Correlation Networks Provide New Insights into the Architecture of Testicular Steroid Pathways in Pigs

Steroid metabolism is a fundamental process in the porcine testis to provide testosterone but also estrogens and androstenone, which are essential for the physiology of the boar. This study concerns boars at an early stage of puberty. Using a RT-qPCR approach, we showed that the transcriptional activities of several genes providing key enzymes involved in this metabolism (such as CYP11A1) are correlated. Surprisingly, HSD17B3, a key gene for testosterone production, was absent from this group. An additional weighted gene co-expression network analysis was performed on two large sets of mRNA-seq to identify co-expression modules. Of these modules, two containing either CYP11A1 or HSD17B3 were further analyzed. This comprehensive correlation meta-analysis identified a group of 85 genes with CYP11A1 as hub gene, but did not allow the characterization of a robust correlation network around HSD17B3. As the CYP11A1-group includes most of the genes involved in steroid synthesis pathways (including LHCGR encoding for the LH receptor), it may control the synthesis of most of the testicular steroids. The independent expression of HSD17B3 probably allows part of the production of testosterone to escape this control. This CYP11A1-group contained also INSL3 and AGT genes encoding a peptide hormone and an angiotensin peptide precursor, respectively.

Influences of Immunocastration on Endocrine Parameters, Growth Performance and Carcass Quality, as Well as on Boar Taint and Penile Injuries

Castration of male pigs without anesthesia is a significant welfare issue. Improvac®, a GnRH vaccine induces an endogenous immune response leading to a decrease in testicular steroids. Consequences of different vaccination schemes on testicular function and carcass quality were evaluated in immunocastrated boars (IC), surgical castrates (SC), and entire males (EM). Therefore, 128 male piglets were assigned to five treatment-groups and a long term follow-up group. IC groups received two vaccinations (V1, V2) with Improvac® at 8 and 12, 12 and 16, or 12 and 18 weeks. Testosterone-concentrations decreased significantly two weeks after V2 in feces and dropped in serum from V2 to slaughter (S) except IC-8/12 without differing significantly. GnRH-binding results indicated the highest values for IC-12/18 animals. While IC-12/16 and IC-12/18 animals showed boar taint compounds below the threshold levels, two IC-8/12 animals had concentrations above the threshold level. Feed-efficiency was higher in EM than in SC with IC in between. In IC compared to EM, a decreasing amount of polyunsaturated-fatty-acids was obvious and GnRH-vaccination reduced penile injuries. The examined vaccination protocols reduce penile injuries, improve feed efficiency and carcass quality, and reliably prevents boar taint, if manufacturer’s recommendations concerning vaccination schedules are applied. Therefore immunocastration offers a reliable, animal friendly alternative to surgical castration.

PGE2 inhibits spermatogonia differentiation in zebrafish: interaction with Fsh and an androgen

Changes in zebrafish testicular gene expression induced by follicle-stimulating hormone (Fsh) or anti-Mullerian hormone (Amh) suggested that Amh inhibition and Fsh stimulation of spermatogenesis involved up and downregulation, respectively, of prostaglandin (PG) signaling. We found that Sertoli cells contacting type A undifferentiated (Aund) and differentiating (Adiff) spermatogonia expressed a key enzyme of PG production (Ptgs2); previous work showed that Sertoli cells contacting Adiff and B spermatogonia and spermatocytes showed ptges3b expression, an enzyme catalyzing PGE2 production. In primary testis tissue cultures, PGE2, but not PGD2 or PGF2α, reduced the mitotic activity of Adiff and their development into B spermatogonia. Vice versa, inhibiting PG production increased the mitotic activity of Adiff and B spermatogonia. Studies with pharmacological PG receptor antagonists suggest that an Ep4 receptor mediates the inhibitory effects on the development of spermatogonia, and cell-sorting experiments indicated this receptor is expressed mainly by testicular somatic cells. Combined inhibition of PG and steroid production moreover reduced the mitotic activity of Aund spermatogonia and led to their partial depletion, suggesting that androgens (and/or other testicular steroids), supported by PGE2, otherwise prevent depletion of Aund. Androgens also decreased testicular PGE2 production, increased the transcript levels of the enzyme-catabolizing PGs and decreased PGE2 receptor ptger4b transcript levels. Also Fsh potentially reduced, independent of androgens, PGE2 production by decreasing ptges3b transcript levels. Taken together, our results indicate that PGE2, via Ep4 receptors, favors self-renewal in conjunction with androgens and, independent of Fsh and androgens, inhibits differentiating divisions of spermatogonia.

Effects of chronic administration of the phosphodiesterase inhibitor Vardenafil on serum levels of adrenal and testicular steroids in men with type 2 diabetes mellitus

Background. Steroidogenesis is a complex enzymatic process in which cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) play an important role. Phosphodiesterase-5 inhibitors(PDE5i) increase cGMP, improving NO availability.Objective. to investigate whether long-term, chronic treatment with the PDE5i Vardenafil affects adrenal and testicular steroidogenesis in diabetic men, using liquid chromatography-mass spectrometry (LC-MS/MS).Design. A longitudinal, prospective, investigator-started, randomized, placebo-controlled, double-blind, clinical-trial was carried out.Setting and Participants. 54 male patients affected by T2DM diagnosed within the last 5 years were enrolled. 26 and 28 patients were assigned to the verum and placebo-group, respectively.Interventions. The study consisted of an enrolment phase, a treatment phase (24weeks) (Vardenafil/placebo 10mg twice-daily), and a follow-up phase (24weeks).Outcome measurements: progesterone (P), 17-hydroxyprogesterone (17OHP), androstenedione (A), testosterone (T), dehydroepiandrosterone (DHEA), DHEA sulphate (DHEAS), corticosterone, 11-deoxycortisol and cortisol (C), were evaluated using LC-MS/MS.Results. No differences were seen in sex testicular steroids between study and control group. For the adrenal gland, steroids were considered according to the zona in which they are produced. Considering steroids produced in the zona fasciculata, no significant differences were seen in 11-deoxycortisol and C among visits, both in the study and in the control group. For the zona reticularis, DHEA significantly decreased during treatment only in the study group (p=0.007). At post-hoc test DHEA showed higher levels at visit 2 and 8 than in other visits. The DHEAS/DHEAS ratio significantly increased during treatment only in the verum group. Considering the adrenal zona glomerulosa, corticosterone significantly changed among visits both in the study and in the control group (p<0.001). At post-hoc test, in both groups, corticosterone was significantly higher at visit 2 (p=0.028), 8 (p=0.003), and 10 (p=0.044), i.e. in coincidence with the complete clinical and instrumental examination performed only at these visits according to the study protocol.Conclusions. This is the first double-blind, placebo-controlled clinical-trial in which steroidogenesis is extensively investigated by LC-MS/MS in T2DM men chronically treated with Vardenafil for 6 months, and followed-up for 6 months after therapy-withdrawal. Chronically administered Vardenafil reduces DHEA levels and increases DHEAS/DHEA ratio as possible consequences of modulation of steroidogenic enzymes by tissue changes in cGMP and/or cAMP availability. A possibly stress-related increase in corticosterone is suggested for the first time.

In vitro inhibition of porcine cytochrome P450 by 17β-estradiol and 17α-estradiol

In vitro inhibition of porcine cytochrome P450 by 17β-estradiol and 17α-estradiol Sexually mature pigs are known to possess high concentrations of testicular steroids, which have been shown to change the activities of cytochrome P450 in vitro. The aim of the present study was to evaluate the regulation of CYP1A and CYP2E1 activity by the steroids dihydrotestosterone (DHT), 3β-androstenol, 17β-estradiol and 17α-estradiol. Catalytic activities of 7-ethoxyresorufin O-deethylase (EROD) and 7-methoxyresorufin O-demethylase (MROD) were used as markers of CYP1A activities, while p-nitrophenol hydroxylase (PNPH) was used as a marker of CYP2E1 activities. Of the steroids tested, only 17β-estradiol and 17α-estradiol inhibited EROD and MROD activities. This inhibition was observed when a steroid concentration of 100 μM was used, while lower concentrations showed no inhibitory effect. PNPH activities were inhibited only by 100 μM of 17β-estradiol. The significance of these results in vivo is unknown because inhibition was only found when concentrations of estrogens higher than physiological levels were used. Nevertheless, the results provided further evidence on the important role of estrogens in regulation of porcine cytochrome P450 activities.