The unusual convergence of steroid catabolic pathways in Mycobacterium abscessus.

Mycobacterium abscessus, an opportunistic pathogen responsible for pulmonary infections, contains genes predicted to encode two steroid catabolic pathways: a cholesterol catabolic pathway similar to that of Mycobacterium tuberculosis and a 4-androstenedione (4-AD) catabolic pathway. Consistent with this prediction, M. abscessus grew on both steroids. In contrast to M. tuberculosis, Rhodococcus jostii RHA1, and other Actinobacteria, the cholesterol and 4-AD catabolic gene clusters of the M. abscessus complex lack genes encoding HsaD, the meta-cleavage product (MCP) hydrolase. However, M. abscessus ATCC 19977 harbors two hsaD homologs elsewhere in its genome. Only one of the encoded enzymes detectably transformed steroid metabolites. Among tested substrates, HsaDMab and HsaDMtb of M. tuberculosis had highest substrate specificities for MCPs with partially degraded side chains thioesterified with coenzyme A (kcat/KM = 1.9 × 104 and 5.7 × 103 mM-1s-1, respectively). Consistent with a dual role in cholesterol and 4-AD catabolism, HsaDMab also transformed nonthioesterified substrates efficiently, and a ΔhsaD mutant of M. abscessus grew on neither steroid. Interestingly, both steroids prevented growth of the mutant on acetate. The ΔhsaD mutant of M. abscessus excreted cholesterol metabolites with a fully degraded side chain, while the corresponding RHA1 mutant excreted metabolites with partially degraded side chains. Finally, the ΔhsaD mutant was not viable in macrophages. Overall, our data establish that the cholesterol and 4-AD catabolic pathways of M. abscessus are unique in that they converge upstream of where this occurs in characterized steroid-catabolizing bacteria. The data further indicate that cholesterol is a substrate for intracellular bacteria and that cholesterol-dependent toxicity is not strictly dependent on coenzyme A sequestration.

Sources and control of impurity during one-pot enzymatic production of dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA) has a promising market due to its capacity to regulate human hormone levels as well as preventing and treating various diseases. We have established a chemical esterification coupled biocatalytic-based scheme by lipase-catalyzed 4-androstene-3,17-dione (4-AD) hydrolysis to obtain the intermediate product 5-androstene-3,17-dione (5-AD), which was then asymmetrically reduced by a ketoreductase from Sphingomonas wittichii (SwiKR). Co-enzyme required for KR is regenerated by a glucose dehydrogenase (GDH) from Bacillus subtilis. This scheme is more environmentally friendly and more efficient than the current DHEA synthesis pathway. However, a significant amount of 4-AD as by-product was detected during the catalytic process. Focused on the control of by-products, we investigated the source of 4-AD and identified that it is mainly derived from the isomerization activity of SwiKR and GDH. Increasing the proportion of glucose in the catalytic system as well as optimizing the catalytic conditions drastically reduced 4-AD from 24.7 to 6.5% of total substrate amount, and the final yield of DHEA achieved 40.1 g/L. Furthermore, this is the first time that both SwiKR and GDH have been proved to be promiscuous enzymes with dehydrogenase and ketosteroid isomerase (KSI) activities, expanding knowledge of the substrate diversity of the short-chain dehydrogenase family enzymes. KEY POINTS: • A strategy of coupling lipase, ketoreductase, and glucose dehydrogenase in producing DHEA from 4-AD • Both SwiKR and GDH are identified with ketosteroid isomerase activity. • Development of catalytic strategy to control by-product and achieve highly selective DHEA production.

Characterization of eight types of 17ß-hydroxysteroid dehydrogenases from the Japanese sardine Sardinops melanostictus: The probable role of type 12a in ovarian estradiol synthesis.

17ß-hydroxysteroid dehydrogenases (Hsd17bs) play a critical role in sex steroid biosynthesis. Although multiple types of Hsd17b have been found in fish, there is limited research on their expression and function. Recently, we succeeded in identifying eight types of Hsd17b (types 3, 4, 7, 8, 10, 12a, 12b, and 14) by RNA sequencing in the Japanese sardine Sardinops melanostictus, a commercially important clupeoid fish; however, a homologous sequence of Hsd17b1, which catalyzes the key reaction of estradiol-17ß (E2) synthesis, was absent. Here, we aimed to identify the Hsd17b type that plays a major role in E2 synthesis during ovarian development in Japanese sardine. The cDNAs encoding those eight types of Hsd17b were cloned and sequenced. The expressions of hsd17b3, hsd17b12a, and hsd17b12b were higher in ovary than in testis. In particular, hsd17b12a was predominantly expressed in the ovary. Expression of hsd17b3, hsd17b4, hsd17b12a, and hsd17b12b in the ovary increased during ovarian development. The enzymatic activities of Hsd17b3, Hsd17b12a, and Hsd17b12b were evaluated by expressing their recombinants in human embryonic kidney 293T cells. Hsd17b12a and Hsd17b12b catalyzed the conversion of androstenedione (AD) to testosterone (T) and estrone (E1) to E2. The results of in vitro bioassays using sardine ovaries indicated that E2 is synthesized from pregnenolone via AD and T, but not E1. These results suggest that Hsd17b12a plays a major role in E2 synthesis in sardine ovary by catalyzing the conversion of AD to T.

Roles of Ferric Peroxide Anion Intermediates (Fe3+O2 -, Compound 0) in Cytochrome P450 19A1 Steroid Aromatization and a Cytochrome P450 2B4 Secosteroid Oxidation Model.

Cytochrome P450 (P450, CYP) 19A1 is the steroid aromatase, the enzyme responsible for the 3-step conversion of androgens (androstenedione or testosterone) to estrogens. The final step is C-C bond scission (removing the 19-oxo group as formic acid) that proceeds via a historically controversial reaction mechanism. The two competing mechanistic possibilities involve a ferric peroxide anion (Fe3+O2 -, Compound 0) and a perferryl oxy species (FeO3+, Compound I). One approach to discern the role of each species in the reaction is with the use of oxygen-18 labeling, i.e., from 18O2 and H2 18O of the reaction product formic acid. We applied this approach, using several technical improvements, to study the deformylation of 19-oxo-androstenedione by human P450 19A1 and of a model secosteroid, 3-oxodecaline-4-ene-10-carboxaldehyde (ODEC), by rabbit P450 2B4. Both aldehyde substrates were sensitive to non-enzymatic acid-catalyzed deformylation, yielding 19-norsteroids, and conditions were established to avoid issues with artifactual generation of formic acid. The Compound 0 reaction pathway predominated (i.e., Fe3+O2 -) in both P450 19A1 oxidation of 19-oxo-androstenedione and P450 2B4 oxidation of ODEC. The P450 19A1 results contrast with our prior conclusions (J. Am. Chem. Soc. 2014, 136, 15016-16025), attributed to several technical modifications.

3ßHSD activity saturates at physiological substrate concentrations in intact cells.

BACKGROUND: Conversion of adrenally produced dehydroepiandrosterone (DHEA) to the potent androgen dihydrotestosterone (DHT) is an important mechanism by which prostate cancer reaches castration resistance. At the start of this pathway is a branch point at which DHEA can be converted to Δ4 -androstenedione by the enzyme 3ß-hydroxysteroid dehydrogenase (3ßHSD) or to Δ5 -androstenediol by 17ßHSD. To better understand this process, we studied the kinetics of these reactions in cells. METHODS: Prostate cancer cells (LNCaP cell line) were incubated with steroids (DHEA and Δ5 -androstenediol) over a range of concentrations and the steroid metabolism reaction products were measured by mass spectrometry or by high-performance liquid chromatography to determine reaction kinetics. To confirm the generalizability of results, experiments were also performed in JEG-3 placental choriocarcinoma cells. RESULTS: The two reactions displayed very different saturation profiles, with only the 3ßHSD-catalyzed reaction beginning to saturate within a physiological substrate concentration range. Strikingly, incubating LNCaP cells with low (in the ~10 nM range) concentrations of DHEA resulted in a large majority of the DHEA undergoing 3ßHSD-catalyzed conversion to Δ4 -androstenedione, whereas high concentrations of DHEA (in the 100s of nM range) resulted in most of the DHEA undergoing 17ßHSD-catalyzed conversion to Δ5 -androstenediol. CONCLUSION: Contrary to expectations from previous studies that used purified enzyme, cellular metabolism of DHEA by 3ßHSD begins to saturate in the physiological concentration range, suggesting that fluctuations in DHEA concentrations could be buffered at the downstream active androgen level.

Embryonic heart rate is affected by yolk androgens and egg laying sequence, and correlates with embryonic tissue growth: A study in rock pigeons.

Maternal androgen exposure can have crucial effects on offspring development. Bird eggs are frequently used for studying these effects and virtually all research in this field has focused on post-hatching offspring traits. Yet, much of the yolk, in which the maternal hormones are deposited, is consumed during the embryonic phase. Here, we studied the effects of yolk androgens during this prenatal period. As there is evidence that androgens stimulate post-hatching traits such as increased growth, we measured heart rate throughout incubation as a proxy for prenatal metabolism. Rock pigeons (Columba livia) typically lay 2-egg clutches with yolk androgen levels in second-laid eggs being consistently higher than in first-laid eggs. We investigated whether embryonic heart rate was higher in second- than first-laid eggs. Additionally, we increased yolk androgen levels (testosterone and androstenedione) with the mean difference between those in first- and second-laid eggs, to investigate whether the effects of androgens are egg sequence dependent. As expected, embryonic heart rate predicted body embryo organ- and body mass, and body dimensions, with body mass being significantly higher in second- than first-laid eggs. Androgen treated first-laid eggs increased heart rate to that of second-laid control eggs only temporally, yet it had an overall positive effect on embryo body dimensions but not on tissue mass. Our findings indicate that embryos from different egg laying sequence differed in heart rate and prenatal development outcomes but this can only partially be explained by their difference in maternal androgen levels.

Co-culture of human cryopreserved fragmented ovarian tissue with theca progenitor cells derived from theca stem cells.

PURPOSE: Despite the significant advances in the in vitro development of human primordial follicles, it is still a challenging approach with great potential for improvements. Therefore, the present study aimed to investigate the effect of a feeder layer of human theca progenitor cells (hTPCs) on the development of primordial follicles embedded in human ovarian tissue. METHODS: Fragments of frozen-thawed ovarian tissue were activated using the vanadate-derivative dipotassium bisperoxo (5-hydroxy-pyridine-2-carboxylic) oxovanadate (V) and kit ligand for 24 h. Then, the specimens were divided into the co-culture and mono-culture groups and were cultured with and without a hTPC feeder layer for 6 days, respectively. Afterward, the follicles were counted and classified, and the hormone levels and expression levels of apoptosis- and folliculogenesis-related genes were assessed. RESULTS: Both culture groups showed significant follicle growth (P < 0.05). However, the co-culture group had a significantly higher number of growing follicles compared to the other group (P < 0.05). Moreover, the expression levels of ZP1, ZP2, ZP3, BMP-7, AMH, and GDF9 were significantly higher in the co-culture group compared to the other group (P < 0.05), while the expression levels of P53 and CASP3 were significantly lower (P < 0.05). Also, the concentrations of estradiol, progesterone, testosterone, and androstenedione were significantly higher in the co-culture group compared to the other group (P < 0.05). CONCLUSION: The present study results provided novel evidence on the direct role of hTPCs in the growth and development of human primordial follicles. However, there is a need for future studies to illustrate the underlying mechanisms. Schematic summary of the results. According to our results, the expression of ZP1, ZP2, ZP3, and GDF9 in the oocytes, AMH in the granulosa cells, and BMP4 in the theca cells of the co-culture group were significantly higher than those of the mono-culture and non-culture groups, while the expression of apoptotic genes (BAX, CASP3, and P53) was significantly lower. Moreover, the co-culture group showed significantly increased levels of estradiol, progesterone, testosterone, and androstenedione in its culture media compared to the mono-culture groups.

VEGFA165 can rescue excess steroid secretion, inflammatory markers, and follicle arrest in the ovarian cortex of High A4 cows†.

A population of cows with excess androstenedione (A4; High A4) in follicular fluid, with follicular arrest, granulosa cell dysfunction, and a 17% reduction in calving rate was previously identified. We hypothesized that excess A4 in the ovarian microenvironment caused the follicular arrest in High A4 cows and that vascular endothelial growth factor A would rescue the High A4 phenotype. In trial 1, prior to culture, High A4 ovarian cortex (n = 9) had greater numbers of early stage follicles (primordial) and fewer later-stage follicles compared to controls (n = 11). Culture for 7 days did not relieve this follicular arrest; instead, High A4 ovarian cortex had increased indicators of inflammation, anti-Mullerian hormone, and A4 secretion compared to controls. In trial 2, we tested if vascular endothelial growth factor A isoforms could rescue the High A4 phenotype. High A4 (n = 5) and control (n = 5) ovarian cortex was cultured with (1) PBS, (2) VEGFA165 (50 ng/mL), (3) VEGFA165B (50 ng/mL), or (4) VEGFA165 + VEGFA165B (50 ng/mL each) for 7 days. Follicular progression increased with VEGFA165 in High A4 cows with greater early primary, primary, and secondary follicles than controls. Similar to trial 1, High A4 ovarian cortex secreted greater concentrations of A4 and other steroids and had greater indicators of inflammation compared to controls. However, VEGFA165 rescued steroidogenesis, oxidative stress, and fibrosis. The VEGFA165 and VEGFA165b both reduced IL-13, INFα, and INFß secretion in High A4 cows to control levels. Thus, VEGFA165 may be a potential therapeutic to restore the ovarian steroidogenic microenvironment and may promote folliculogenesis.

Follicle isolation methods reveal plasticity of granulosa cell steroidogenic capacity during mouse in vitro follicle growth.

Follicles are the functional unit of the ovary and several methods have been developed to grow follicles ex vivo, which recapitulate key events of oogenesis and folliculogenesis. Enzymatic digestion protocols are often used to increase the yield of follicles from the ovary. However, the impact of these protocols on the outermost theca and granulosa cells, and thereby follicle function, is not well defined. To investigate the impact of enzymatic digestion on follicle function, we collected preantral follicles from CD1 mice either by enzymatic digestion (Enzy-FL) or mechanical isolation (Mech-FL) and compared follicle growth, steroidogenesis and cell differentiation within an encapsulated in vitro follicle growth system which maintains the 3D architecture of the oocyte and its surrounding somatic cells. Follicles were encapsulated in 0.5% alginate and cultured for 8 days. Compared with Enzy-FL, Mech-FL grew more rapidly and produced significantly higher levels of androstenedione, estradiol and progesterone. The expression of theca-interstitial cell marker genes, Cyp17a1, which encodes 17-hydroxylase/17, 20-lyase and catalyzes the hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, and the conversion of these products into dehydroepiandrosterone and androstenedione, and Star, which encodes a transport protein essential for cholesterol entry into mitochondria, were also higher in Mech-FL than in Enzy-FL. Mech-FL maintained an intact theca-interstitial layer on the outer edge of the follicle that phenocopied in vivo patterns as confirmed by alkaline phosphatase staining, whereas theca-interstitial cells were absent from Enzy-FL from the onset of culture. Therefore, preservation of the theca cell layer at the onset of culture better supports follicle growth and function. Interestingly, granulosa cells in the outermost layers of Enzy-FL expressed CYP17A1 by Day 4 of culture while maintaining inhibin α-subunit expression and a cuboidal nucleus. Thus, in the absence of theca-interstitial cells, granulosa cells have the potential to differentiate into androgen-producing cells. This work may have implications for human follicle culture, where enzymatic isolation is required owing to the density of the ovarian cortex.

High androgen concentrations in follicular fluid of polycystic ovary syndrome women.

BACKGROUND: According to current definitions of Polycystic Ovary Syndrome (PCOS), hyperandrogenism is considered as a key element in the pathogenesis of this common endocrinopathy. However, until now, studies about ovarian androgen profile in women are very rare. Our aim was then to characterise the expression profile of the androgens in follicular fluid of 30 PCOS patients, and compare it to those of 47 Control women and 29 women with only polycystic ovary morphology on ultrasounds (ECHO group). METHODS: A retrospective, single-centre cohort study was performed. The intrafollicular concentrations of the key androgens were assessed and correlated with the intrafollicular levels of some adipokines of interest. Androgens were quantified by mass spectrophotometry combined with ultra-high-performance liquid chromatography, while adipokine concentrations were measured by ELISA assays. RESULTS: In PCOS patients, the intrafollicular concentrations of the androgens synthesised by ovarian theca cells, i.e., 17OH-pregnenolone, dehydroepiandrosterone, Δ4-androstenedione and testosterone, were significantly higher than those of the androgens of adrenal origin, and positively correlated with the main PCOS clinical and biological features, as well as with the adipokines mostly expressed in the follicular fluid of PCOS patients, i.e. resistin, omentin, chemerin and apelin. Conversely, Control women showed the highest levels of 17OH-progesterone, deoxycorticosterone and 11-deoxycortisol. Confirming these results, apelin levels were negatively associated with pregnenolone and deoxycorticosterone concentrations, while visfatin levels, which were higher in the Control group, negatively correlated with the Δ4-androstenedione and testosterone ones. CONCLUSIONS: PCOS is characterised by a selective increase in the intrafollicular levels of the androgens synthesised by theca cells, strengthening the hypothesis that ovarian hyperandrogenism plays a central role in its pathogenesis. Further, the significant correlation between the intrafollicular concentrations of the androgens and most of the adipokines of interest, including apelin, chemerin, resistin and omentin, confirms the existence of a close relationship between these two hormonal systems, which appear deeply involved in ovarian physiology and PCOS physiopathology.

Integrating the female masculinization and challenge hypotheses: Female dominance, male deference, and seasonal hormone fluctuations in adult blue-eyed black lemurs (Eulemur flavifrons).

In the decades since female social dominance was first described in strepsirrhine primates, researchers have sought to uncover the proximate and ultimate explanations for its development. In the females of various female-dominant species, androgens have been implicated as regulators of behavior and/or predictors of seasonal fluctuations in aggression (the 'Female Masculinization Hypothesis'). Males, more generally, respond to changing social demands via seasonal fluctuations in androgen-mediated behavior (the 'Challenge Hypothesis'), that may also entail changes in activation of the hypothalamic-pituitary-adrenal axis. Here, we explore if androgens, glucocorticoids, and intersexual behavior fluctuate seasonally in the female-dominant, blue-eyed black lemur (Eulemur flavifrons), with potential consequences for understanding female aggression and male deference. Across two studies conducted during the breeding and nonbreeding seasons, we assessed rates of mixed-sex, dyadic social behavior (aggression and affiliation) and concentrations of fecal glucocorticoid metabolites (Study 1) and serum sex hormones (androstenedione, testosterone, and estradiol; Study 2). Our results align with several predictions inspired by the Female Masculinization and Challenge Hypotheses for intersexual relations: During the breeding season, specifically, both aggression and androstenedione peaked in females, while female-initiated affiliation decreased, potentially to facilitate female resource access and reproductive control. By comparison, all target hormones (androgens, estrogen, and glucocorticoids) peaked in males, with glucocorticoid concentrations potentially increasing in response to the surge in female aggression, and unusually high estrogen concentrations year-round potentially facilitating male deference via male-initiated affiliation. These results suggest complex, seasonally and hormonally mediated behavior in Eulemur flavifrons.

High expression of CFTR in cumulus cells from mature oocytes is associated with high-quality of oocyte and subsequent embryonic development.

OBJECTIVE: The purpose of this study was to explore the association of expression of cystic fibrosis transmembrane conductance regulator (CFTR) in cumulus cells (CCs) from mature oocytes with oocyte quality and embryonic development. METHODS: A total of 338 infertile women who underwent ovarian stimulation cycle of oocyte retrieval in Zhejiang University School of Medicine were retrospectively enrolled in this study. The relative mRNA expression levels of CFTR, bone morphogenetic protein 15 (BMP15), and growth differentiation factor 9 (GDF9) in CCs were detected by qPCR technology. ROC curve was applied for the diagnosis of oocyte maturation. The serum levels of anti-Müllerian hormone (AMH), E2, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and androstenedione were measured. Oocyte maturation rate, fertilization rate, cleavage rate, high-quality embryo formation rate, and implantation rate after embryo transfer were also determined. RESULTS: The mRNA expression levels of CFTR in CCs were significantly increased in metaphase II (MII) oocytes compared to that in metaphase I (MI) or germinal vesicle (GV) oocytes. The ROC curve analysis illustrated that CFTR mRNA expression could efficiently discriminate MII oocytes from MI or GV oocytes (AUC = 0.954), and revealed that 0.695 RQU is the optimal cut-off value for diagnosis. So the cut-off value of 2-ΔΔCT = 0.70 was used to divide the patients into two groups: low- (n = 114) and high-CFTR group (n = 224). The mRNA expression of CFTR in CCs was positively correlated with the antral follicular count (AFC), number of oocytes retrieved, number of MII oocytes, serum E2 level on hCG day, and BMP15 and GDF9 expression in CCs. Under continuous stimulation with the same dose of recombinant follicle-stimulating hormone (rFSH), the number of follicles, average recovered oocytes, recovered oocytes, MII oocytes, as well as the oocyte recovery rate, fertilization rate, oocyte cleavage rate, high-quality embryo formation rate, and implantation rate were decreased in patients with lower CFTR. CONCLUSIONS: This study suggests that CFTR expression in CCs is associated with the developmental potential of human oocytes.

Biotransformation of Phytosterols into Androstenedione-A Technological Prospecting Study.

Androstenedione (AD) is a key intermediate in the body's steroid metabolism, used as a precursor for several steroid substances, such as testosterone, estradiol, ethinyl estradiol, testolactone, progesterone, cortisone, cortisol, prednisone, and prednisolone. The world market for AD and ADD (androstadienedione) exceeds 1000 tons per year, which stimulates the pharmaceutical industry's search for newer and cheaper raw materials to produce steroidal compounds. In light of this interest, we aimed to investigate the progress of AD biosynthesis from phytosterols by prospecting scientific articles (Scopus, Web of Science, and Google Scholar databases) and patents (USPTO database). A wide variety of articles and patents involving AD and phytosterol were found in the last few decades, resulting in 108 relevant articles (from January 2000 to December 2021) and 23 patents of interest (from January 1976 to December 2021). The separation of these documents into macro, meso, and micro categories revealed that most studies (articles) are performed in China (54.8%) and in universities (76%), while patents are mostly granted to United States companies. It also highlights the fact that AD production studies are focused on "process improvement" techniques and on possible modifications of the "microorganism" involved in biosynthesis (64 and 62 documents, respectively). The most-reported "process improvement" technique is "chemical addition" (40%), which means that the addition of solvents, surfactants, cofactors, inducers, ionic liquids, etc., can significantly increase AD production. Microbial genetic modifications stand out in the "microorganism" category because this strategy improves AD yield considerably. These documents also revealed the main aspects of AD and ADD biosynthesis: Mycolicibacterium sp. (basonym: Mycobacterium sp.) (40%) and Mycolicibacterium neoaurum (known previously as Mycobacterium neoaurum) (32%) are the most recurrent species studied. Microbial incubation temperatures can vary from 29 °C to 37 °C; incubation can last from 72 h to 14 days; the mixture is agitated at 140 to 220 rpm; vegetable oils, mainly soybean, can be used as the source of a mixture of phytosterols. In general, the results obtained in the present technological prospecting study are fundamental to mapping the possibilities of AD biosynthesis process optimization, as well as to identifying emerging technologies and methodologies in this scenario.

Mechanism of the Clinically Relevant E305G Mutation in Human P450 CYP17A1.

Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon-carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C17-C20 bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon-carbon bond scission.

Effects of neonatal exposure to methoxychlor on corpus luteum in gilts: A transcriptomic analysis.

This study investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities, on luteal function in pigs. Piglets were injected subcutaneously with MXC (20 µg/kg body weight) or corn oil (control) between postnatal Days 1 and 10 (N = 5/group). Corpora lutea from sexually mature gilts were examined for luteal steroid and prostaglandin concentrations and processed for total RNA isolation and subsequent RNA sequencing. Intra-luteal concentrations of androstenedione and prostaglandin E2 were greater, while that of estrone was lower when compared to control. Fifty-three differentially expressed (DE) microRNAS (miRNAs) (p-adjusted <.05 and log2(fold change) ≥.5) and 359 DE genes (p-adjusted <.05 and log2(fold change) ≥1) were identified in luteal tissue in response to neonatal MXC treatment. MXC was found to affect the expression of genes related to lipogenesis, steroidogenesis, membrane transport, immune response, cell signaling and adhesion. These results suggest an earlier onset of structural luteolysis in pigs caused by MXC actions in neonates. Since negative correlation analysis showed the potential interactions of miRNAs with specific messenger RNAs, we propose that these miRNAs are potential mediators of the long-term MXC effect on the CL function in pigs.

Functional differences in the hypothalamic-pituitary-gonadal axis are associated with alternative reproductive tactics based on an inversion polymorphism.

The evolution of social behavior depends on genetic changes, yet, how genomic variation manifests itself in behavioral diversity is still largely unresolved. Chromosomal inversions can play a pivotal role in producing distinct behavioral phenotypes, in particular, when inversion genes are functionally associated with hormone synthesis and signaling. Male ruffs exhibit alternative reproductive tactics (ARTs) with an autosomal inversion determining two alternative morphs with clear behavioral and hormonal differences from the ancestral morph. We investigated hormonal and transcriptomic differences in the pituitary and gonads. Using a GnRH challenge, we found that the ability to synthesize testosterone in inversion carriers is severely constrained, whereas the synthesis of androstenedione, a testosterone precursor, is not. Inversion morphs were able to produce a transient increase in androstenedione following the GnRH injection, supporting the view that pituitary sensitivity to GnRH is comparable to that of the ancestral morph. We then performed gene expression analyses in a second set of untreated birds and found no evidence of alterations to pituitary sensitivity, gonadotropin production or gonad sensitivity to luteinizing hormone or follicle-stimulating hormone across morphs. Inversion morphs also showed reduced progesterone receptor expression in the pituitary. Strikingly, in the gonads, inversion morphs over-expressed STAR, a gene that is located outside of the inversion and responsible for providing the cholesterol substrate required for the synthesis of sex hormones. In conclusion, our results suggest that the gonads determine morph-specific differences in hormonal regulation.

Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria.

4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2Δkstd211 + ΔkshB122, showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2kstd2 + Δkstd211+ΔkshB122 and HGMS2kshA51 + Δkstd211+ΔkshA226, by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

Occult symptomatic bilateral pure Leydig cell tumors in a postmenopausal woman: a case report.

BACKGROUND: Pure Leydig cell tumors (LCTs) represent 0.1% of ovarian masses. Postmenopausal patients typically present with virilization. Although LCTs can be challenging to locate on conventional imaging, positron emission tomography (PET) has been demonstrated to be effective. CASE: A 64-year-old postmenopausal woman presented with alopecia, facial hirsutism, and clitoromegaly. Laboratory findings included elevated testosterone and androstenedione. Ultrasound, computed tomography, and magnetic resonance imaging showed no adnexal masses. PET did not demonstrate ovarian fludeoxyglucose-avidity. Histopathology after bilateral salpingo-oophorectomy revealed bilateral Leydig cell tumors. Her testosterone normalized 2 weeks postoperatively. CONCLUSION: We describe the occult, symptomatic, bilateral ovarian Leydig cell tumors, an occurrence that has not been described in the literature. Virilizing tumors must be considered in patients with evidence of hyperandrogenism, even without pelvic masses on imaging.

Extracellular vesicles of bovine small follicular fluid promote ovarian cortical stromal cell proliferation and steroidogenesis.

The aim of this study was to investigate the effects of extracellular vesicles (EVs) on the proliferation and steroid hormone synthesis of bovine ovarian cortical stromal cells in vitro. The release and uptake of EVs are the new mechanisms of cell-to-cell communication. Using reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, TUNEL and other experiments, we found that EVs in bovine follicular fluid can promote the proliferation and synthesis of androstenedione and progesterone in ovarian cortical stromal cells. Moreover, 100 µg/ml EVs caused the most significant effect. We conclude that EVs at 100 µg/ml can significantly promote the proliferation and synthesis of androstenedione and progesterone in ovarian cortical stromal cells. This research is of great significance for further elucidating the regulatory role of follicular fluid EVs in follicular development and atresia and for research on the interaction of ovarian stromal cells, granulosa cells and oocytes.

Theca cell-conditioned medium enhances steroidogenesis competence of buffalo (Bubalus bubalis) granulosa cells.

Theca cells (TCs) play a crucial role in follicular development and atresia. TCs synthesize androgens that act as substrate for granulosa cells (GCs) aromatization to oestrogens needed for follicular growth. However, the effects of TCs in the form of conditioned medium on steroidogenesis in buffalo GCs remain unclear. In the present study, the impacts of TC-conditioned medium (TCCM) on oestrogen synthesis in buffalo GCs were examined. The results showed that TCs secreted principally testosterone, but almost no androstenedione or oestradiol into TCCM. TCs at passage 3 had a stronger secretion capacity of testosterone in TCCM. Furthermore, TCCM collected at 72 hr improved both the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1, 3ß-HSD and 17ß-HSD) and the secretion levels of estradiol in GCs. The treatment of 72 hr in TCCM promoted both the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1 and 3ß-HSD) and the secretion levels of estradiol in GCs. Besides, TCCM that was collected at 72 hr and applied to GCs for 72 hr (72 & 72 hr) improved the sensitivity of buffalo GCs to FSH. This study indicates that TCCM (72 & 72 hr) enhances the steroidogenesis competence of GCs mainly through facilitating the responsiveness of GCs to FSH in buffalo.