Gonadotropin inhibitory hormone downregulates steroid hormone secretion and genes expressions in duck granulosa cells.

The mechanisms by which GnIH regulates the steroid synthesis pathway in duck granulosa cells remain poorly understood. In this study, we measured steroid hormone secretion by ELISA and reproduction-associated gene expression by quantitative real-time Polymerase Chain Reaction (qPCR) in duck granulosa cells treated with different concentrations of GnIH (0, 0.1, 1, 10, and 100 ng/mL) for 24 h. The genome-wide expression profiles of GnIH-treated cells (0 and 10 ng/mL) were evaluated by high-throughput RNA sequencing. Compared with untreated cells, the secretion of the steroid hormones E2, E1, P4, and T was downregulated, with that of E1 and P4 reaching statistical significance (P<0.05); in contrast, the secretion of ACV and INH was significantly upregulated (P<0.05) after treatment with 10 and 100 ng/mL GnIH. The expression of encoding steroidogenic proteins and enzymes genes (STAR, CYP11A1, CYP17A1, CYP19A1, and 3-ß-HSD) and encoding gonadotropin receptors genes (FSHR, LHR) were significantly declined (P<0.05) in the 10 and 100 ng/mL GnIH treatments. Transcriptome sequencing identified 348 differentially expressed genes (DEGs), including 253 upregulated and 95 downregulated genes. The DEGs were mainly involved in cell growth and death, immune response, and steroid biosynthesis pathways. We identified four novel DEGs (MROH5, LOC113840576, SDR42E1, and LOC113841457) with key roles in the regulation of steroid hormone biosynthesis. Our study revealed changes in gonadal steroid hormone secretion and steroid biosynthesis pathway-related gene expression in duck granulosa cells under the inhibitory effect of GnIH. These data contribute to our understanding of the molecular and genetic mechanisms underlying reproduction in ducks.

Gonadotropin inhibitory hormone downregulates steroid hormone secretion and genes expressions in duck granulosa cells

The mechanisms by which GnIH regulates the steroid synthesis pathway in duck granulosa cells remain poorly understood. In this study, we measured steroid hormone secretion by ELISA and reproduction-associated gene expression by quantitative real-time Polymerase Chain Reaction (qPCR) in duck granulosa cells treated with different concentrations of GnIH (0, 0.1, 1, 10, and 100 ng/mL) for 24 h. The genome-wide expression profiles of GnIH-treated cells (0 and 10 ng/mL) were evaluated by high-throughput RNA sequencing. Compared with untreated cells, the secretion of the steroid hormones E2, E1, P4, and T was downregulated, with that of E1 and P4 reaching statistical significance (P<0.05); in contrast, the secretion of ACV and INH was significantly upregulated (P<0.05) after treatment with 10 and 100 ng/mL GnIH. The expression of encoding steroidogenic proteins and enzymes genes (STAR, CYP11A1, CYP17A1, CYP19A1, and 3-β-HSD) and encoding gonadotropin receptors genes (FSHR, LHR) were significantly declined (P<0.05) in the 10 and 100 ng/mL GnIH treatments. Transcriptome sequencing identified 348 differentially expressed genes (DEGs), including 253 upregulated and 95 downregulated genes. The DEGs were mainly involved in cell growth and death, immune response, and steroid biosynthesis pathways. We identified four novel DEGs (MROH5, LOC113840576, SDR42E1, and LOC113841457) with key roles in the regulation of steroid hormone biosynthesis. Our study revealed changes in gonadal steroid hormone secretion and steroid biosynthesis pathway-related gene expression in duck granulosa cells under the inhibitory effect of GnIH. These data contribute to our understanding of the molecular and genetic mechanisms underlying reproduction in ducks.(AU)

Gonadotropin inhibitory hormone downregulates steroid hormone secretion and genes expressions in duck granulosa cells

Abstract The mechanisms by which GnIH regulates the steroid synthesis pathway in duck granulosa cells remain poorly understood. In this study, we measured steroid hormone secretion by ELISA and reproduction-associated gene expression by quantitative real-time Polymerase Chain Reaction (qPCR) in duck granulosa cells treated with different concentrations of GnIH (0, 0.1, 1, 10, and 100 ng/mL) for 24 h. The genome-wide expression profiles of GnIH-treated cells (0 and 10 ng/mL) were evaluated by high-throughput RNA sequencing. Compared with untreated cells, the secretion of the steroid hormones E2, E1, P4, and T was downregulated, with that of E1 and P4 reaching statistical significance (P<0.05); in contrast, the secretion of ACV and INH was significantly upregulated (P<0.05) after treatment with 10 and 100 ng/mL GnIH. The expression of encoding steroidogenic proteins and enzymes genes (STAR, CYP11A1, CYP17A1, CYP19A1, and 3-β-HSD) and encoding gonadotropin receptors genes (FSHR, LHR) were significantly declined (P<0.05) in the 10 and 100 ng/mL GnIH treatments. Transcriptome sequencing identified 348 differentially expressed genes (DEGs), including 253 upregulated and 95 downregulated genes. The DEGs were mainly involved in cell growth and death, immune response, and steroid biosynthesis pathways. We identified four novel DEGs (MROH5, LOC113840576, SDR42E1, and LOC113841457) with key roles in the regulation of steroid hormone biosynthesis. Our study revealed changes in gonadal steroid hormone secretion and steroid biosynthesis pathway-related gene expression in duck granulosa cells under the inhibitory effect of GnIH. These data contribute to our understanding of the molecular and genetic mechanisms underlying reproduction in ducks.

The synthesis of steroids by Taenia crassiceps WFU cysticerci and tapeworms is related to the developmental stages of the parasites.

Taeniids tapeworms are hermaphroditic helminths that gradually develop testis and ovaries in their reproductive units. The larval stage of the tapeworms named cysticercus is a vesicle that contains the scolex and proliferates asexually in the abdominal cavity of mice. Once in the host, they evaginate, attach to the gut and develop into an adult organism, the tapeworm. We have previously reported reported that T. crassiceps ORF and solium cysticerci transform steroid precursors to androgens and estrogens. Taenia crassiceps WFU cysticerci can also synthesize corticosteroids. The aim of the present work is to investigate the relationship between steroid synthesis ability and the developmental stage of the parasite T. crassiceps WFU. To this purpose, cysticerci were obtained from the abdominal cavity of female mice, manually separated in invaginated (IC) and evaginated parasites (EC) and preincubated for 24 h in DMEM plus antibiotics/antimycotics. Next step consisted in incubation for different periods in the fresh media added with tritiated androstenedione (3H-A4) or progesterone (3H-P4) and incubated for different periods. Taenia crassiceps WFU tapeworms were recovered from the intestine of golden hamsters that had been orally infected with cysticerci. The worms were pre-cultured in DMEM plus FBS and antibiotics, and then incubated without FBS for different time periods, in the presence of 3H-A4 or 3H-P4. At the end of the experiments the media from cysticerci and tapeworms were analyzed by thin layer chromatography. Results showed that testosterone synthesis was significantly higher in the evaginated cysticerci and increased with time in culture. The invaginated and evaginated cysticerci also synthesized small quantities of 17ß-estradiol (E2) and estrone. The evaginated cysticerci synthesized twice more 3H-deoxycorticosterone (3H-DOC) than the invaginated parasites, the production increased significantly with time in culture. Taenia crassiceps WFU tapeworms synthesized significant quantities of 3H-testosterone and small amounts of estrone after only 3 h of culture in the presence of 3H-A4. The tapeworms also transformed 3H-P4 to 3H-DOC and increased its synthesis after 24 h in culture. In summary, our data show the pathways that T. crassiceps WFU cysticerci use to synthesize sexual steroids in both larval developmental stages and reveals the steroidogenic capacity of the tapeworms.

17,20 beta-P and cortisol are the main in vitro metabolites of 17-hydroxy-progesterone produced by spermiating testes of Micropogonias furnieri (Desmarest, 1823) (Perciformes Sciaenidae)

The aim was to investigate the major C21 steroids produced by spermiating white croaker Micropogonias furnieri (Sciaenidae) in order to establish the potential mediator of gamete maturation in males of this species. The testes steroid production at the spawning season was identified incubating the 3H-17-hydroxy-4-pregnene-3,20-dione precursor through thin layer chromatography, high pressure liquid chromatography, enzymatic oxydation, acetylation and immunochemistry analyses. 17,20β-Dihydroxy-4-pregnen-3-one (17,20β-P) and 11β,17,21-Trihydroxy-4-pregnene-3,20-dione (cortisol) were the main metabolites produced. Contrary to what we expected, 17,20β,21-Trihydroxy-4-pregnen-3-one was not detected. Circulating levels of 17,20β-P were undetectable in immature testes and in those at the first spermatogenesis stages, while a clear increase was observed during the whole spermatogenesis and spermiation phases (from undetectable to 1047 pg mL-1). In vitro studies together with plasma detection suggest that 17,20β-P is a good steroid candidate involved in M. furnieri testes maturation. The role of cortisol during late phases of testes development needs further studies.(AU)

El objetivo fue investigar cuales eran los esteroides C21 más importantes producidos por los testículos en espermiación de la corvina blanca Micropogonias furnieri (Sciaenidae) para poder identificar los potenciales mediadores de la maduración gamética de los machos de esta especie. Los esteroides producidos por los testículos en espermiación fueron identificados después de su incubación con el precursor 3H-17-hidroxi-4-pregnene-3,20-diona a través de cromatografía de capa fina y cromatografía líquida de alta presión y posteriormente por oxidación enzimática, acetilación e inmunoquímica. Los principales metabolitos producidos por los testículos en espermiación fueron la 17,20β-Dihidroxi-4-pregnen-3-ona (17,20β-P) y la 11β,17,21-Trihidroxi-4-pregnene-3,20-diona (cortisol). Contrariamente a lo esperado, no se encontró el derivado tri-hidroxilado de la progesterona llamado 17,20β,21-Trihidroxi-4-pregnen-3-ona. Los niveles circulantes de 17,20β-P fueron no detectable en los testículos inmaduros y en aquellos en inicios de espermatogénesis, mientras que un aumento claro en las concentraciones circulantes fue detectada en corvinas en plena espermatogénesis y en espermiación (desde no detectable a 1047 pg mL-1). Los resultados obtenidos in vitrojunto a los cambios a nivel plasmático sugieren que la 17,20β-P es un buen candidato a proponer como esteroide involucrado en la regulación del proceso de maduración testicular de la corvina. La función del cortisol a nivel testicular debería ser mejor estudiada en las etapas finales del desarrollo testicular de esta especie.(AU)

17,20 beta-P and cortisol are the main in vitro metabolites of 17-hydroxy-progesterone produced by spermiating testes of Micropogonias furnieri (Desmarest, 1823) (Perciformes Sciaenidae)

The aim was to investigate the major C21 steroids produced by spermiating white croaker Micropogonias furnieri (Sciaenidae) in order to establish the potential mediator of gamete maturation in males of this species. The testes steroid production at the spawning season was identified incubating the 3H-17-hydroxy-4-pregnene-3,20-dione precursor through thin layer chromatography, high pressure liquid chromatography, enzymatic oxydation, acetylation and immunochemistry analyses. 17,20β-Dihydroxy-4-pregnen-3-one (17,20β-P) and 11β,17,21-Trihydroxy-4-pregnene-3,20-dione (cortisol) were the main metabolites produced. Contrary to what we expected, 17,20β,21-Trihydroxy-4-pregnen-3-one was not detected. Circulating levels of 17,20β-P were undetectable in immature testes and in those at the first spermatogenesis stages, while a clear increase was observed during the whole spermatogenesis and spermiation phases (from undetectable to 1047 pg mL-1). In vitro studies together with plasma detection suggest that 17,20β-P is a good steroid candidate involved in M. furnieri testes maturation. The role of cortisol during late phases of testes development needs further studies.

El objetivo fue investigar cuales eran los esteroides C21 más importantes producidos por los testículos en espermiación de la corvina blanca Micropogonias furnieri (Sciaenidae) para poder identificar los potenciales mediadores de la maduración gamética de los machos de esta especie. Los esteroides producidos por los testículos en espermiación fueron identificados después de su incubación con el precursor 3H-17-hidroxi-4-pregnene-3,20-diona a través de cromatografía de capa fina y cromatografía líquida de alta presión y posteriormente por oxidación enzimática, acetilación e inmunoquímica. Los principales metabolitos producidos por los testículos en espermiación fueron la 17,20β-Dihidroxi-4-pregnen-3-ona (17,20β-P) y la 11β,17,21-Trihidroxi-4-pregnene-3,20-diona (cortisol). Contrariamente a lo esperado, no se encontró el derivado tri-hidroxilado de la progesterona llamado 17,20β,21-Trihidroxi-4-pregnen-3-ona. Los niveles circulantes de 17,20β-P fueron no detectable en los testículos inmaduros y en aquellos en inicios de espermatogénesis, mientras que un aumento claro en las concentraciones circulantes fue detectada en corvinas en plena espermatogénesis y en espermiación (desde no detectable a 1047 pg mL-1). Los resultados obtenidos in vitrojunto a los cambios a nivel plasmático sugieren que la 17,20β-P es un buen candidato a proponer como esteroide involucrado en la regulación del proceso de maduración testicular de la corvina. La función del cortisol a nivel testicular debería ser mejor estudiada en las etapas finales del desarrollo testicular de esta especie.

Parasites and steroid hormones: corticosteroid and sex steroid synthesis, their role in the parasite physiology and development.

In many cases parasites display highly complex life cycles that include the penetration and permanence of the larva or adults within host organs, but even in those that only have one host, reciprocal, intricate interactions occur. Evidence indicates that steroid hormones have an influence on the development and course of parasitic infections. The host gender's susceptibility to infection, and the related differences in the immune response are good examples of the host-parasite interplay. However, the capacity of these organisms to synthesize their own steroidogenic hormones still has more questions than answers. It is now well-known that many parasites synthesize ecdysteroids, but limited information is available on sex steroid and corticosteroid synthesis. This review intends to summarize some of the existing information in the field. In most, but not all parasitosis the host's hormonal environment determines the susceptibility, the course, and severity of parasite infections. In most cases the infection disturbs the host environment, and activates immune responses that end up affecting the endocrine system. Furthermore, sex steroids and corticosteroids may also directly modify the parasite reproduction and molting. Available information indicates that parasites synthesize some steroid hormones, such as ecdysteroids and sex steroids, and the presence and activity of related enzymes have been demonstrated. More recently, the synthesis of corticosteroid-like compounds has been shown in Taenia solium cysticerci and tapeworms, and in Taenia crassiceps WFU cysticerci. In-depth knowledge of the parasite's endocrine properties will contribute to understand their reproduction and reciprocal interactions with the host, and may also help designing tools to combat the infection in some clinical situations.

Mycobacterium smegmatis synthesizes in vitro androgens and estrogens from different steroid precursors.

Fast-growing mycobacteria such as Mycobacterium sp. and Mycobacterium smegmatis degrade natural sterols. They are a model to study tuberculosis. Interestingly, M. smegmatis has been found in river effluents derived from paper production, and therefore, it would be important to gain further insight into its capacity to synthesize steroids that are potential endocrine disruptors affecting the development and reproduction of fishes. To our knowledge, the capacity of M. smegmatis to synthesize estrogens and even testosterone has not been previously reported. Therefore, the objective of this study was to investigate the capacity of M. smegmatis to synthesize in vitro testosterone and estrogens from tritiated precursors and to investigate the metabolic pathways involved. Results obtained by thin-layer chromatography showed that (3)H-progesterone was transformed to 17OH-progesterone, androstenedione, testosterone, estrone, and estradiol after 6, 12, or 24 h of incubation. (3)H-androstenedione was transformed into testosterone and estrogens, mainly estrone, and (3)H-testosterone was transformed to estrone and androstenedione. Incubation with (3)H-dehydroepiandrosterone rendered androstenediol, testosterone, and estrogens. This ability to transform less potent sex steroids like androstenedione and estrone into other more active steroids like testosterone and estradiol or vice versa suggests that M. smegmatis can influence the amount of self-synthesized strong androgens and estrogens and can transform those found in the environment.

Taenia solium tapeworms synthesize corticosteroids and sex steroids in vitro.

Cysticercosis is a disease caused by the larval stage of Taenia solium cestodes that belongs to the family Taeniidae that affects a number of hosts including humans. Taeniids tapeworms are hermaphroditic organisms that have reproductive units called proglottids that gradually mature to develop testis and ovaries. Cysticerci, the larval stage of these parasites synthesize steroids. To our knowledge there is no information about the capacity of T. solium tapeworms to metabolize progesterone or other precursors to steroid hormones. Therefore, the aim of this paper was to investigate if T. solium tapeworms were able to transform steroid precursors to corticosteroids and sex steroids. T. solium tapeworms were recovered from the intestine of golden hamsters that had been orally infected with cysticerci. The worms were cultured in the presence of tritiated progesterone or androstenedione. At the end of the experiments the culture media were analyzed by thin layer chromatography. The experiments described here showed that small amounts of testosterone were synthesized from (3)H-progesterone by complete or segmented tapeworms whereas the incubation of segmented tapeworms with (3)H-androstenedione, instead of (3)H-progesterone, improved their capacity to synthesize testosterone. In addition, the incubation of the parasites with (3)H-progesterone yielded corticosteroids, mainly deoxicorticosterone (DOC) and 11-deoxicortisol. In summary, the results described here, demonstrate that T. solium tapeworms synthesize corticosteroid and sex steroid like metabolites. The capacity of T. solium tapeworms to synthesize steroid hormones may contribute to the physiological functions of the parasite and also to their interaction with the host.

Cloning, characterization and functional expression of Taenia solium 17 beta-hydroxysteroid dehydrogenase.

The 17ß-hydroxysteroid dehydrogenases (17ß-HSD) are key enzymes involved in the formation (reduction) and inactivation (oxidation) of sex steroids. Several types have been found in vertebrates including fish, as well as in invertebrates like Caenorhabditis elegans, Ciona intestinalis and Haliotis diversicolor supertexta. To date limited information is available about this enzyme in parasites. We showed previously that Taenia solium cysticerci are able to synthesize sex steroid hormones in vitro when precursors are provided in the culture medium. Here, we identified a T. solium 17ß-HSD through in silico blast searches in the T. solium genome database. This coding sequence was amplified by RT-PCR and cloned into the pcDNA 3.1(+) expression vector. The full length cDNA contains 957bp, corresponding to an open reading frame coding for 319 aa. The highest identity (84%) at the protein level was found with the Echinococcus multilocularis 17ß-HSD although significant similarities were also found with other invertebrate and vertebrate 17ß-HSD sequences. The T. solium Tsol-17ßHSD belongs to the short-chain dehydrogenase/reductase (SDR) protein superfamily. HEK293T cells transiently transfected with Tsol17ß-HSD induced expression of Tsol17ß-HSD that transformed 3H-androstenedione into testosterone. In contrast, 3H-estrone was not significantly transformed into estradiol. In conclusion, T. solium cysticerci express a 17ß-HSD that catalyzes the androgen reduction. The enzyme belongs to the short chain dehydrogenases/reductase family and shares motifs and activity with the type 3 enzyme of some other species.

Trilostane therapy for treatment of spontaneous hyperadrenocorticism in cats: 15 cases (2004-2012).

BACKGROUND: Medical treatment with trilostane improves clinical signs, causes unclear insulin requirement changes, and variable survival times in cats. OBJECTIVES/HYPOTHESIS: To characterize the long-term efficacy of trilostane in treating cats with hyperadrenocorticism (HAC). ANIMALS: Fifteen client-owned cats with spontaneous HAC. METHODS: Multicenter descriptive retrospective study with a search performed on all medical records for cats diagnosed with spontaneous HAC. RESULTS: Clinical signs (13 of 15 cats) and ACTH stimulation testing results (13 of 15) improved with trilostane therapy. Diabetes mellitus was reported in 9/15 cases. Insulin requirements decreased by 36% within 2 months in 6/9 diabetic cats. Median survival time was 617 days for all cats (range 80-1,278 days). Complications included weight loss, urinary tract infections, chronic kidney disease, seizures, and recurrent pancreatitis. Hypocortisolemia was documented in 1 case. Cause of death occurred as a result of nonadrenal or nondiabetic illnesses (renal failure, seizures [caused by hypoglycemia or unknown]), or lymphoma. CONCLUSIONS AND CLINICAL IMPORTANCE: Trilostane ameliorates clinical signs of HAC in cats, is tolerated well in the long term, and can lead to improved regulation of diabetes.