Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout Oncorhynchus mykiss spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?

Background: In rainbow trout (Oncorhynchus mykiss), the endocrine control of spermiation is not fully understood. Besides IIketotestosterone (IIKT) and 17alpha, 20beta-dihydroxyprogesterone (MIS), the potential physiological ligand of the mineralocorticoid receptor (MR) II-deoxycorticosterone (DOC), is a credible candidate in O. mykiss spermiation regulation as spermiation is accompanied with changes in aqueous and ionic flows. Methods: In this study, we investigated potential roles of DOC during spermiation 1) by describing changes in blood plasma DOC level, MR mRNA abundance during the reproductive cycle and MR localization in the reproductive tract 2) by investigating and comparing the effects of DOC (10 mg/kg) and MIS (5 mg/kg) supplementations on sperm parameters 3) by measuring the in vitro effect of DOC on testis MIS production. Results: The plasma concentration of DOC increased rapidly at the end of the reproductive cycle to reach levels that were 10-50 fold higher in mature males than in immature fish. MR mRNA relative abundance was lower in maturing testes when compared to immature testes, but increased rapidly during the spermiation period, immediately after the plasma rise in DOC. At this stage, immunohistochemistry localized MR protein to cells situated at the periphery of the seminiferous tubules and in the efferent ducts. Neither DOC nor MIS had significant effects on the mean sperm volume, although MIS treatment significantly increased the percentage of males producing milt. However, a significant reduction in the spermatocrit was observed when DOC and MIS were administrated together. Finally, we detected an inhibitory effect of DOC on testis MIS production in vitro. Conclusion: These results are in agreement with potential roles of DOC and MR during spermiation and support the hypothesis that DOC and MIS mechanisms of action are linked during this reproductive stage, maybe controlling milt fluidity. They also confirm that in O. mykiss MIS is involved in spermiation induction

Aging Male Questionnaire in normal and complaining men.

International audienceINTRODUCTION: Detection of androgen deficiency is at least, based on specific questionnaires, defined by sexual, psychological, and somatic variables. Their relationships with sexual hormone levels are poorly understood. AIM: To assess the Aging Male Symptoms (AMS) score and sex hormone levels in normal and complaining men in order to define the relationship between the key parameters related to androgen deficiency. METHODS: Nine hundred and three men were interviewed via phone by a trained interviewer who completed the questionnaire; 539 men consulting for a checkup in a health center and 471 complaining men, who completed the AMS scale in clinical setting, were selected, after excluding subjects with major and/or chronic diseases, endocrine disorders, psychological dysfunctions, and metabolic syndrome. MAIN OUTCOME MEASURES: Total AMS score and psychological, somatic and sexual subscores, as a function of age. RESULTS: The AMS questionnaires the were completed in a clinical setting or via calling-up line were comparable. In both cases, total AMS scores and subscores were significantly dependent of age and were correlated to income. In normal men, the only two parameters that significantly changed with age were the AMS sexual subscore and bioavailable testosterone (BT). Complaining men aged more than 50 years old had a significantly higher total AMS scores, subscores, and BT level than normal men up to 60 years old, and these differences weakened with increasing age. In normal and complaining men, whatever the AMS sexual subscore, any variation in testosterone (T) and BT levels was observed. CONCLUSIONS: The AMS scale could be defined as a screening test for androgen deficiency symptoms in men between 50 and 65 years of age. The sexual AMS subscore and BT level are the key variables to identify those symptoms; the severity of sexual symptoms can not be explained by a BT level decrease

Plasma and hepatic reaction to acute estrogen exposure.

<p>A. Representative scheme of E2 biological activity and detoxification pathways. E2 can go through oxidative metabolism and be converted in E1 by 17bHsd2 or in hydroxyl-metabolites by enzymes of the Cyp family. Then it can be methylated by COMT, reduced by P450 reductase (resulting in DNA damage) or excreted after conjugative metabolism by GST. E2 can also be directly sulfo-conjugated by SULT or gluco-conjugated by UGT, and excreted. B–E. Plasma estradiol (B) and derivatives E2-sulfate (E2-S; C), estrone (D) and estrone-sulfate (E1-S; E) were measured by GC/MS from the day of birth (P0) to PND6 (P6). Each point represents the mean ± SEM of at least four pools of two animals each. A two-way ANOVA indicated that there was a significantly different profile for the 10 µg/d E2 treatment than for all other treated or control groups (p<0.001), E1 (p<0.001), E1-S (p = 0.002). Hormonal levels varied according to time, independent of treatment, for each metabolite tested: E1 (p<0.001), E2 (p<0.001), E1-S (p<0.001) and E2-S (p<0.001) (n = 4–5 pools of 3 to 4 animals at PND0, 4–5 pools of 2–4 animals at PND1, 4 pools of 2–3 animals at PND2, 4 pools of 2 animals at PND3 and 8 animals at PND6). F–J. Quantitative RT-PCR for <i>Hsd17b2</i> (F), <i>Ugt1a1</i> (G), <i>Cyp1b1</i> (H), <i>Cyp2b1/2</i> (I) and <i>Gsta2</i> (J) using liver samples of controls (white bars) and animals treated with 10 µg E2 at PND0 and PND1 (black bars) shows E2 impairment on post-natal <i>Hsd17b2</i>, <i>Cyp2b1/2</i> and <i>Gsta2</i> expression dynamics. Each bar represents mean ± SEM of the fold-change in target gene expression relative to a reference gene Snx17 and calibrator sample. Each point represents mRNA from 3 pools of ovaries from 3 animals. *p<0.05 (two-way ANOVA, followed by Tukey test). <b>a</b> shows an increase from PND0, <b>b</b> shows a decrease from PND1, <b>c</b> shows a decrease from PND3 and <b>*</b> shows a difference from the age-matched control group.</p

Implication of the mineralocorticoid axis in rainbow trout osmoregulation during salinity acclimation

Cortisol and glucocorticoid receptors (GRs) play an important role in fish osmoregulation, whereas the involvement of the mineralocorticoid receptor (MR) and its putative ligand 11-deoxycorticosterone (DOC) is poorly investigated. In this study, we assessed the implication of DOC and MR in rainbow trout (Oncorhynchus mykiss) osmoregulation during hypo- and hypersaline acclimation in parallel with the cortisol-GR system. A RIA for DOC was developed to measure plasma DOC levels, and a MR-specific antibody was developed to localize MR protein in the gill, intestine, and kidney. This is the first study to report DOC plasma levels during salinity change and MR localization in fish osmoregulatory tissue. Corticosteroid receptor mRNA abundance was investigated in osmoregulatory tissue during salinity acclimation, and the effect of cortisol and DOC on ionic transporters gene expression was assayed using an in vitro gill incubation method. Differential tissue-, salinity-, and time-dependent changes in MR mRNA levels during both hyper- and hyposaline acclimations and the ubiquitous localization of MR in osmoregulatory tissue suggest a role for the MR in osmoregulation. Presumably, DOC does not act as ligand for MR in osmoregulation because there were no changes in plasma DOC levels during either freshwater-seawater (FW-SW) or SW-FW acclimation or any effect of DOC on gill ionic transporter mRNA levels in the gill. Taken together, these results suggest a role for MR, but not for DOC, in osmoregulation and confirm the importance of cortisol as a major endocrine regulator of trout osmoregulation

Primers used for qPCR and <i>in situ</i> hybridization (*).

<p>Primers used for qPCR and <i>in situ</i> hybridization (*).</p

Ovarian reaction to acute estrogen exposure.

<p>A. A false-color heatmap shows cases of increasing, decreasing, detectable (but without differential expression) and undetectable transcript signal intensities across the replicates for different total ovary samples at the time points given (top). Each line corresponds to a probe set and each column to a sample replicate. A color scale is shown for signal intensity percentiles (bottom). Gene symbols and numbers of transcripts are shown at the right. B. Conventional RT-PCR screening of the expression of various enzymes involved in E2 metabolism in PND0, control (C) and E2-treated (E) PND1 and adult female ovaries, and PND0 livers reveals changes in expression of <i>Ugt1a1</i>, <i>Gsta2</i>, <i>Gstm5</i> between newborn and adult ovaries, stable expression of <i>Hsd17b2</i>, <i>Cyp1b1</i>, <i>Gstp1</i>, and the faint expression of <i>Sult1e1</i> and <i>Cyp2b1/2</i> by contrast to control <i>Snx17</i> RNAs. C–H. Quantitative RT-PCR for <i>Hsd17b2</i> (C), <i>Cyp1b1</i> (D), <i>Cyp2b1/2</i> (E), <i>Gsta2</i> (F), <i>Ugt1a1</i> (G) and the <i>Rbp4</i> E2 target gene (H) in ovaries of controls (white bars) and animals treated with 10 µg E2 (black bars) at PND0 (2 h after injection) and PND1 shows E2 impairment of post-natal <i>Gper</i> up-regulation. Each bar represents mean ± SEM of the fold-change in target gene expression relative to a Snx17 reference gene and calibrator sample. Each point represents mRNAs from 3 to 6 pools of 6–16 and 10–26 ovaries, respectively. *p<0.05 (two-way ANOVA, followed by Tukey test).</p

Perinatal ovarian receptivity to estrogens.

<p>A–D: Quantitative RT-PCR for Esr1 (A), Esr2 (B), Gper (C) and Nr1i2 (D) performed on control ovaries at e18.5, day of birth (PND0) and PND12. Each point is constituted by three pools of at least four animals. Data points represent the mean ± SEM of the fold-change in target gene expression relative to a Snx17 reference gene and calibrator sample. Each point represents mRNA from 3 pools of ovaries from 3 animals. *p<0.05 (ANOVA, followed by PLSD test). <b>x</b> shows a statistically significant difference from e18.5 and <b>y</b> shows a statistically significant difference from PND0. E–P. <i>In situ</i> hybridizations for Esr1 (E, H), Esr2 (F, I, K), and Gper (G, J, L) in PND1 (E–G), PND6 (H-K) and PND2 (L-P) control ovaries show lower Esr1 expression in the ovary than in the oviduct epithelium (Ovd), higher expression of Esr2 in granulosa cells with follicle growth, and expression of Gper in the oocytes and granulosa cells. Inset in F shows a higher magnification of a group of follicles boxed in F. K shows a higher magnification of a primary (Iary and a secondary (IIary) follicles boxed in I. Inset in G shows another section of the ovary containing the oviduct. A comparison of <i>Gper</i> mRNA profile by <i>in situ</i> hybridization (L) with Esr2 (N, red) and Ybx2 (M, cytoplasmic, green) (and merged pictures O) by immunofluorescence revealed co-expression of both receptors in oocytes at the time of treatment. P shows hybridization with <i>Gper</i> sense probe. Scale bar: 100 µm except in insert in F and K (50 µm). Q-T. Quantitative RT-PCR for <i>Esr1</i> (Q), <i>Esr2</i> (R), <i>Gper</i> (S) and <i>Nr1i2</i> (T) using ovarian samples of controls (white bars) and animals treated with 10 µg E2 at PND0 (2 h after injection) and PND1 (black bars) shows E2 impairment of post-natal <i>Gper</i> up-regulation. Each bar represents mean ± SEM of the fold-change in target gene expression relative to a Snx17 reference gene and calibrator sample. Each point represents mRNAs from 3 to 6 pools of 6–16 and 10–26 ovaries, respectively. *p<0.05 (ANOVA, followed by PLSD test).</p

Characterization of oocyte depletion.

<p>A. Time-course showing the decline in oocyte number per ovary in control (white points and dotted lines) and 10 µg E2-treated ovaries (black points and continuous lines) from PND0 to PND3. Data are expressed as mean ± SEM of 5–14 ovaries. *p<0.01 <i>vs.</i> age-matched controls (ANOVA followed by PLSD test). B. Time-course of apoptotic oocyte number per ovary in control (white points and dotted lines) and 10 µg E2-treated ovaries (black points and continuous lines) from PND0 to PND2. Data are expressed as mean ± SEM of 4–8 ovaries. C–D. Quantitative RT-PCR for <i>Bax</i> (C), <i>Bcl2</i> (D), <i>Nobox</i> (E), <i>Figla</i> (F), <i>Scp1</i> (G), <i>Hnrnpk</i> (H), <i>Foxo3a</i> (I) and <i>Eif4e</i> (J) in ovarian samples of controls (white bars) and animals treated with 10 µg E2 (black bars) at PND0 (2 h after injection) and PND1 shows transient down-regulation of <i>Bcl2</i> mRNA at PND0 and the absence of <i>Nobox</i> mRNA up-regulation between PND1 and PND2 in E2-treated ovaries. Each bar represents mean ± SEM of the fold-change in target gene expression relative to a Snx17 reference gene and calibrator sample). Data from 3–6 pools of 6–14 ovaries. *p<0.05 <i>vs.</i> age-matched control (two-way ANOVA, followed by t-test).</p