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Endogenous opioids are found in the central nervous system and multiple peripheral organs and affect the hypothalamus–pituitary–gonad axis at several levels in vertebrates. However, the influence of these opioid peptides on fish spermatogenesis is poorly studied. This investigation examined the impact of the opioid peptide dynorphin-A (DYN) on the pituitary–testicular axis in the tilapia Oreochromis mossambicus. Intraperitoneal injections of 5 µg DYN/day/fish for 21 days did not affect the early developmental stages of germ cells, but caused a significant decrease in the numbers of secondary spermatocytes and early and late spermatids compared with the controls. Although the mean area of the lobule was significantly decreased, there was a significant increase in the areas of the lumen and the interstitium in the DYN-treated group relative to controls. These results were concomitant with significantly lower serum levels of 11-ketotestosterone (11-KT) and luteinizing hormone (LH) in DYN-treated fish than in controls. However, the serum level of cortisol was significantly higher in the DYN-treated fish group. Overall, these results, for the first time, indicate that DYN interferes with spermatogenesis at the late stage of meiosis, leading to impairment in spermiogenesis, possibly mediated through the suppression of 11-KT secretion in the testis and LH release from the pituitary. This study also suggests that DYN may bring about this inhibitory effect via activation of the stress axis in teleosts. [ABSTRACT FROM AUTHOR]

The most suitable biochemical markers for therapy adjustment in patients with congenital adrenal hyperplasia are controversial. 11-Oxygenated androgens are a promising new approach. The objective of this study was to investigate the diurnal rhythm of 11-ketotestosterone in children and adolescents in saliva and to correlate it with salivary 17-hydroxyprogesterone. Fifty-one samples of steroid day-profiles from 17 patients were additionally analysed for 11-ketotestosterone, retrospectively. All patients were treated in our university outpatient clinic for paediatric endocrinology between 2020 and 2022. Steroid day-profiles of 17 patients could be examined. The cohort showed a balanced sex ratio. The median age was 13 years. The measurements for 17-hydroxyprogesterone were carried out during routine care by immunoassay. The measurements of 11-ketotestosterone were performed from frozen saliva samples using an implemented in-house protocol for liquid chromatography–tandem mass spectrometry (LC-MS/MS). The most important outcome were the absolute values for 11-ketotestosterone, their diurnal rhythmicity and the correlation with 17-hydroxyprogesterone. Both steroids show a circadian diurnal rhythm. 17-hydroxyprogesterone and 11-ketotestosterone correlate significantly. 11-Ketotestosterone showed a positive correlation with BMI at all times of the day. 11-Ketotestosterone shows circadian rhythmicity in our cohort and correlates with 17-hydroxyprogesterone. These findings serve as an important basis for prospective research into 11-oxygenated androgens as therapeutic markers in paediatrics. However, 11-ketotestosterone appears to be very dependent on BMI. [ABSTRACT FROM AUTHOR]

Aldo‐keto reductase 1C3 (AKR1C3) is a key enzyme in the activation of both classic and 11‐oxygenated androgens. In adipose tissue, AKR1C3 is co‐expressed with 11β‐hydroxysteroid dehydrogenase type 1 (HSD11B1), which catalyzes not only the local activation of glucocorticoids but also the inactivation of 11‐oxygenated androgens, and thus has the potential to counteract AKR1C3. Using a combination of in vitro assays and in silico modeling we show that HSD11B1 attenuates the biosynthesis of the potent 11‐oxygenated androgen, 11‐ketotestosterone (11KT), by AKR1C3. Employing ex vivo incubations of human female adipose tissue samples we show that inhibition of HSD11B1 results in the increased peripheral biosynthesis of 11KT. Moreover, circulating 11KT increased 2–3 fold in individuals with type 2 diabetes after receiving the selective oral HSD11B1 inhibitor AZD4017 for 35 days, thus confirming that HSD11B1 inhibition results in systemic increases in 11KT concentrations. Our findings show that HSD11B1 protects against excess 11KT production by adipose tissue, a finding of particular significance when considering the evidence for adverse metabolic effects of androgens in women. Therefore, when targeting glucocorticoid activation by HSD11B1 inhibitor treatment in women, the consequently increased generation of 11KT may offset beneficial effects of decreased glucocorticoid activation. [ABSTRACT FROM AUTHOR]

Adrenosterone (Androst-4-ene-3,11,17-trione, 11OXO) is forbidden in sports according to the Prohibited List of the World Anti-Doping Agency. The administration of 11OXO may be detected by monitoring the urinary concentrations of its main human metabolites 11β-hydroxy-androsterone and 11β-hydroxy-etiocholanolone. Preliminary urinary concentration and concentration ratio thresholds have been established for sports drug testing purposes, but adaptations are desirable as the suggested limits would result in numerous suspicious findings due to naturally elevated concentrations and ratios. Recently, the metabolism of 11-oxo-testosterone (KT) was investigated in the context of anti-doping research, resulting in a preliminary urinary concentration threshold and a confirmation procedure based on the determination of carbon isotope ratios (CIRs). Gas chromatography coupled to isotope ratio mass spectrometry was employed to investigate the CIRs of selected steroids. As KT is also a metabolite of 11OXO, the developed protocols for KT have been tested to elucidate their potential to detect the administration of 11OXO after a single oral dose of 100 mg. In order to further improve the analytical approach, the threshold for urinary concentrations of KT was re-investigated by employing a reference population of n = 5232 routine doping control samples. Quantification of urinary steroids was conducted by employing gas chromatography coupled to triple quadrupole mass spectrometry. Derived from these, a subset of n = 106 samples showing elevated concentrations of KT was investigated regarding their CIRs. By means of this, potentially positive samples due to the illicit administration of 11OXO or KT could be excluded, and the calculation of reference population-derived thresholds for the concentrations and CIR of KT was possible. Based on the results, the urinary concentration threshold for KT is suggested to be established at 130 ng/mL. [ABSTRACT FROM AUTHOR]

Adrenosterone (Androst-4-ene-3,11,17-trione, 11OXO) is forbidden in sports according to the Prohibited List of the World Anti-Doping Agency. The administration of 11OXO may be detected by monitoring the urinary concentrations of its main human metabolites 11β-hydroxy-androsterone and 11β-hydroxy-etiocholanolone. Preliminary urinary concentration and concentration ratio thresholds have been established for sports drug testing purposes, but adaptations are desirable as the suggested limits would result in numerous suspicious findings due to naturally elevated concentrations and ratios. Recently, the metabolism of 11-oxo-testosterone (KT) was investigated in the context of anti-doping research, resulting in a preliminary urinary concentration threshold and a confirmation procedure based on the determination of carbon isotope ratios (CIRs). Gas chromatography coupled to isotope ratio mass spectrometry was employed to investigate the CIRs of selected steroids. As KT is also a metabolite of 11OXO, the developed protocols for KT have been tested to elucidate their potential to detect the administration of 11OXO after a single oral dose of 100 mg. In order to further improve the analytical approach, the threshold for urinary concentrations of KT was re-investigated by employing a reference population of n = 5232 routine doping control samples. Quantification of urinary steroids was conducted by employing gas chromatography coupled to triple quadrupole mass spectrometry. Derived from these, a subset of n = 106 samples showing elevated concentrations of KT was investigated regarding their CIRs. By means of this, potentially positive samples due to the illicit administration of 11OXO or KT could be excluded, and the calculation of reference population-derived thresholds for the concentrations and CIR of KT was possible. Based on the results, the urinary concentration threshold for KT is suggested to be established at 130 ng/mL.

To induce oocyte development, eels are weekly injected with salmon or carp pituitary extract (CPE). The weekly handling and hormone peaks result in inferior oocyte quality; therefore, alternative treatments that improve oocyte quality and reproductive success require investigation. The enhancement of early sexual maturation by a single injection with human chorionic gonadotropin (hCG), administered prior to CPE treatment, was investigated. Fifty feminized eels were subjected to simulated migration, after which eels received either a hCG or a sham injection. After two months, the hCG-treated eels showed an increase in eye size, gonadosomatic index (GSI), and plasma 11-ketotestosterone concentration, when compared with the sham-injected controls. The hCG-treated eels showed increases in oocyte diameter and lipid area, and in ovarian expression of aromatase (cyp19), follicle stimulating hormone receptor (fshr) and lipoprotein lipase (lpl). Yolk was present in the oocytes of the hCG-treated eels, not yet in the oocytes of the controls. The hCG-induced deposition of yolk may relate to early-life treatment with 17β-estradiol during feminization. hCG-treated eels required four CPE injections less to mature than the controls. hCG treatment may benefit reproductive success in feminized eels by initiating vitellogenesis and reducing the hypophysation period, although larvae were obtained from most females in both groups. [ABSTRACT FROM AUTHOR]

The anabolic properties of 11‐hydroxyandrostenedione (OHA4) and its physiologically active metabolites 11‐ketotestosterone (KT) and 11‐ketodihydrotestosterone (KDHT) have been discussed in several recent publications. Especially KT has become readily available via internet‐based providers. No doping control methods for the detection of KT or KDHT exist, neither on the initial testing procedure level nor as confirmatory assay. Probing for the misuse of adrenosterone, the prohormone of OHA4, has already been addressed, and the suggested marker for its misuse was mainly the urinary concentration of 11‐hydroxyandrosterone (OHA). In addition, for confirmation purposes, the carbon isotope ratios (CIR) were taken into consideration. The urinary concentration of OHA is highly variable, and the endogenous dilution after exogenous administration may therefore be considerable; hence, described approaches resulted in short detection times. In this study, the human metabolism of KT was investigated in order to provide additional means for the detection of KT and its prohormone OHA4. Two volunteers (one female and one male) orally administered 20 mg of KT each, and urine samples were collected for 5 days. Urinary concentrations of KT and its metabolites were investigated, and a reference population encompassing 220 male and female athletes was investigated in order to elucidate preliminary thresholds. As confirmation procedure, an isotope ratio mass spectrometry‐based method was developed in order to determine the CIR of KT and relevant metabolites. The developed methods enabled the detection of exogenous KT for more than 20 h after a single oral administration, which is comparable to a single oral testosterone administration. [ABSTRACT FROM AUTHOR]

For many decades, the prevailing paradigm in endocrinology was that testosterone and 5a-dihydrotestosterone are the only potent androgens in the context of human physiology. The more recent identification of adrenal derived 11-oxygenated androgens and particularly 11-ketotestosterone have challenged these established norms, prompting a revaluation of the androgen pool, particularly in women. Since being recognized as bone fide androgens in humans, numerous studies have focused their attention on understanding the role of 11-oxygenated androgens in human health and disease and have implicated them as role players in conditions such as castration resistant prostate cancer, congenital adrenal hyperplasia, polycystic ovary syndrome, Cushing's syndrome, and premature adrenarche. This review therefore provides an overview of our current knowledge on the biosynthesis and activity of 11-oxygenated androgens with a focus on their role in disease states. We also highlight important analytical considerations for measuring this unique class of steroid hormone. [ABSTRACT FROM AUTHOR]

Prolactin and 11‐ketotestosterone (11‐KT) are important reproductive hormones in fishes, which may also influence immunocompetence. The immunocompetence handicap hypothesis states that higher androgen concentrations that support secondary sex traits are traded off against a decrease in immune system function. To test the relationships between these hormones and immunocompetence, we experimentally manipulated 11‐ketotestosterone and prolactin in the freshwater fish, bluegill (Lepomis macrochirus) during parental care using implants that contained either 11‐KT, prolactin, or an inert control. We vaccinated individuals to stimulate the acquired immune response, then measured immunocompetence as the number of granulocytes, lymphocytes and monocytes, and the expression of interleukin 8 in each sample. We did not observe any significant differences in the immune measures among the hormone treatments. Our results indicate that in bluegill, there is no trade‐off between androgens or prolactin and immunocompetence. Research Highlights: Prolactin or androgen (11‐KT) treatment did not affect immune responses in bluegill sunfish.We find no support the immunocompetence handicap hypothesis in fish.Increased 11‐KT affected behavior, but not the immune response to a vaccine. [ABSTRACT FROM AUTHOR]

In order to provide reliable indicators of the spawning season of captive helicopter catfish (Wallago attu), this study evaluated the temporal variation in gonadosomatic index (GSI), plasma levels of testosterone (T), and 11-ketotestosterone (11-KT) in male broodstock in captivity. GSI was estimated as the percentage of the relative weight of testis to total body weight. Plasma levels of sex steroids were determined by enzyme-linked immunosorbent assay (EIA). Testis samples were dehydrated and embedded in paraffin, then sectioned at 5 μm thickness. The highest level of T (402.1 ± 16.7 pg/mL) was found in June, followed by a peak in 11-KT level (76.9 ± 4.7 pg/mL) in May. Testes containing the highest concentrations of spermatozoa were observed from June to August. The GSI of males increased significantly from January to June and peaked in July (2.14%). Taken together, we conclude that the spawning season of captive helicopter catfish occurs from June to August. These results will contribute to the basic knowledge of the reproductive biology of helicopter catfish, which can be useful in artificial breeding.

Introduction: 11-ketotestosterone (11KT), which is derived by the bioconversion of testosterone via 11b-hydroxytestosterone (11OHT), is a potent agonist of the human androgen receptor. The adrenal gland is considered an important organ in 11KT production because CYP11B1, which catalyzes testosterone to 11OHT, is expressed in the adrenal glands. The present study aimed to demonstrate adrenal gland involvement in 11KT production in prepubertal children, a topic which has not yet been addressed by any previous studies. Methods: Three, retrospective, observational studies were performed. Study 1 enrolled patients aged 8 months to 7 years with severe Kawasaki disease (KD) who were treated with mPSL pulse. Studies 2 and 3 included patients who had received a corticotropin-releasing hormone (CRH) stimulation test and adrenocorticotropic hormone (ACTH) stimulation test, respectively. Samples were collected before and after treatment or drug administration, and serum 11KT, 11OHT, and other 11- oxygenated androgens were measured by LC-MS/MS. Steroid hormone values before and after medication were analyzed using the Wilcoxon signed rank test. Results: Studies 1, 2, and 3 included twenty patients with severe KD, eight patients with a CRH stimulation test, and eight patients with an ACTH stimulation test, respectively. Study 1 demonstrated that the median (IQR) 11KT level was significantly higher before, than after, mPSL pulse (0.39 (0.28-0.47) nmol/L versus 0.064 (0.012-0.075) nmol/L; P < 0.001). Studies 2 and 3 indicated no significant difference in the median 11KT value before and after the CRH or ACTH stimulation test while the 11OHT value was significantly higher after the test. Conclusion: In conclusion, the mediation of 11KT production by ACTH demonstrated the importance of the adrenal glands in the synthesis of this androgen in prepubertal children. [ABSTRACT FROM AUTHOR]

Introduction11-ketotestosterone (11KT), which is derived by the bioconversion of testosterone via 11β-hydroxytestosterone (11OHT), is a potent agonist of the human androgen receptor. The adrenal gland is considered an important organ in 11KT production because CYP11B1, which catalyzes testosterone to 11OHT, is expressed in the adrenal glands. The present study aimed to demonstrate adrenal gland involvement in 11KT production in prepubertal children, a topic which has not yet been addressed by any previous studies.MethodsThree, retrospective, observational studies were performed. Study 1 enrolled patients aged 8 months to 7 years with severe Kawasaki disease (KD) who were treated with mPSL pulse. Studies 2 and 3 included patients who had received a corticotropin-releasing hormone (CRH) stimulation test and adrenocorticotropic hormone (ACTH) stimulation test, respectively. Samples were collected before and after treatment or drug administration, and serum 11KT, 11OHT, and other 11-oxygenated androgens were measured by LC-MS/MS. Steroid hormone values before and after medication were analyzed using the Wilcoxon signed rank test.ResultsStudies 1, 2, and 3 included twenty patients with severe KD, eight patients with a CRH stimulation test, and eight patients with an ACTH stimulation test, respectively. Study 1 demonstrated that the median (IQR) 11KT level was significantly higher before, than after, mPSL pulse (0.39 (0.28-0.47) nmol/L versus 0.064 (0.012-0.075) nmol/L; P < 0.001). Studies 2 and 3 indicated no significant difference in the median 11KT value before and after the CRH or ACTH stimulation test while the 11OHT value was significantly higher after the test.ConclusionIn conclusion, the mediation of 11KT production by ACTH demonstrated the importance of the adrenal glands in the synthesis of this androgen in prepubertal children.

Context: Wiedemann-Steiner Syndrome (WSS) is a genetic disorder associated with an array of clinical phenotypes, including advanced bone age and short stature. 11-ketotestosterone (11KT) is a member of the group known as 11-oxygenated C19 androgens that are implicated in premature adrenarche. Case description: Case 1: The patient is a 3 year and 11-month-old female diagnosed with WSS due to deletion of KMT2A detected on CGH microarray. At two years and 11 months, imaging revealed an advanced bone age. We obtained an 11KT level on this patient. 11KT in case 1 was elevated at 26.3 ng/dL, while the normal reference range is 7.3-10.9 ng/dL and the reference interval for premature adrenarche is 12.3-22.9 ng/dL, The repeat 11KT at follow up (chronological age 4 years and 6 months) was still elevated at 33.8 ng/dL Case 2: A second child with WSS and a 5kb intragenic KMT2A deletion was evaluated at 11 months of age; his 11KT was 4.5 ng/dL. Conclusions: The elevated 11KT may indicate maturational changes related to increasing adrenal gland androgenic activation and may explain the advanced bone age seen in some patients with WSS. To our knowledge, this is the first case report that describes 11KT as a bioactive androgen potentially causing bone age advancement in WSS. Lack of elevation of 11KT in the second child who is an infant suggests increasing androgenic precursors and metabolites related to premature adrenarche may need to be longitudinally followed. [ABSTRACT FROM AUTHOR]

Ghrelin, a peptide found in the brain and gut, is predicted to play a significant role in the control of various physiological systems in fish. The objective of this study was to examine the impact of ipamorelin acetate (IPA), a ghrelin agonist, on the reproductive axis of the tilapia Oreochromis mossambicus. The administration of either 5 or 30 µg of IPA for 21 days led to a significant and dose-dependent rise in food intake concomitant with a significant increase in the numbers of primary spermatocytes, secondary spermatocytes, and early spermatids compared to the control group. There was a significant rise in the number of late spermatids, as well as the areas of the lobule and lumen, in fish treated with 30 µg of IPA, compared to the control group. Moreover, there was no significant difference in the percentage of gonadotropin-releasing hormone (GnRH)-immunoreactive fibres in the hypothalamus and anterior pituitary gland across different groups. However, a significant elevation in the expression of androgen receptor protein was observed in fish treated with 30 µg of IPA. Furthermore, the concentrations of luteinizing hormone (LH) and 11-ketotestosterone (11-KT) in the serum of fish treated with either 5 or 30 µg of IPA were significantly elevated in comparison to the control group. Collectively, these findings suggest that the administration of ghrelin enhances the development of germ cells during the meiosis-I phase and that this effect might be mediated via the stimulation of 11-KT and androgen receptors at the testicular level and LH at the pituitary level in the tilapia. • IPA treatment stimulates secretion of LH and 11-KT in tilapia. • Treatment of IPA stimulates germ cells progression at the meiosis stage. • IPA treatment increases androgen receptor protein expression in the testis. • Food consumption rate shows strong positive correlation with LH and 11-KT. [ABSTRACT FROM AUTHOR]

ContextWiedemann-Steiner Syndrome (WSS) is a genetic disorder associated with an array of clinical phenotypes, including advanced bone age and short stature. 11-ketotestosterone (11KT) is a member of the group known as 11-oxygenated C19 androgens that are implicated in premature adrenarche.Case descriptionCase 1: The patient is a 3 year and 11-month-old female diagnosed with WSS due to deletion of KMT2A detected on CGH microarray. At two years and 11 months, imaging revealed an advanced bone age. We obtained an 11KT level on this patient. 11KT in case 1 was elevated at 26.3 ng/dL, while the normal reference range is 7.3-10.9 ng/dL and the reference interval for premature adrenarche is 12.3-22.9 ng/dL, The repeat 11KT at follow up (chronological age 4 years and 6 months) was still elevated at 33.8 ng/dL Case 2: A second child with WSS and a 5kb intragenic KMT2A deletion was evaluated at 11 months of age; his 11KT was 4.5 ng/dL.ConclusionsThe elevated 11KT may indicate maturational changes related to increasing adrenal gland androgenic activation and may explain the advanced bone age seen in some patients with WSS. To our knowledge, this is the first case report that describes 11KT as a bioactive androgen potentially causing bone age advancement in WSS. Lack of elevation of 11KT in the second child who is an infant suggests increasing androgenic precursors and metabolites related to premature adrenarche may need to be longitudinally followed.

In the present study, we aimed to examine the cellular structure of the testes, the hormonal changes occurring throughout the reproductive cycle, and the fatty acid composition in rainbow trout. An electron microscopic analysis was conducted to investigate the morphology and cellular changes during the development of the testes. Germ cells were observed in different stages of differentiation: primary spermatogonia (PSGs), secondary spermatogonia (SSGs), primary spermatocytes (PSCs), secondary spermatocytes (SSCs), spermatids (SPDs) and spermatozoa (SZs). Differentiated spermatozoa were observed within the cysts. Regarding steroid hormones, the maximum level of testosterone was in the mature stage, and the minimum level was in the spent stage; for 11‐ketotestosterone, the highest level was in the maturing stage and the lowest level was in the spent stage. In terms of the fatty acid composition, the levels of C14, C15, C16, C18, C16:1n7, C18:1n7, C18:1n9, C20:4n6, n‐3 PUFAs, n‐6 PUFAs, C20:5n‐3 (EPA), C22:5n‐3 (DPA) and C22:6n3 (DHA) (g−1 of gonad wet weight) were the lowest during the immature stage and the highest during the mature stage of the testes. The major significance of the study is the sex steroid hormonal profile, the primary regulators of spermatogenesis and the fatty acid composition, which is important for the development of gonads and sperm. These data should contribute to the knowledge regarding the reproductive biology of male rainbow trout, for which research data remain scarce. [ABSTRACT FROM AUTHOR]

Pituitary gonadotropins, metabolic hormones, and sex steroids are known factors affecting the advanced stages of ovarian development in teleost fish. However, the effects of these hormones and of the interactions between them on the growth of previtellogenic ovarian follicles are not known. In order to address this void in understanding, previtellogenic ovarian fragments from eel, Anguilla australis, were incubated in vitro with recombinant Japanese eel follicle-stimulating hormone (rec-Fsh), human chorionic gonadotropin (hCG), or 11-ketotestosterone (11-KT) in the presence or absence of recombinant human insulin-like growth factor-1 (IGF1). The results of long-term in vitro culture (21 days) demonstrated that rec-Fsh and 11-KT, rather than hCG, caused significant increases in the diameter of previtellogenic oocytes. Meanwhile, only 11-KT induced a significant increase in lipid accumulation. Moreover, a greater effect on oocyte growth was observed when IGF1 supplementation was combined with 11-KT rather than with rec-Fsh or hCG. For short-term culture (24 h), treatment with 11-KT in the presence or absence of IGF1 had no significant effects on mRNA levels of target genes (lhr, cyp19, cyp11b, lpl, and ldr) except for upregulation of fshr. There were no significant effects of rec-Fsh on expression of any target gene, whereas hCG downregulated the expression of these genes. There was no evidence for any interaction between the gonadotropins and IGF1 that resulted in growth of previtellogenic oocytes. Taken together, these results suggest that hormones from both the reproductive and the metabolic axes regulate the growth of previtellogenic oocytes in Anguilla australis. [ABSTRACT FROM AUTHOR]

11-Oxyandrogens, such as 11-ketotestosterone (11-KT), 11-ketodihydrotestosterone (11-KDHT), 11β-hydroxytestosterone (11-OHT), 11β-hydroxyandrostenedione (11-OHA4), and 11-KA4, are newly specified human androgens. These 11-oxyandrogens are present in the cord blood and placenta, as well as in the blood of men and women of various ages, and are produced primarily in the adrenal gland. Accumulating evidence suggests that these steroids contribute to androgen excess in patients with 21-hydroxylase deficiency or polycystic ovary syndrome. More importantly, unlike classic androgens, 11-oxyandrogens produced in maternal tumors can pass through the placenta without being converted into estrogens, and cause severe virilization of female fetuses. Thus, overproduction of 11-oxyandrogens represents a new mechanism of 46,XX disorders of sex development. On the other hand, the physiological roles of 11-oxyandrogens remain to be clarified. This mini-review introduces the current understanding of 11-oxyandrogens, from the perspective of pediatric endocrinology [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) and idiopathic hirsutism (IH) are androgen excess disorders requiring the determination of classic androgen levels for diagnosis. 11-oxygenated androgens have high androgenic potential, yet their clinical value in those disorders is not clear. Additionally, the role of endocrine disruptors (EDs), particularly in IH, remains understudied. We analyzed 25 steroids and 18 EDs in plasma samples from women with IH, PCOS, and controls using LC-MS/MS. Cytokine levels and metabolic parameters were assessed. Comparisons included non-obese women with PCOS (n = 10), women with IH (n = 12) and controls (n = 20), and non-obese versus obese women with PCOS (n = 9). Higher levels of 11-oxygenated androgens were observed in women with PCOS compared to those with IH, but not controls. Conversely, 11-oxygenated androgen levels were lower in women with IH compared to controls. Cytokine levels did not differ between women with IH and controls. Bisphenol A (BPA) levels were higher in obese women with PCOS compared to non-obese women with PCOS. Bisphenol S occurrence was higher in women with PCOS (90%) compared to controls (65%) and IH (50%). Significant correlations were found between androgens (11-ketotestosterone, androstenedione, testosterone) and insulin and HOMA-IR, as well as between immunomodulatory 7-oxygenated metabolites of DHEA and nine interleukins. Our data confirms that PCOS is a multiendocrine gland disorder. Higher BPA levels in obese women might exacerbate metabolic abnormalities. IH was not confirmed as an inflammatory state, and no differences in BPA levels suggest BPA does not play a role in IH pathogenesis.

Simple Summary: Endocrine regulation has been thought to play a major role in the onset of migration. Anguillid eels provide a good model for studying the onset mechanisms of migrations to breeding areas, because the process of the onset of migration occurs in inland waters. In this review, we summarize information about the silvering process in anguillid eels and the dynamics of mRNA expression of neurohormones and pituitary hormones, thyroid hormones, and sex steroids associated with the onset of the spawning migration. We also provide new results. Because 11-KT drastically increases during silvering, the role of 11-KT in the onset of spawning migration was discussed in detail. Anguillid eels are the iconic example of catadromous fishes, because of their long-distance offshore spawning migrations. They are also a good model for research on the onset mechanisms of migrations to breeding areas, because the migrations begin in inland waters. When eels transform from yellow eels to silver eels, it is called silvering. Silver eels show various synchronous external and internal changes during silvering, that include coloration changes, eye-size increases, and gonadal development, which appear to be pre-adaptations to the oceanic environment and for reproductive maturation. A strong gonadotropic axis activation occurs during silvering, whereas somatotropic and thyrotropic axes are not activated. Among various hormones, 11-ketotestosterone (11-KT) drastically increases during spawning migration onset. Gradual water temperature decreases simulating the autumn migratory season, inducing 11-KT increases. Administration of 11-KT appeared to cause changes related to silvering, such as early-stage oocyte growth and eye enlargement. Moreover, 11-KT may be an endogenous factor that elevates the migratory drive needed for the spawning migration onset. These findings suggested that water temperature decreases cause 11-KT to increase in autumn and this induces silvering and increases migratory drive. In addition, we newly report that 11-KT is associated with a corticotropin-releasing hormone that influences migratory behavior of salmonids. This evidence that 11-KT might be among the most important factors in the spawning migration onset of anguillid eels can help provide useful knowledge for understanding endocrinological mechanisms of the initiation of spawning migrations. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disease in women of childbearing age, with an incidence of 5–10%. This study compared the traits of zebrafish with three diagnostic criteria for human PCOS, and the diagnostic criteria for zebrafish PCOS were proposed: decreased fecundity, elevated testosterone (T) or 11-ketotestosterone (11-KT) levels and increased cortical-alveolar oocyte (CO) ratio, enhancing the zebrafish PCOS model's accuracy. According to the mammalian PCOS classification, the type of zebrafsh PCOS is divided into four phenotypes (A, B, C and D), but the four phenotypes of zebrafish PCOS are not fully covered in the existing studies (A and D). In this study, we successfully induced phenotype B zebrafish PCOS model using the aromatase inhibitor, letrozole (LET). That is, wild-type female zebrafish were exposed to 1000 μg/L LET for 30 days. Reproductive tests showed decreased fecundity in female zebrafish exposed to LET (Control: 132.63, 146.00, 173.00; LET: 29.20, 90.00, 82.71). Hormone analysis showed that female zebrafish exposed to LET had significantly lower 17β-estradiol/testosterone (E2/T) ratios, indicating elevated T levels. Meanwhile, levels of 11-KT in the ovaries exposed to LET were significantly up-regulated (Control: 0.0076 pg/μg; LET: 0.0138 pg/μg). Pathological sections of the ovary showed fewer CO in the LET-exposed group (Control: 16.27%; LET: 8.38%). In summary, the zebrafish PCOS model summarized and studied in this study provide a reliable and economical tool for the screening of therapeutic drugs, as well as for the etiology research and treatment strategies of PCOS. [Display omitted] • Letrozole (LET) exposure led to reduced fecundity in female zebrafish. • LET exposure led to elevated levels of 11-ketotestosterone and testosterone in female zebrafish. • LET exposure affected ovary maturation in female zebrafish. • LET exposure induced phenotype B polycystic ovary syndrome (PCOS) model in zebrafish. [ABSTRACT FROM AUTHOR]

In vertebrates, opioid peptides are thought to be involved in the regulation of reproduction; however, the significance of enkephalins in testicular function remains unclear. We examined the influence of δ-opioid receptor agonist leucine enkephalin (L-ENK) on the hypophysial-testicular axis of the cichlid fish Oreochromis mossambicus. Treatment with a low dose of L-ENK (60 µg) caused a significant increase in the numbers of primary and secondary spermatocytes and early and late spermatids, concomitant with intense immunolabelling of testicular androgen receptors, but did not significantly alter serum luteinizing hormone (LH) and 11-ketotestosterone (11-KT) levels compared to those of controls. Nevertheless, treatment with a high dose of L-ENK (200 µg) caused a significant reduction in the numbers of secondary spermatocytes as well as late spermatids associated with marginal immunolabelling of androgen receptors and significantly lower concentrations of serum 11-KT and LH compared to controls. In addition, the serum cortisol level was not affected in low-dose L-ENK-treated fish, but its level was significantly increased in the high-dose L-ENK-treated group. Together, these findings indicate that a low dose of L-ENK stimulates the germ cells at the meiosis stage and promotes further stages of spermatogenesis, whereas a high concentration of L-ENK inhibits spermatogenesis at the advanced stages. This effect appears to be mediated through the suppression of testicular steroidogenesis and the reduction of LH release in the pituitary gland of tilapia. The findings also suggest that elevated L-ENK levels in teleosts may exert their inhibitory influence on the hypophysial-testicular axis via glucocorticoids. • L-ENK exerts both stimulatory and inhibitory effects on spermatogenesis. • A high dose of L-ENK suppresses 11-KT and LH secretion and androgen receptors. • A low dose of L-ENK promotes spermatogonial recruitment for meiosis. • L-ENK at a high dose augments serum cortisol level. [ABSTRACT FROM AUTHOR]

To efficiently produce seedlings in aquaculture farms, it is essential to control the ratio of males and females by sex distinguishing. On the other hand, the concentration of 11-ketotestosterone (11-KT), which is contained in the plasma, would be an excellent indicator that is significantly different between males and female fish. However, the concentration of 11-KT is challenging to measure due to its low concentration. In this study, we attempted to develop a high-sense biosensor system enhanced by single-walled carbon nanotubes (SWCNTs) with high conductivity and improved the efficiency and accuracy of fish gender determination. First, a self-assembled monolayer (SAM)was formed on the surface of a disk-type gold electrode, and an anti-rabbit IgG was immobilized on the SAM end. Next, an anti-11-KT antibody was immobilized on this IgG antibody, and then SWCNTs were immobilized on the electrode surface to fabricate the biosensor. Then, we attempted to quantify 11-KT by analyzing the minor electrochemical changes on the electrode surface before and after the reaction of an anti-11-KT antibody with 11-KT as an antigen by cyclic voltammetry. Next, after examining the influence of various conditions (temperature, pH, reaction time) on the response value of this sensor, the specificity of the sensor using other hormones was confirmed. Furthermore, as an application of this sensor to the actual sample, the 11-KT concentration in a Nile tilapia (Oreochromis niloticus) plasma was measured and correlated with the measurement value obtained by the conventional method (ELISA) was compared.

The histological process of gonadal differentiation, together with the endocrine changes of sex steroid hormones and some of their precursors, was studied in hatchery-produced greater amberjack Seriola dumerili from 101 until 408 days post-hatching (dph), with samplings conducted every 50 days. Histological processing showed that sex differentiation began at 101 dph with the formation of the ovarian cavity in females, while the presumptive males did not yet contain any germ cells in their gonad. At 150 dph, we observed the first germ cells in the developing testes. Sex differentiation in almost all sampled individuals was complete at 408 dph. No size dimorphism was observed between the sexes, and the sex ratio was 1:1, suggesting that there was no influence of early rearing in captivity on sex differentiation. Plasma concentrations of adrenosterone (Ad), androstenedione (Δ4), 11-ketotestosterone (11ΚΤ), testosterone (Τ), estradiol (Ε2), progesterone (P4) and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) were measured in males and females with the use of liquid chromatography tandem mass spectrometry (LC–MS/MS) to examine their role in the sex differentiation process. From the seven hormones, the only one that exhibited differences between the sexes was 11-KT and the plasma 11-KT concentration was found to be a useful indication of greater amberjack sex. Variations were observed in the mean values of Ad, Δ4, 11-KT, T, P4 and 17,20βP over time in one or both sexes, indicating their involvement in the sex differentiation process. [ABSTRACT FROM AUTHOR]

It is imperative to discriminate between male and female fish during the juvenile stage and pre-adjust the required numbers of males and females to efficiently raise parent fish in aquaculture farms. One method to discriminate between males and females is to measure a fishspecific steroid hormone such as 11-ketotestosterone (11-KT) in fish plasma. However, conventional methods such as enzyme-linked immunosorbent assay (ELISA) are complicated to operate and require a long time for analysis. Here, we introduce a novel immunosensor system that combines immunological reaction and electrochemical measurement with the aim of achieving a rapid and straightforward measurement method in preparation for gender determination of fish. Using our system, we observed stepwise decreases in the oxidation peak current value with increasing concentration of 11-KT in the sample. Furthermore, a strong linear relationship was obtained between the standard solution concentrations of 0.21 and 3.30 pmol ml-1 (R = 0.9869) in only 20 min. Next, the optimum operating conditions of the sensor were evaluated. It was also confirmed that the sensor did not respond to other hormones. Moreover, a strong linear relationship was obtained between the values and those obtained by the conventional ELISA method (R = 0.9927). [ABSTRACT FROM AUTHOR]

Context: Several studies have highlighted the importance of the 11-oxygenated 19-carbon (11oxC19) adrenal-derived steroids as potential biomarkers for monitoring patients with 21-hydroxylase deficiency (21OHD).Objective: To analyze circadian rhythmicity of 11oxC19 steroids in saliva profiles and evaluate their relevance as potential monitoring parameters in 21OHD.Design, Setting, and Participants: Cross-sectional single-center study including 59 patients with classic 21OHD (men = 30; women = 29) and 49 body mass index- and age-matched controls (men = 19; women = 30).Outcome Measures: Salivary concentrations of the following steroids were analyzed by liquid chromatography-tandem mass spectrometry: 17-hydroxyprogesterone (17OHP), androstenedione (A4), testosterone (T), 11β-hydroxyandrostenedione (11OHA4), and 11-ketotestosterone (11KT).Results: Similar to the previously described rhythmicity of 17OHP, 11OHA4 and 11KT concentrations followed a distinct diurnal rhythm in both patients and controls with highest concentrations in the early morning and declining throughout the day (11-OHA4: mean reduction of hormone concentrations between timepoint 1 and 5 (Δ mean) in male patients = 66%; male controls Δ mean = 83%; female patients Δ mean = 47%; female controls Δ mean = 86%; 11KT: male patients Δ mean = 57%; male controls Δ mean = 63%; female patients Δ mean = 50%; female controls Δ mean = 76%). Significant correlations between the area under the curve for 17OHP and 11KT (rpmale = 0.773<0.0001; rpfemale = 0.737<0.0001), and 11OHA4 (rpmale = 0.6330.0002; rpfemale = 0.5640.0014) were observed in patients but not present or reduced in controls.Conclusions: Adrenal 11oxC19 androgens are secreted following a diurnal pattern. This should be considered when evaluating their utility for monitoring treatment control. [ABSTRACT FROM AUTHOR]

Context: Androstenedione (A4) and testosterone (T) are produced by both the adrenal glands and the gonads. The adrenal enzyme 11β-hydroxylase (CYP11B1) executes the final step in cortisol synthesis; CYP11B1 also uses A4 and T as substrates, generating 11-hydroxyandrostenedione and 11-hydroxytestosterone, respectively. It has been suggested that CYP11B1 is expressed in the gonads, yet the circulating levels of all 11-oxygenated androgens (11-oxyandrogens) are similar in males and females of reproductive ages, despite enormous differences in T., Objective: To assess the gonadal contribution to the circulating pool of 11-oxyandrogens., Methods: We used liquid chromatography-tandem mass spectrometry to measure 13 steroids, including traditional and 11-oxyandrogens in: (I) paired gonadal and peripheral vein blood samples obtained during gonadal venograms from 11 patients (7 women), median age 37 (range 31-51 years); and (II) 17 women, median age 57 (range 41-81 years) before and after bilateral salpingo-oophorectomy (BSO). We also compared CYP11B1, 17α-hydroxylase/17,20-lyase (CYP17A1), and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) mRNA expression in adrenal, ovarian, and testicular tissue., Results: A4, T, estradiol, estrone, progesterone, 17α- and 16α-hydroxyprogesterone were all higher in gonadal veins vs. periphery (p < 0.05 for all), while four 11-oxyandrogens were similar between matched gonadal and peripheral vein samples. Equally, in women who underwent BSO, A4 (median [interquartile range]: 59.7 [47.7-67.6] ng/dL vs. 32.7 [27.4-47.8] ng/dL, p < 0.001) and T (24.1 [16.4-32.3] vs.15.5 [13.7-19.0] ng/dL, p < 0.001) declined, while 11-oxyandrogens remained stable. Gonadal tissue displayed negligible CYP11B1 mRNA., Conclusion: Despite producing substantial amounts of A4 and T, human gonads are not relevant sources of 11-oxyandrogens., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Gynogenetic progeny of sterlet Acipenser ruthenus were obtained by inseminating eggs with UV-inactivated sperm followed by meiotic restoration of ploidy by heat shock. Gynogenetic origin of progeny was assessed by parentage assignment using 15 microsatellite markers. Sex ratio was investigated by gonad histology in 15 3-year-old fish, eight of which were confirmed gynogenotes, with five of those containing primary oocytes (62.5%). Blood plasma concentration of 11-ketotestosterone (KT-11) was analyzed in 63 4-year-old juveniles, 46 of which were genetically confirmed gynogenotes. Measurement of KT-11 showed the proportion of female sterlet to be 65.2%. This sex ratio conformed to the current hypothesis of female heterogametic sex determination but was at the low end of the reported female ratio in sturgeon gynogenetic progeny. [ABSTRACT FROM AUTHOR]

Anguillid eels are the iconic example of catadromous fishes, because of their long-distance offshore spawning migrations. They are also a good model for research on the onset mechanisms of migrations to breeding areas, because the migrations begin in inland waters. When eels transform from yellow eels to silver eels, it is called silvering. Silver eels show various synchronous external and internal changes during silvering, that include coloration changes, eye-size increases, and gonadal development, which appear to be pre-adaptations to the oceanic environment and for reproductive maturation. A strong gonadotropic axis activation occurs during silvering, whereas somatotropic and thyrotropic axes are not activated. Among various hormones, 11-ketotestosterone (11-KT) drastically increases during spawning migration onset. Gradual water temperature decreases simulating the autumn migratory season, inducing 11-KT increases. Administration of 11-KT appeared to cause changes related to silvering, such as early-stage oocyte growth and eye enlargement. Moreover, 11-KT may be an endogenous factor that elevates the migratory drive needed for the spawning migration onset. These findings suggested that water temperature decreases cause 11-KT to increase in autumn and this induces silvering and increases migratory drive. In addition, we newly report that 11-KT is associated with a corticotropin-releasing hormone that influences migratory behavior of salmonids. This evidence that 11-KT might be among the most important factors in the spawning migration onset of anguillid eels can help provide useful knowledge for understanding endocrinological mechanisms of the initiation of spawning migrations.

The reproductive physiology of fish can be changed by the presence of pollutants in the water, which act as endocrine disrupting compounds (EDC). We evaluated the impacts of water contaminants in polluted reservoirs acting as possible EDC on the reproductive physiology of Astyanax fasciatus and Hoplias malabaricus males. We used biomarkers with different levels of biological organization. Hoplias malabaricus adult males were collected in the summer and winter at five different sites in the Tietê River Basin: the Ponte Nova reservoir (PN), considered a reference site due to the low anthropogenic influence; the Billings reservoir (BIL) at two different branches; and the Guarapiranga reservoir (GUA) at two different branches. Astyanax fasciatus adult males were collected at PN and BIL. BIL and GUA are subjected to great anthropogenic action. We analyzed gonadal histomorphology, testosterone (T), 11-ketotestosterone (11-KT), estradiol (E2) plasma levels, and gene expression of hepatic vitellogenin (vtgA) and pituitary follicle stimulating hormone (fshβ). In the PN reservoir (reference), the biomarkers analyzed in both species did not differ between the periods analyzed. This is an evidence that the animals keep the same reproductive activity during both seasons. The changes in the plasma concentration of gonadal steroids in both species in polluted reservoirs suggest the presence of EDC compounds in the water and/or adjusts of the physiological setpoint to allow the reproduction in such adverse conditions. The use of vtgA as biomarker suggests the presence of estrogenic compounds, mainly in BIL, but with a more evident response of H. malabaricus. However, even considering physiological changes, both species present testes during the maturation phase that allow the reproduction in an environment with a high degree of pollution.

Androgens are the obligatory precursors of estrogens. In humans, classic androgen biosynthesis yields testosterone, thought to represent the predominant circulating active androgen both in men and women. However, recent work has shown that 11-ketotestosterone, derived from the newly described 11-oxygenated androgen biosynthesis pathway, makes a substantial contribution to the active androgen pool in women. Considering that classic androgens are the obligatory substrates for estrogen biosynthesis catalyzed by cytochrome P450 aromatase, we hypothesized that 11-oxygenated androgens are aromatizable. Here we use steroid analysis by tandem mass spectrometry to demonstrate that human aromatase generates 11-oxygenated estrogens from 11-oxygenated androgens in 3 different cell-based aromatase expression systems and in human ex vivo placenta explant cultures. We also show that 11-oxygenated estrogens are generated as a byproduct of the aromatization of classic androgens. We show that 11β-hydroxy-17β-estradiol binds and activates estrogen receptors α and β and that 11β-hydroxy-17β-estradiol and the classic androgen pathway-derived active estrogen, 17β-estradiol, are equipotent in stimulating breast cancer cell line proliferation and expression of estrogen-responsive genes. 11-oxygenated estrogens were, however, not detectable in serum from individuals with high aromatase levels (pregnant women) and elevated 11-oxygenated androgen levels (patients with congenital adrenal hyperplasia or adrenocortical carcinoma). Our data show that while 11-oxygenated androgens are aromatizable in vitro and ex vivo, the resulting 11-oxygenated estrogens are not detectable in circulation, suggesting that 11-oxygenated androgens function primarily as androgens in vivo. [ABSTRACT FROM AUTHOR]

Androgen(s) is one of the sex steroids that are involved in many physiological phenomena of vertebrate species. Although androgens were originally identified as male sex hormones, it is well known now that they are also essential in females. As in the case of other steroid hormones, androgen is produced from cholesterol through serial enzymatic reactions. Although testis is a major tissue to produce androgens in all species, androgens are also produced in ovary and adrenal (interrenal tissue). Testosterone is the most common and famous androgen. It represents a major androgen both in males and females of almost vertebrate species. In addition, testosterone is a precursor for producing significant androgens such as11-ketotestosterone, 5α-dihydrotestosterone, 11-ketodihydrotestosterones and 15α-hydroxytestosterone in a species- or sex-dependent manner for their homeostasis. In this article, we will review the significance and characteristics of these androgens, following a description of the history of testosterone discovery and its synthetic pathways.

The deficiency or insufficiency of androgen can trigger a range of reproductive diseases as well as other symptoms. Stem Leydig cells (SLCs) are critical for the formation and maintenance of a functional androgen-producing cell (Leydig cell, LC) population throughout adult male life. However, to date, our knowledge about SLCs is poor. Here we report the derivation and characterisation of a clonal stem LC line (designated as TSL) capable of 11- ketotestosterone (11-KT) production from a 3-month-old Nile tilapia (Oreochromis niloticus) testis. The cells retained stable proliferation after 77 generations with normal karyotype and growth factor dependency. They expressed platelet-derived growth factor receptor-α (pdgfr α), nestin and chicken ovalbumin upstream promoter transcription factor II (coup - tfIIa), which are characteristic of SLCs. Upon induction in defined medium, TSLs could undergo differentiation into steroidogenically active LCs and produce 11-KT. When implanted into recipient Nile tilapia testes from which endogenous LCs had been eliminated by ethane dimethanesulphonate (EDS) treatment, the PKH26-labelled TSLs could colonise the interstitium, subsequently express steroidogenic genes and restore 11-KT production. Taken together, our data suggest that TSLs possess the ability of continuous proliferation and potential of differentiation into functional LCs in vitro and in vivo. To the best of our knowledge TSL might represent the first stem LC line capable of 11-KT production to date. Our study may offer new opportunities for investigating the self-renewal of SLCs and steroidogenesis in vitro , and provide an invaluable in vitro model for investigating endocrine disruptors. Stem Leydig cells are critical for the formation and maintenance of a functional androgen-producing cell (Leydig cell) population throughout adult male life. A clonal stem Leydig cell line capable of androgen production from 3-month-old Nile tilapia testis has been successfully established. This cell line may offer new opportunities for investigating the self-renewal of stem Leydig cells and steroidogenesis in vitro , and also provide an invaluable in vitro model for screening and investigating endocrine disruptors. [ABSTRACT FROM AUTHOR]

11-ketotestosterone (11-KT) is a non-aromatizable and the most potent androgen in a few teleost. It has been reported that 11-KT in serum had a high concentration and increased sharply before the period of yolk deposition in females of few fishes. The aim of this study was to analyze the role of 11-KT both in vivo and in vitro on ovarian development, related gene expression levels, Vitellogenin (Vtg) synthesis, and serum sex steroid concentrations in previtellogenic cultured sterlet (Acipenser ruthenus). Silastic strips embedded with 11-KT (5 or 25 mg/kg) were implanted in vivo for 30 days. Ovarian masculinization or sex reversal was not observed. Histological analysis showed that 11-KT promoted sterlet ovarian development in a dose-dependent manner. Vtg and testosterone (T) increased significantly, while 17β-estradiol (E2) decreased with no significant difference among groups. The expression of genes androgen receptor (ar), vtg and lipoprotein lipase (lpl) were significantly increased in liver. However, 11-KT had no effect on the expression of foxl2 and cyp19a1 in ovary. In vitro, after incubation with 11-KT (10 and 100 μM) for 5 days, both T and E2 concentrations increased in both hepatic explants and ovarian explants culture medium; the concentration of Vtg also increased in hepatic explants culture medium. The expression of ar, era, vtg, and lpl increased significantly in hepatic explants. However, only the expression of era significantly increased in cultured ovarian explants. Altogether, these results suggest that 11-KT induced ovarian development, as well as Vtg and lipid synthesis, and could be an important factor facilitating the initiation of Vtg synthesis in the liver of the previtellogenic sterlet.

The in vivo effect of 11‐ketotestosterone (11KT) on transcript levels of the gonadotropin receptors (fshr and lhr) and sex differentiation‐related genes (dmrt1 and foxl2) was examined in the ovaries of immature female beluga. For this purpose, six fish were treated with implants containing 2.5 mg 11KT and a placebo group of six females of the same age and gametogenic stage were given a blank implant. The implants were intraperitoneally inserted into 4‐year‐old females at the previtellogenic stage (mean body weight 5580 ± 165 g) and maintained under culture conditions for 8 weeks. Ovary samples for gene expression analysis of lhr, fshr, dmrt1 and foxl2 were collected by biopsy at 3 and 8 weeks post implantation. Diameters of oocytes increased in response to 11KT treatment, both at 3 and at 8 weeks post implantation, but no obvious changes were evident in cytology. Three weeks of 11KT treatment did not affect target gene expression, but a tendency for a time‐dependent decrease of lhr and dmrt1 mRNA levels was observed in both treatment and placebo groups. By 8 weeks of treatment, however, 11KT implants provoked the upregulation of fshr and foxl2 transcript levels. Furthermore, lhr and dmrt1 transcript abundances recovered by 8 weeks of exposure in both blank‐ and 11KT‐implanted beluga. These results suggest that 11KT, either directly or indirectly, may affect gametogenesis and regulate some key components of the reproductive axis in female beluga. [ABSTRACT FROM AUTHOR]

Different evolutionary interests between males and females can lead to the evolution of sexual dimorphism. However, intersex genetic correlations due to the shared genome can constrain the evolution of sexual dimorphism, resulting in intra‐locus sexual conflict. One of the mechanisms resolving this conflict is sex linkage, which allows males and females to carry different alleles on sex chromosomes. Another is a regulatory mutation causing sex‐biased gene expression, which is often mediated by gonadal steroids in vertebrates. How do these two mechanisms differ in the contributions to the resolution of intra‐locus sexual conflict? The magnitude of sexual conflict often varies between the juvenile and adult stages. Because gonadal steroids change in titre during development, we hypothesized that gonadal steroids play a role in sexual dimorphism expression only at certain developmental stages, whereas sex linkage is more important for sexual dimorphism expressed throughout life. Our brain transcriptome analysis of juvenile and adult threespine sticklebacks showed that the majority of genes that were sex‐biased in both stages were sex‐linked. The relative contribution of androgen‐dependent regulation to the sex‐biased transcriptome increased and that of sex linkage declined in adults compared to juveniles. The magnitude of the sex differences was greater in sex‐linked genes than androgen‐responsive genes, suggesting that sex linkage is more effective than androgen regulation in the production of large sex differences in gene expression. Overall, our data are consistent with the hypothesis that sex linkage is effective in resolving sexual conflict throughout life, whereas androgen‐dependent regulation can contribute to temporary resolution of sexual conflict. [ABSTRACT FROM AUTHOR]

Background: Classically, serum testosterone (T) and androstenedione (A4) have been the mainstay for the biochemical assessment of hyperandrogenism. However, recent evidence suggests 11β-hydroxyandrostenedione (11OHA4) and 11-ketotestosterone (11KT) may also be important. Here, we describe the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for quantitation of total serum T, A4, 17-hydroxyprogesterone (17OHP), 11OHA4 and 11KT. In addition, we applied the method to assess pre-analytical stability. Methods: An isotopically labelled internal standard was added to samples prior to supported liquid extraction (SLE). Extracts were analysed using LC-MS/MS to detect T/A4/17OHP/11OHA4 and 11KT along with their corresponding internal standards. Samples (n = 7) were collected from healthy volunteers (n = 14) and left incubated at 20 °C for up to 72 h. Tubes were retrieved at select time points, centrifuged, separated and frozen prior to analysis. Results: The total run time was 4 min. For all analytes, intra- and inter-assay imprecision did not exceed 7.9% and 5.3%, respectively; matrix effects were negligible and mean recoveries ranged from 95.3 to 111.6%. The limits of quantitation (LOQs) were 0.25 nmol/L for T, A4 and 11OHA4, 0.50 nmol/L for 17OHP, and 0.24 nmol/L for 11KT. No significant change was observed in pre-centrifugation A4 or female T concentrations over 72 h. Significant increases (p < 0.01) in concentrations of 11KT, 17OHP, 11OHA4 and male T were observed after 2, 8, 12 and 24 h, respectively. Conclusions: We developed a robust LC-MS/MS assay for the quantitation of total serum T/A4/17OHP/11OHA4 and 11KT. Applying the method to determine pre-analytical stability suggests samples requiring 11KT need separating from the cells within 2 h. [ABSTRACT FROM AUTHOR]

11-ketotestosterone (11-KT) is a non-aromatizable and the most potent androgen in a few teleost. It has been reported that 11-KT in serum had a high concentration and increased sharply before the period of yolk deposition in females of few fishes. The aim of this study was to analyze the role of 11-KT both in vivo and in vitro on ovarian development, related gene expression levels, Vitellogenin (Vtg) synthesis, and serum sex steroid concentrations in previtellogenic cultured sterlet (Acipenser ruthenus). Silastic strips embedded with 11-KT (5 or 25 mg/kg) were implanted in vivo for 30 days. Ovarian masculinization or sex reversal was not observed. Histological analysis showed that 11-KT promoted sterlet ovarian development in a dose-dependent manner. Vtg and testosterone (T) increased significantly, while 17β-estradiol (E2) decreased with no significant difference among groups. The expression of genes androgen receptor (ar), vtg and lipoprotein lipase (lpl) were significantly increased in liver. However, 11-KT had no effect on the expression of foxl2 and cyp19a1 in ovary. In vitro , after incubation with 11-KT (10 and 100 μM) for 5 days, both T and E2 concentrations increased in both hepatic explants and ovarian explants culture medium; the concentration of Vtg also increased in hepatic explants culture medium. The expression of ar, era, vtg , and lpl increased significantly in hepatic explants. However, only the expression of era significantly increased in cultured ovarian explants. Altogether, these results suggest that 11-KT induced ovarian development, as well as Vtg and lipid synthesis, and could be an important factor facilitating the initiation of Vtg synthesis in the liver of the previtellogenic sterlet. [ABSTRACT FROM AUTHOR]

The impacts of androgens and glucocorticoids on spermatogenesis have intrigued scientists for decades. 11ß-hydroxylase, encoded by cyp11c1, is the key enzyme involved in the synthesis of 11-ketotestosterone and cortisol, the major androgen and glucocorticoid in fish, respectively. In the present study, a Cyp11c1 antibody was produced. Western blot and immunohistochemistry showed that Cyp11c1 was predominantly expressed in the testicular Leydig cells and head kidney interrenal cells. A mutant line of cyp11c1 was established by CRISPR/Cas9. Homozygous mutation of cyp11c1 caused a sharp decrease of serum cortisol and 11-ketotestosterone, and a delay in spermatogenesis which could be rescued by exogenous 11-ketotestosterone or testosterone, but not cortisol treatment. Intriguingly, this spermatogenesis restored spontaneously, indicating compensatory effects of other androgenic steroids. In addition, loss of Cyp11c1 led to undersized testes with a small er efferent duct and disordered spermatogenic cysts in adult males. However, a small amount of viable sperm was produced. Taken together, our results demonstrate that cyp11c1 is important for testicular development, especially for the initiation and proper progression of spermatogenesis. 11-ketotestosterone is the most efficient androgen in tilapia. [ABSTRACT FROM AUTHOR]

The aquaculture of greater amberjack Seriola dumerili is of considerable research interest worldwide. The larviculture methods employed to culture this species, however, are still under development, and the majority of farms still rely on wild-caught juveniles. One of the problems associated with the hatchery production of this species is the optimal selection of broodstock to ensure a stable supply of high-quality eggs. Specifically, no reliable low-stress sex-discrimination technique is currently available for selecting broodstock of this species. This study investigated the efficacy of a hormone-based sex-discrimination method in full-cycle cultured S. dumerili, ranging in age from 412 to 1150 days after hatching (DAH). Plasma concentrations of the female hormone 17β-estradiol (E2) and the male hormone 11-ketotestosterone (11-KT) were measured in both spawning and non-spawning seasons, and the optimal threshold levels for sex discrimination were estimated using a receiver operating characteristic curve. Sex discrimination using E2 produced several false positives in younger fish, and had an overall accuracy of 78.7%. However, sex discrimination using 11-KT had an accuracy of 96.7%, even in 412 DAH fish. This study demonstrated that sex discrimination using 11-KT is a reliable method for optimizing the sex ratio of S. dumerili broodstock, even before the broodstock mature. [ABSTRACT FROM AUTHOR]

The seasonal dynamics of the northern largemouth bass (Micropterus salmoides salmoides) reproductive cycle was investigated in a Midwestern United States population. Mature largemouth bass (n = five males and five females) were collected each month over a one‐year period. Environmental cues (water temperature and photoperiod) were recorded in situ using data loggers. Temporal changes in plasma sex steroid hormones (testosterone [T], 11‐ketotestosterone [11‐KT], and estradiol‐17β [E2]) and vitellogenin (Vtg), gamete development (histology), and organosomatic indices were highly associated with temporal water temperature and photoperiod dynamics. Female plasma concentrations of T and E2 and organosomatic indices exhibited similar temporal patterns, with peaks occurring in early summer. Gamete development in females was associated with increases in gonadosomatic index (GSI) and Vtg. In males, plasma concentrations of T and organosomatic indices exhibited bimodal peaks; high peaks occurred in the spring for T and GSI and in the fall for viscerosomatic and hepatosomatic indices. Male gamete development and GSI closely tracked temporal patterns in plasma T, with GSI lagging behind the T cycle by approximately one sampling period. Plasma concentrations of 11‐KT were elevated in late winter and spring and were considerably higher than T throughout the reproductive cycle. Unlike the Florida largemouth bass (M. salmoides floridanus) advanced gamete development was observed in both sexes before winter, which may reduce the immediate energy required for gamete development in the spring. Results from this study also provide baseline information necessary for off‐season spawning procedures and endocrine‐disruption studies in northern largemouth bass. Research Highlight: Advanced gamete development occurred in both sexes prior to winterFemale plasma vitellogenin production was bimodal and did not coincide with peak concentrations of estradiol‐17βMales exhibited bimodal peaks of plasma testosterone [ABSTRACT FROM AUTHOR]

Lead (Pb) is an emerging contaminant widely distributed in aquatic environments, which has serious effects on human and animal health. In this study, we determined whether Pb exposure affects gametogenesis, sex steroids, estrogen (ERα and ERβ), and androgen (AR) receptors. Adult specimens of Astyanax bimaculatus were exposed in duplicate to 15, 50, and 100 μg/L of lead acetate, whereas the control group was not exposed. After 28 days of exposure, fish were euthanized and samples of the gonads, liver, and blood were collected for analysis. The results indicated a reduction in the gonadosomatic index as well as the diameters of the vitellogenic follicles and seminiferous tubules in the exposed groups. Morphometry of gametogenesis revealed inhibition of the secondary oocyte growth and a reduction in the number of spermatozoa in the 50 and 100 μg/L Pb-treated groups. In females, plasma 17β-estradiol (E2) increased following 15 and 50 μg/L Pb treatment, whereas males exhibited an increase in E2 and 11-ketotestosterone following treatment with 15 and 100 μg/L Pb, respectively. Vitellogenin was significantly reduced in females exposed to 100 μg/L Pb, but metallothionein levels were unchanged. ERα, ERβ, and AR were immunolocalized in the somatic and germ cells, with increased ovarian expression of ERα and Erβ in the 100 μg/L Pb-treated group, but no significant difference in AR among the groups. In males, only ERα increased in the 100 μg/L Pb-treated group. These results indicate that Pb exposure impairs gametogenesis, disrupts estrogen receptor signaling, and affects the expression of major reproductive biomarkers in A. bimaculatus. [Display omitted] • Fish exposure to lead (Pb) affects E2 and 11-KT levels, and gametogenesis. • In females, endocrine disruption reduces VTG and impairs the oocyte growth. • In males, dysregulated spermatogenesis leads to reduction of spermatozoa. • ERα and ERβ are differently affected by Pb exposure in the ovaries and testes. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is defined by androgen excess and ovarian dysfunction in the absence of a specific physiological diagnosis. The best clinical marker of androgen excess is hirsutism, while the best biochemical parameter is still a matter of debate. Current consensus guidelines recommend, among other hormones, serum free testosterone as an important serum parameter to measure androgen excess. Recently, however, novel active androgens and androgen metabolic pathways have been discovered. To assess the contribution of novel androgens and related steroid biosynthetic pathways to the serum steroid pool in PCOS women in comparison to healthy controls. This is a case control study, wherein PCOS was diagnosed according to the AE-PCOS 2009 criteria. Serum steroid profiling was performed by liquid chromatography high-resolution mass spectrometry. Yeditepe University and associated clinics in Istanbul, Turkey, together with Bern University Hospital Inselspital, Bern, Switzerland. 42 PCOS women and 42 matched, healthy control women. Assessment of 34 steroids compartmentalized in four androgen related pathways: the classic androgen pathway, the backdoor pathway, the C11-oxy backdoor pathway, and the C11-oxy (11β-hydroxyandrostenedione) pathway. Metabolites of all four pathways were identified in healthy and PCOS women. Highest concentrations were found for progesterone in controls and androstenedione in PCOS. Lowest levels were found for 11-ketotestosterone in controls compared to PCOS, and for 20α-hydroxyprogesterone in PCOS compared to controls. PCOS also had higher serum testosterone levels compared to the controls. PCOS women had overall higher levels of steroid metabolites of all four androgen pathways compared to healthy controls. Novel alternative pathways contribute to the androgen production in healthy and PCOS women. Hyperandrogenism in PCOS is characterized by an overall increase of serum androgens in the classic, backdoor and C11-oxy pathways. While monogenetic disorders of steroid biosynthesis can be recognized by a specific pattern in the steroid profile, no diagnostic pattern or classifier was found in the serum for PCOS. [Display omitted] • Classical androgen levels, androstenedione and testosterone, were higher in PCOS. • The downstream androgen metabolite, androsterone, was higher in PCOS. • 11-Ketotestosterone, produced in the C11-oxy androgen pathway, was higher in PCOS. • Active androgen metabolic pathways were identified in PCOS. • Steroid pathway analysis hints at the involvement of even more androgen pathways. [ABSTRACT FROM AUTHOR]

Background: Classical and 11-oxygenated androgens both contribute to the androgen pool. Regular monitoring of the androgen status is required in disorders of steroidogenesis, and multiplexing of androgens improves the diagnostic ability of an assay. Due to the cheap non-invasive collection, saliva is advantageous when multiple samples are required. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers sensitive, simultaneous quantification of steroids with short run times. Here, we have developed an LC-MS/MS assay for the simultaneous measurement of 17-hydroxyprogesterone, androstenedione, testosterone, 11β-hydroxyandrostenedione and 11-ketotestosterone in saliva. Methods: Samples (300 μ L unstimulated whole saliva) were prepared by supported liquid extraction with dichloromethane and were reconstituted in 40% methanol. After online solid phase extraction with C18 cartridges, liquid chromatography was performed on a C8 column using a water/methanol gradient containing 0.1% formic acid and 2 mmol/L ammonium acetate. A Waters TQ-S mass spectrometer was used for quantification. Results: Total run time was 6.4 min. For all analytes, recovery was between 89% and 109%, ion suppression between 86% and 105%. Intra- and inter-assay comparisons showed a coefficient of variation <10% and the bias between measured and nominal concentration varied between –8% and 10%. Interference with a large set of natural and synthetic steroids was excluded. The assay was applied for the measurement of the androgen profile in healthy men (n = 17) and women (n = 10) which confirmed the sensitivity of the assay to be appropriate. Conclusion: We present a novel LC-MS/MS assay for the comprehensive profiling of classical and 11-oxygenated androgens with potential for routine clinical application. [ABSTRACT FROM AUTHOR]

• Testicular ar expression decreases following hCG treatment, despite increases in 11-KT production. • 11-KT augments testicular Fsh sensitivity via increased fshr transcript abundance. • hCG induced spermatogenesis proceeds due to a positive feedback loop. Receptors for follicle-stimulating hormone (Fshr), luteinizing hormone (Lhcgr1 and Lhcgr2) and androgens (Ara and Arb) transduce the hormonal signals that coordinate spermatogenesis, but the factors that regulate the abundance of these transducers in fish testes remain little-understood. To mend this paucity of information, we first determined changes in transcript abundance for these receptors (fshr, lhcgr1, ara and arb) during spermatogenesis induced by human chorionic gonadotropin (hCG) injection in the eel, Anguilla australis. We related our findings to testicular production of the fish androgen, 11-ketotestosterone (11-KT), and to the levels of the transcripts encoding steroidogenic acute regulatory protein (star) and 11β-hydroxylase (cyp11b), and subsequently evaluated the effects of hCG or 11-KT on mRNA levels of these target genes in vitro. Testicular 11-KT production was greatly increased by hCG treatment, both in vivo and in vitro , and associated with up-regulation of star and cyp11b transcripts. In situ hybridization indicated that testicular fshr mRNA levels were higher in the early stages of hCG-induced spermatogenesis, while lhcgr1 transcripts were most abundant later, once spermatids were observed. In vitro experiments further showed that hCG and its steroidal mediator 11-KT significantly increased fshr transcript abundance. These data provide new angles on the interactions between gonadotropin and androgen signaling during early spermatogenesis. Increases in levels of 11-KT following hCG injection elevated testicular fshr mRNA levels augmenting Fsh sensitivity in the testis. This evidence is suggestive of a positive feedback loop between gonadotropins and 11-KT that may be key to regulating early spermatogenesis in fish. [ABSTRACT FROM AUTHOR]

Abstract Low concentrations of nonylphenol (NP) in aquatic environment can induce drastic effects on the endocrine system in animals. In this study, we examined the modulatory effects of NP on reproductive and physiological parameters in juveniles of the red seabream and black rockfish following waterborne NP exposure (0, 1, 10, and 50 μg L−1) for 60 days. In red seabream exposed to 50 μg L−1 NP, plasma levels of 17β-estradiol (E2) and 11-ketotestosterone (11-KT) were significantly lower at 30 and 60 days, while E2 levels were slightly higher in 10 μg L−1-exposed individuals at day 30. Similarly, significantly lower levels of E2 and 11-KT were observed in 10 and 50 μg L−1-exposed black rockfish at 60 days, whereas the E2 level was higher in 1 μg L−1-exposed individuals at day 30. After exposure to NP, plasma and mRNA levels of vitellogenin (VTG) were significantly higher in both species at 30 and 60 days, similar to the inducible effects from synthetic estrogen. Plasma cortisol levels were significantly elevated by relatively higher concentrations of NP (10 and 50 μg L−1) at 30 and 60 days. Finally, 60 days of exposure of 50 μg L−1 NP significantly decreased the gonadosomatic index (GSI) and increased the hepatosomatic index (HSI) in both species. The results obtained from this study provide an evidence of the endocrine disrupting potential of waterborne NP on early stages of economically important marine fish. The NP-triggered endocrine modulation can induce effects on the development of reproductive and metabolic organs in fish species. Graphical abstract Unlabelled Image Highlights • Direct evidence on accumulation of waterborne NP in marine fish blood. • Strong endocrine disruption potential of NP on juvenile stage of marine fish. • Analysis of NP-triggered stress induction and modulation of somatic parameters. [ABSTRACT FROM AUTHOR]

Present study was undertaken to explore the possibilities of inducing maturity and spawning of T. putitora under captivity through manipulation of environmental cues i.e. photoperiod and temperature. In experiment I, adult T. putitora were subjected to three photoperiod (8L:16D, 12L:12D and 16L:12D) regimes for 100 days. In females, darker photoperiod (8L:16D) significantly decreased the plasma estradiol levels compared to 12L:12D and 16L:8D photoperiods. Plasma 17α, 20β-diOH-P was significantly ( p  < .05) changed both in females as well as males due to different photoperiods. Highest level was observed at 12L:12D in females and 16L:8D in males whereas the lowest level was found in 8L:16D. There was no significant effect of photoperiod on plasma 11-ketotestosterone (11-KT) levels and gonado-somatic index (GSI) of both males and females. In experiment II, effect of temperature (ambient, 21.2 ± 1.4 °C and elevated, 23.7 ± 1.3 °C; Mean ± SD) on inducing maturity under captive conditions was explored. The level of 17β-estradiol and 17α, 20β-diOH-P was significantly increased in females kept at elevated temperature compared to its ambient counterpart. Correspondingly, GSI of females increased 6-fold at elevated temperature. In fact in the ovary of females reared at elevated temperature, oocytes were in vitellogenic and maturing stage, whereas it was only in cortical alveolar stages in the fishes kept at ambient temperature. On the other hand, there was no marked change found in testicular histology. However, there was presence of some macrophage aggregations on the efferent duct region of testes at elevated temperature. No significant ( p  > .05) effect of temperature was observed on plasma levels of 11-KT of both sexes. Experiment III was meant to explore the possibility of achieving captive spawning by simulating natural spawning ground using a gravel bed bio-filter. Natural spawning occurred after four months of rearing in the simulated facility. Subsequently, fishes further spawned seven times in a period of 4 months on an interval of 15–20 days. Overall, our results suggested that captive maturity and spawning of golden mahseer can be achieved by manipulating the environmental cues. The results solved the prevailing decadal issue of captive maturation of golden mahseer which otherwise has been a major bottleneck for its wide spread rehabilitation and conservation programmes. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • 11-oxygenated androgen precursors are the preferred substrates for AKR1C3. • Oxidative 17βHSD enzymes regulate androgen activation. • Increased AKR1C3:17βHSD2 ratios favour the activation of 11-oxygenated androgens. • Computational modelling provides a valuable tool to study androgen metabolism. Abstract The progression of castration resistant prostate cancer (CRPC) is driven by the intratumoral conversion of adrenal androgen precursors to potent androgens. The expression of aldo-keto reductase 1C3 (AKR1C3), which catalyses the reduction of weak androgens to more potent androgens, is significantly increased in CRPC tumours. The oxidation of androgens to their inactive form is catalysed by 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2), but little attention is given to the expression levels of this enzyme. In this study, we show that the 11-oxygenated androgen precursors of adrenal origin are the preferred substrate for AKR1C3. In particular we show that the enzymatic efficiency of AKR1C3 is 8- and 24-fold greater for 11-ketoandrostenedione than for the classic substrates androstenedione and 5α-androstanedione, respectively. Using three independent experimental systems and a computational model we subsequently show that increased ratios of AKR1C3:17βHSD2 significantly favours the flux through the 11-oxygenated androgen pathway as compared to the classical or 5α-androstanedione pathways. Our findings reveal that the flux through the classical and 5α-androstanedione pathways are limited by the low catalytic efficiently of AKR1C3 towards classical androgens combined with the high catalytic efficiency of 17βHSD2, and that the expression of the oxidative enzyme therefore plays a vital role in determining the steady state concentration of active androgens. Using microarray data from prostate tissue we confirm that the AKR1C3:17βHSD2 ratio is significantly increased in patients undergoing androgen deprivation therapy as compared to benign tissue, and further increased in patients with CRPC. Taken together this study therefore demonstrates that the ratio of AKR1C3:17βHSD2 is more important than AKR1C3 expression alone in determining intratumoral androgen levels and that 11-oxygenated androgens may play a bigger role in CRPC than previously anticipated. [ABSTRACT FROM AUTHOR]