties of heterobifunctional photoaffinity reagents for the identification and isolation of receptors

A method employing isotopically- and photoaffinity-labeled probes and polyclonal and monoclonal antibody to the probes for the identification, isolation and recovery of protein receptors is described. Antibody was raised against N-(3-(p-azido-m-($\sp{125}$I) -iodophenyl)) propionate (AIPP) coupled to and photolyzed to BSA. The antibodies specifically bound AIPP-derivatized proteins. An isolation system was developed utilizing this probe and two antigenically identical reversible analogues. N-(3-((p-azido-m-($\sp{125}$I) -iodo-phenyl)propionyl)amidoethyl-1,3-dithiopropionyl) succinimide (Reversible $\sp{125}$I-AIPPS) reacts with primary amines and N-(((3-p-azido-m-($\sp{125}$I) -iodophenyl)propionyl)amidoethyl)dithiopyridine ($\sp{125}$I-AIPP-PDA) reacts with reduced thiols. The applicability of the system was established by derivatizing known ligands (Transferrin and Interferon-alpha) with one of the probes. The ligand-probe was then allowed to interact with its receptor by incubation with SS5 lymphoma cells and cross-linked by photolysis at 300 nm. The photolyzed ligand/probe/receptor preparation was then recovered with AIPP antibody. Utilization of N-(3-((p-azido-m-($\sp{125}$I) -iodo-phenyl-propionyl)-amidoethyl-1,3-dithiopropionyl) succinimide (Reversible $\sp{125}$I-AIPPS) allowed the components of the photolyzed complex to be separated by treatment with 2-mercaptoethanol in the SDS-PAGE solubilization buffer. Ligand and receptor labeling were then assessed by Coomassie staining and autoradiography. Results of receptor assays suggest that $\sp{125}$I-AIPP was, indeed, transferred to moieties that represent the receptors for both Transferrin and Interferon-alpha. ^

Citric acid cycle flux and pressure-volume work in the isolated working rat heart

It has been demonstrated previously that the mammalian heart cannot sustain physiologic levels of pressure-volume work if ketone bodies are the only substrates for respiration. In order to determine the metabolic derangement responsible for contractile failure in hearts utilizing ketone bodies, rat hearts were prefused at a near-physiologic workload in a working heart apparatus with acetoacetate and competing or alternate substrates including glucose, lactate, pyruvate, propionate, leucine, isoleucine, valine and acetate. While the pressure-volume work for hearts utilizing glucose was stable for 60 minutes of perfusion, performance fell by 30 minutes for hearts oxidizing acetoacetate as the sole substrate. The tissue content of 2-oxoglutarate and its transamination product, glutamate, were elevated in hearts utilizing acetoacetate while succinyl-CoA was decreased suggesting impaired flux through the citric acid cycle at the level of 2-oxoglutarate dehydrogenase. Further studies indicated that the inhibition of 2-oxoglutarate dehydrogenase developed prior to the onset of contractile failure and that the inhibition of the enzyme may be related to sequestration of the required cofactor, coenzyme A, as the thioesters acetoacetyl-CoA and acetyl-CoA. The contractile failure was not observed when glucose, lactate, pyruvate, propionate, valine or isoleucine were present together with acetoacetate, but the addition of acetate or leucine to acetoacetate did not improve performance indicating that improved performance is not mediated through the provision of additional acetyl-CoA. Furthermore, addition of competing substrates that improved function did not relieve the inhibition of 2-oxoglutarate dehydrogenase and actually resulted in the further accumulation of citric acid cycle intermediates "upstream" of 2-oxoglutarate dehydrogenase (2-oxoglutarate, glutamate, citrate and malate). Studies with (1-$\sp{14}$C) pyruvate indicate that the utilization of ketone bodies is associated with activation of NADP$\sp+$dependent malic enzyme and enrichment of the C4 pool of the citric acid cycle. The results suggest that contractile failure induced by ketone bodies in rat heart results from inhibition of 2-oxoglutarate dehydrogenase and that reversal of contractile failure is dissociated from relief of the inhibition, but rather is due to the entry of carbon units into the citric acid cycle as compounds other than acetyl-CoA. This mechanism of enrichment (anaplerosis) provides oxaloacetate for condensation with acetyl-CoA derived from ketone bodies allowing continued energy production by sustaining flux through a span of the citric acid cycle up to the point of inhibition at 2-oxoglutarate dehydrogenase for energy production thereby producing the reducing equivalents necessary to sustain oxidative phosphorylation. ^

Hormonal risk factors and testicular cancer

The cause of testicular cancer is not known and recent hypotheses have suggested an altered hormonal milieu may increase the risk of testis cancer. This study examined modulation of testicular cancer risk by hormonal factors, specifically: environmental xenoestrogens (e.g. organochlorines), prenatal maternal estrogens, testosterone indices (age at puberty, severe adolescent acne, self-reported balding), sedentary lifestyle and dietary consumption of fats and phytoestrogens.^ A hospital based friend matched case-control study was conducted at the University of Texas M. D. Anderson Cancer Center in Houston, Texas, between January 1990 and October 1996. Cases had a first primary testis tumor diagnosed between age 18 to 50 years and resided in Texas, Louisiana, Oklahoma or Arkansas.^ Cases and friend controls completed a mail questionnaire and case/control mothers were contacted by phone regarding pregnancy related variables. The study population comprised 187 cases, 148 controls, 147 case mothers and 86 control mothers. Odds ratios were virtually identical whether the match was retained or dissolved, thus the analyses were conducted using unconditional logistic regression.^ Cryptorchidism was a strong risk factor for testis cancer with an age-adjusted odds ratio (OR) of 7.7 (95% confidence interval (CI): 2.3-26.3). In a final model (adjusted for age, education, and cryptorchidism), history of severe adolescent acne and self-reported balding were both significantly protective, as hypothesized. For acne (yes vs. no) the OR was 0.5 (CI: 0.3-1.0) and for balding (yes vs. no) the OR was 0.6 (CI: 0.3-1.0). Marijuana smoking was a risk factor among heavy, regular users (17 times/week, OR = 2.4; CI: 0.9-6.4) and higher saturated fat intake increased testis cancer risk (saturated fat intake $>$ 15.2 grams/day vs. $<$ 11.8 grams/day, OR = 3.3; CI: 1.5-7.1). Early puberty, xenoestrogen exposure, elevated maternal estrogen levels, sedentary lifestyle and dietary phytoestrogen intake were not associated with risk of testicular cancer.^ In conclusion, testicular cancer may be associated with endogenous androgen metabolism although environmental estrogen exposure can not be ruled out. Further research is needed to understand the underlying hormonal mechanisms and possible dietary influences. ^

A novel form of CYP3A from rat brain: cDNA cloning, protein characterization and gene regulation of cytochrome P450 3A9

One full length cDNA clone, designated 3aH15, was isolated from a rat brain cDNA library using a fragment of CYP3A2 cDNA as a probe. 3aH15 encoded a protein composed of 503 amino acid residues. The deduced amino acid sequence of 3aH15 was 92% identical to mouse Cyp3a-13 and had a 68.4% to 76.5% homology with the other reported rat CYP3A sequences. Clone 3aH15 was thus named CYP3A9 by Cytochrome P450 Nomenclature Committee. CYP3A9 seems to the major CYP3A isozyme expressed in rat brain. Sexual dimorphism of the expression of CYP3A9 was shown for the first time in rat brain as well as in rat liver. CYP3A9 appears to be female specific in rat liver based on the standards proposed by Kato and Yamazoe who defined sex specific expression of P450s as being a 10-fold or higher expression level in one sex compared with the other. CYP3A9 gene expression was inducible by estrogen treatment both in male and in female rats. Male rats treated with estrogen had a similar expression level of CYP3A9 mRNA both in the liver and brain. Ovariectomy of adult female rats drastically reduced the mRNA level of CYP3A9 which could be fully restored by estrogen replacement. On the other hand, only a two-fold induction of CYP3A9 expression by dexamethasone was observed in male liver and no significant induction of CYP3A9 mRNA was observed in female liver or in the brains. These results suggest that estrogen may play an important role in the female specific expression of the CYP3A9 gene and that CYP3A9 gene expression is regulated differently from other CYP3A isozymes. ^ P450 3A9 recombinant protein was expressed in E. coli using the pCWOri+ expression vector and the MALLLAVF amino terminal sequence modification. This construct gave a high level of expression (130 nmol P450 3A9/liter culture) and the recombinant protein of the modified P450 3A9 was purified to electrophoretic homogeneity (10.1 nmol P450/mg protein) from solubilized fractions using two chromatographic steps. The purified P450 3A9 protein was active towards the metabolism of many clinically important drugs such as imipramine, erythromycin, benzphetamine, ethylmorphine, chlorzoxazone, cyclosporine, rapamycin, etc. in a reconstituted system containing lipid and rat NADPH-P450 reductase. Although P450 3A9 was active towards the catabolism of testosterone, androstenedione, dehydroepiandrosterone (DHEA) and 17β-estradiol, P450 3A9 preferentially catalyzes the metabolism of progesterone to form four different hydroxylated products. Optimal reconstitution conditions for P450 3A9 activities required a lipid mixture and GSH. The possible mechanisms of the stimulatory effects of GSH on P450 3A9 activities are discussed. Sexually dimorphic expression of P450 3A9 in the brain and its involvement in many neuroactive drugs as well as neurosteroids suggest the possible role of P450 3A9 in some mental disorders and brain functions. ^

AN INVESTIGATION INTO THE MOLECULAR MECHANISMS OF ANDROGEN ACTION IN THE SERTOLI CELL

Steroid hormones regulate target cell function via quantitative and qualitative changes in RNA and protein synthesis. In the testis, androgens are known to play an important role in the regulation of spermatogenesis. The Sertoli cell (SC), whose function is thought to be supportive to the developing germ cell, has been implicated as an androgen target cell. Although cytoplasmic androgen receptors and chromatin acceptor sites for androgen-receptor complexes have been found in SC, effects on RNA synthesis have not previously been demonstrated. In this study, SC RNA synthetic activity was characterized and the effect of testosterone on SC nuclear transcriptional activity in vitro assessed. SC exhibited two fold increases in RNA and ribonucleotide pool concentrations during sexual maturation. These changes appeared to correlate with a previously observed increase in protein concentration per cell over an age span of 15-60 days. Following incubation with ('3)H-uridine, SC from older animals incorporated more label into RNA than SC from younger animals. Since the relative concentration of cytidine nucleotides was higher in SC from older rats, the age-related increase in tritium incorporation may reflect an associated increase in incorporation of ('3)H-CMP into RNA. Alternatively, the enhanced labeling may be the result of either a change in the base composition of the RNA resulting in a higher proportion of CMP and UMP in the RNA, or compartmentalization of the nucleotide pools. The physiologic consequences of these maturational alterations of nucleotide pools remains to be elucidated. RNA polymerase activities were characterized in intact nuclei obtained from cultured rat SC. (alpha)-Amanitin resistant RNA polymerase I+III activity was identical when measured in low or high ionic strength (0.05 M or 0.25 M ammonium sulfate (AS)) in the presence of MnCl(,2) or MgCl(,2), with a divalent cation optimum of 1.6 mM. RNA polymerase II was most active in 0.25 M AS and 1.6 mM MnCl(,2). The apparent Km of RNA polymerase II for UTP was 0.016 mM in 0.05 M AS and 0.037 mM in 0.25 M AS. The apparent Km values for total polymerase activity was 0.008 mM and 0.036 mM at low and high ionic strenghts, respectively. These data indicate that Sertoli cell RNA polymerase activities have catalytic properties characteristic of eukaryotic polymerase activities in general. In the presence of 21 (mu)M testosterone, RNA polymerase II activity increased two fold at 15 minutes, then declined but was still elevated over control values six hours after androgen addition. Polymerase I+III activity was not greatly affected by testosterone. The stimulation of polymerase II measured at 15 minutes was dose-dependent, with a maximum at 0.53 nM and no further stimulation up to 10('-5) M (ED(,50) = 0.25 nM testosterone), and was androgen specific. The results of preliminary RNA isolation and characterization experiments suggested that the synthesis of several species of RNA was enhanced by testosterone administration. These findings have great potential importance since they represent the first demonstration of a direct effect of androgens on the transcriptional process in the Sertoli cell. Furthermore, the results of these studies constitute further evidence that the Sertoli cell is a target for androgen action in the testis. ^

Sex Differences in the Gut Microbiome Drive Hormone-Dependent Regulation of Autoimmunity

Microbial exposures and sex hormones exert potent effects on autoimmune diseases, many of which are more prevalent in women. We demonstrate that early-life microbial exposures determine sex hormone levels and modify progression to autoimmunity in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). Colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipient's microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and robust T1D protection. These effects were dependent on androgen receptor activity. Thus, the commensal microbial community alters sex hormone levels and regulates autoimmune disease fate in individuals with high genetic risk.

Early development of human lymphomas in a prostate cancer xenograft program using triple knock-out immunocompromised mice

BACKGROUND There is an urgent need for preclinical models of prostate cancer; however, clinically relevant patient-derived prostate cancer xenografts (PDXs) are demanding to establish. METHODS Sixty-seven patients who were undergoing palliative transurethral surgery or radical prostatectomy for histologically confirmed, clinically relevant prostate cancer were included in the study. Fresh prostate cancer tissue was identified by frozen analysis in 48 patients. The cancer tissue was transplanted subcutaneously and under the renal capsule of NSG and NOG mice supplemented with human testosterone. All growing PDXs were evaluated by histology and immunohistochemistry. RESULTS Early assessment of the animals at least three months after transplantation included 27/48 (56.3%) eligible PDX cohorts. PDX growth was detected in 10/27 (37%) mouse cohorts. Eight of the ten PDXs were identified as human donor derived lymphomas, including seven Epstein Barr virus (EBV)-positive diffuse large B-cell lymphomas and one EBV-negative peripheral T-cell lymphoma. One sample consisted of benign prostatic tissue, and one sample comprised a benign epithelial cyst. Prostate cancer was not detected in any of the samples. CONCLUSIONS Tumors that arise within the first three months after prostate cancer xenografting may represent patient-derived EBV-positive lymphomas in up to 80% of the early growing PDXs when using triple knockout NSG immunocompromised mice. Therefore, lymphoma should be excluded in prostate cancer xenografts that do not resemble typical prostatic adenocarcinoma. Prostate 9999: XX-XX, 2014. © 2015 Wiley Periodicals, Inc.

Menstrual cycle phase affects discrimination of infant cuteness

Recent studies have shown that women are more sensitive than men to subtle cuteness differences in infant faces. It has been suggested that raised levels in estradiol and progesterone may be responsible for this advantage. We compared young women's sensitivity to computer-manipulated baby faces varying in cuteness. Thirty-six women were tested once during ovulation and once during the luteal phase of their menstrual cycle. In a two alternative forced-choice experiment, participants chose the baby which they thought was cuter (Task 1), younger (Task 2), or the baby that they would prefer to babysit (Task 3). Saliva samples to assess levels of estradiol, progesterone and testosterone were collected at each test session. During ovulation, women were more likely to choose the cuter baby than during the luteal phase, in all three tasks. These results suggest that cuteness discrimination may be driven by cyclic hormonal shifts. However none of the measured hormones were related to increased cuteness sensitivity. We speculate that other hormones than the ones measured here might be responsible for the increased sensitivity to subtle cuteness differences during ovulation.

Steroidogenesis of the testis - new genes and pathways

Defects of androgen biosynthesis cause 46,XY disorder of sexual development (DSD). All steroids are produced from cholesterol and the early steps of steroidogenesis are common to mineralocorticoid, glucocorticoid and sex steroid production. Genetic mutations in enzymes and proteins supporting the early biosynthesis pathways cause adrenal insufficiency (AI), DSD and gonadal insufficiency. The classic androgen biosynthesis defects with AI are lipoid CAH, CYP11A1 and HSD3B2 deficiencies. Deficiency of CYP17A1 rarely causes AI, and HSD17B3 or SRD5A2 deficiencies only cause 46,XY DSD and gonadal insufficiency. All androgen biosynthesis depends on 17,20 lyase activity of CYP17A1 which is supported by P450 oxidoreductase (POR) and cytochrome b5 (CYB5). Therefore 46,XY DSD with apparent 17,20 lyase deficiency may be due to mutations in CYP17A1, POR or CYB5. Illustrated by patients harboring mutations in SRD5A2, normal development of the male external genitalia depends largely on dihydrotestosterone (DHT) which is converted from circulating testicular testosterone (T) through SRD5A2 in the genital skin. In the classic androgen biosynthetic pathway, T is produced from DHEA and androstenedione/-diol in the testis. However, recently found mutations in AKR1C2/4 genes in undervirilized 46,XY individuals have established a role for a novel, alternative, backdoor pathway for fetal testicular DHT synthesis. In this pathway, which has been first elucidated for the tammar wallaby pouch young, 17-hydroxyprogesterone is converted directly to DHT by 5α-3α reductive steps without going through the androgens of the classic pathway. Enzymes AKR1C2/4 catalyse the critical 3αHSD reductive reaction which feeds 17OH-DHP into the backdoor pathway. In conclusion, androgen production in the fetal testis seems to utilize two pathways but their exact interplay remains to be elucidated.