Boars exhibit high concentrations of sulfonated estrogens (SE) mainly originating from the testicular-epididymal compartment. Intriguingly, in porcine Leydig cells, sulfonation of estrogens is colocalized with aromatase and steroid sulfatase (STS), indicating that de novo synthesis of unconjugated estrogens (UE), their sulfonation and hydrolysis of SE occur within the same cell type. So far in boars no plausible concept concerning the role of SE has been put forward. To obtain new information on SE formation and hydrolysis, the porcine testicular-epididymal compartment was screened for the expression of the estrogen-specific sulfotransferase SULT1E1 and STS applying real-time RT-qPCR, Western blot and immunohistochemistry. The epididymal head was identified as the major site of SULT1E1 expression, whereas in the testis, it was virtually undetectable. However, SE tissue concentrations are clearly consistent with the testis as the predominant site of estrogen sulfonation. Results from measurements of estrogen sulfotransferase activity indicate that in the epididymis, SULT1E1 is the relevant enzyme, whereas in the testis, estrogens are sulfonated by a different sulfotransferase with a considerably lower affinity. STS expression and activity was high in the testis (Leydig cells, rete testis epithelium) but also present throughout the epididymis. In the epididymis, SULT1E1 and STS were colocalized in the ductal epithelium, and there was evidence for their apocrine secretion into the ductal lumen. The results suggest that in porcine Leydig cells, SE may be produced as a reservoir to support the levels of bioactive UE via the sulfatase pathway during periods of low activity of the pulsatile testicular steroidogenesis.

Objectives: Demonstration of downregulation of luteinizing hormone (LH) using the gonadotropin-releasing hormone (GnRH)-agonist buserelin as the active ingredient in the form of a slow-release implant. Material and methods: To eliminate any negative feedback mechanisms of endogenous sex steroids, nine bitches were ovariohysterectomized prior to treatment. The applied drug was the slow-release implant Profact Depot((R)) with the active ingredient buserelin; dosages were 3.3, 6.6 or 13.2 mg per dog (n = 3 per group). LH bioavailability was assessed in single blood samples and in sequential blood samples collected over a 6-hour time windows, which allowed determination of the AUC (area under the curve), basal concentration, number of pulses and maximal pulse-amplitude. Results: No dose dependency was observed, leading to the conclusion that maximum efficiency could already be achieved using the lowest dose of 3.3 mg. Therefore, for further evaluation, the dogs were combined as a single group. An increasing downregulatory effect was observed from weeks 2 to 26, with the pharmacodynamic activity lasting approximately 34 weeks. There was a significant increase 1 hour after implantation to almost twice the pre-treatment value; elevated but continuously declining LH concentrations lasted a further 8 hours. Conclusion: Similar as in male dogs, in the bitch buserelin in the form of a slow-release implant also led to downregulation of LH secretion with a preceding initial increase lasting for several hours. This increase must be seen in relation to the unwanted side effects when using this type of drug for down regulation of ovarian function in the bitch. Clinical relevance: Inhibition of the initial LH increase appears to be an important factor to allow for routine clinical use of slow-release GnRH-agonist implants in the bitch.

The mechanisms governing corpus luteum (CL) function in domestic dogs remain not fully elucidated. The upregulated expression of cyclooxygenase 2 and prostaglandin (PG) E2 synthase (PGES) at the beginning of the canine luteal phase indicated their luteotrophic roles, and the steroidogenic activity of PGE2 in the early canine CL has been confirmed in vitro. Recently, by applying a cyclooxygenase 2 (COX2)-specific inhibitor (firocoxib [Previcox]; Merial) from the day of ovulation until the midluteal phase, the luteotrophic effects of PGs have been shown in vivo. This is a follow-up study investigating the underlying endocrine mechanisms associated with the firocoxib-mediated effects on the canine CL. Experimental groups were formed with ovariohysterectomies performed on Days 5, 10, 20, or 30 of firocoxib treatments (10 mg/kg bw/24h; TGs = treated groups). Untreated dogs served as controls. A decrease of steroidogenic acute regulatory (STAR) protein expression was observed in TGs. The expression of PGE2 synthase was significantly suppressed in TGs 5 and 10, and both PGE2 and PGF2α levels were decreased in luteal homogenates, particularly from CL in TG 5. Similarly, expression of the prolactin receptor (PRLR) was diminished in TGs 5 and 20. The expression of PGE2 receptors PTGER2 (EP2) and PTGER4 (EP4), the PG- transporter (PGT), and 15-hydroxy PG dehydrogenase (HPGD) was not affected in TGs. Our results substantiate a direct luteotrophic role of PGs in the early canine CL, i.e., by upregulating the steroidogenic machinery. Additionally, the possibility of an indirect effect on PRL function arises from the increased prolactin receptor expression in response to PGE2 treatment in canine lutein cells observed in vitro.

Sulfated steroids have been traditionally regarded as inactive metabolites. However, they may also serve as precursors for the production of active free steroids in target cells. In this study, we used the boar as a model to study the metabolism, transport, and function of steroid sulfates due to their high production in the porcine testicular-epididymal compartment, of which the role is unknown. To characterize the secretion of free and sulfated steroids, plasma samples were collected from six postpubertal boars over 6  h every 20  min from the jugular vein. Long-term secretion profiles were also established in seven boars stimulated with human chorionic gonadotropin. To directly characterize the testicular output, samples were collected from superficial testicular arterial and venous blood vessels. Testosterone, androstenedione and sulfated pregnenolone, DHEA, estrone (E1), and estradiol-17β (E2) were determined by liquid chromatography-tandem mass spectrometry. Free E1 and E2 were measured by RIA. Irrespective of a high variability between individuals, the results suggest that i) all steroids assessed are primarily produced in the testis, ii) they exhibit similar profiles pointing to a pulsatile secretion with low frequency (three to five pulses per day), and iii) after synthesis at least a major proportion is immediately released into peripheral circulation. The fact that all steroid sulfates assessed are original testicular products and their high correlations with one another suggest their role as being intermediates of testicular steroidogenesis rather than as being inactivated end products. Moreover, a substantial use of sulfated steroids in porcine testicular steroidogenesis would assign a crucial regulatory role to steroid sulfatase, which is highly expressed in Leydig cells.

This study evaluated ovarian activity in late gestation and post-partum in guanacos in captivity. Follicular dynamics was monitored every second day from 40 days before and other 40 after delivery by transrectal sonography and by plasma steroids concentrations. Seven out of eight (87.5%) of gestating females presented ovarian follicular activity under progesterone levels >3 nmol/l with maximum follicular size of 8.42 ± 0.83 mm from days 23 to 1 before delivery. After delivery, all females have follicular wave development from day 0 to 38, with larger follicular size and longer follicular wave phases and interwave interval when compared with pre-partum data. During post-partum period, there was a close relationship between follicle size and estradiol-17β concentration, with r = 0.69 at the beginning of growth phase and r = 0.86 in association with the largest dominant follicle. Plasma estradiol-17β concentration varied from 11.92 to 198.55 pmol/l. Plasma estrone sulfate, free estrone and progesterone returned to baseline concentrations during peripartal period and remained basal thereafter. The results described follicular activity during late gestation and early post-partum period. These findings provide relevant information to understand physiological changes occurring during this reproductive key period in seasonal breeders with long gestation duration as New and Old World camelids.

In order to characterize the steroidogenic capacity of the adult porcine epididymis, the organ was divided into eight segments (from proximal to distal: head 1 – 2, body 1 – 4, tail 1 – 2) and tissue samples from each segment were screened for the expression of mRNA encoding important steroidogenic enzymes using real-time RT-PCR. A surprisingly high expression of HSD3B1-mRNA was found in 6 of the 7 animals investigated. Relative gene expression (RGE) levels (geometric mean [dispersion factor]) increased continuously from 6.7 [6.4] in the proximal part of the epididymal head to 143.8 [8.5] in the hind part of the tail, while the RGE levels determined in the testes were only 4.8 [11.7]. In comparison, measurement in adrenal tissue from an adult boar yielded an RGE level of 1494.6. To test for 3ß-hydroxysteroid dehydrogenase activity in the testis and the hind part of the epididymal tail, the conversion of pregnenolone into progesterone was assessed in an in vitro assay in the presence of a NAD+-regenerating system using microsomal fractions prepared by differential centrifugation as the source of the enzyme (n = 5 animals). With 32.9 [2.77] resp. 14.6 [1.65] ng/mg protein/min formation of progesterone in the epididymal tail vs. testis was much closer than was expected from the mRNA data. Thus, although in pigs so far only HSD3B1 has been identified as a relevant enzyme for the conversion of 3ß-hydroxy-5-ene- into 3-keto-4-ene steroids possibly in the testis a different isoenzyme may be expressed. The physiological substrate and the biological role of HSD3B1 highly expressed in the porcine epididymal tail is currently unclear, especially in the light of a high pregnenolone sulforansferase activity and SULT2B1 expression observed in this tissue in a concomitant study.

In adult boars high levels of various sulfated steroids have been determined with maximum concentrations in the systemic circulation up to 150 ng/ml for dehydroepiandrosterone sulfate (DHEAS), 45 ng/ml for estrone sulfate (E1S) and 20 ng/ml for pregnenolone sulfate (P5S). Measurements in the testicular circulation clearly indicate that they mainly originate from the testis. In order to obtain further information on their formation, the expression of sulfotransferases (SULTs) considered relevant for the sulfation of estrogens (SULT1E1), DHEA (SULT2A1) and of P5 and cholesterol (SULT2B1) was investigated on the mRNA (SULTs 1E1, 2A1 and 2B1) and the protein level (SULTs 1E1 and 2B1) in the porcine testis and epididymis applying real-time RT-PCR, western blot and immunohistochemistry (IHC). Moreover, sulfation of E1, DHEA and P5 was assessed in an in vitro assay using cytosolic fractions in the presence of 3′-phosphoadenosine-5′-phosphosulfate. Relative gene expression (RGE) levels of SULT1E1 were high in the epididymal head (EH), decreased gradually in the epididymal body (EB) and tail (ET) but were at the limit of detection in the testis. Corresponding results were obtained from IHC and determinations of enzyme activity. RGE levels for SULT2B1 were minimal in the testis and EH, increased gradually in EB and were maximal in the hind part of ET. In the epididymis this expression pattern was consistent with sulfation of P5 and with cytosolic immunostaining in epithelial cells. However, in the EH epithelium and in various cell types of the testis distinct nuclear signals occurred in the absence of sulfation of P5. RGE levels of SULT2A1-mRNA were high in the testis and variable in the epididymis. However, sulfation of DHEA was virtually undetectable. The release of high amounts of steroid sulfates from the porcine testis in the virtual absence of relevant sulfotransferase activities remains unclear but might be explained by the extensive utilization of sulfated precursors.

Testicular function in the dog was down-regulated using two different GNRH agonist implants, with adult and juvenile testes serving as controls. Treatment resulted in an increased percentage of the interstitial area and decreased area of Leydig cell nuclei. Expression of StAR and the steroidogenic enzymes cytochrome P450 side-chain cleavage enzyme (P450scc, CYP11A1) and cytochrome P450 17α-hydroxylase-17,20-lyase (P450c17, CYP17A1) in Leydig cells was blocked at the mRNA and protein level, showing no differences between the two agonists. Staining for androgen receptor (AR) by immunohistochemistry was positive in Sertoli, Leydig and peritubular cells and some spermatogonia, with in situ hybridization confirming expression in Sertoli cells. At the mRNA level, expression of AR was not affected; however, translation was blocked (reduced percentage of AR-positive Sertoli cells), with the number of nuclei in basal position being decreased. In the juvenile testes, mRNA expression of StAR, CYP11A1 and CYP17A1 was higher compared with the other groups but distinctly lower for the AR. At the protein level, the expression was at the limit of detection for StAR; AR-positive Sertoli cells were not detected. Our observations show that the down-regulated testis is different from the juvenile one rather resembling the testicular status in seasonal breeders out of season.

Objectives: In cattle no biological role has been definitely identified for placental estrogens and progesterone. However, in the bovine trophoblast androgens may also be produced and have local effects. Thus, the aims of this study were to identify androgen receptor (AR) expressing cells and to monitor testosterone tissue concentrations in bovine placentomes throughout gestation.Methods: Placental AR expression was characterized at the mRNA and protein level applying conventional and real-time RT-qPCR, western blot and immunohistochemistry. Testosterone was measured by radioimmunoassay.Results: AR-mRNA was qualitatively detected from day 50 of gestation until term. Mean relative gene expression levels were constant between day 100 and late gestation. A slight non-significant increase was observed in the prepartal period. With immunohistochemistry distinct nuclear signals were predominantly observed in invasive trophoblast giant cells (TGC) from day 80 until term. In mature TGC of the trophoblast, immature TGC and uninucleated trophoblast cells, stromal cells of the chorionic caruncular epithelial and stromal cells immunoreactive score values were low at early and midgestation but increased significantly (p < 0.01) during late gestation and remained high until parturition. With western blot in placentomal tissue a specific band of approximately 110 kDa was detected as it was the case in epididymis used as a positive control. Testosterone concentrations increased from 0.70 +/- 0.29 pmol/g wet tissue between days 60-220 to 4.22 +/- 1.29 pmol/g during late gestation (p < 0.001).Discussion: The results are consistent with androgens as active products of bovine placental steroidogenesis. The substantial up-regulation of AR expression during TGC differentiation suggests that androgens may be related to this process. (C) 2013 Elsevier Ltd. All rights reserved.

To date, no details are available concerning the restart of steroidogenesis following the downregulation of testicular endocrine and germinative function by gonadotrophin-releasing hormone (GnRH)-agonist implants. This restart was assessed by determining the expression of steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage enzyme (P450scc) and cytochrome P450 17α-hydroxylase,17,20-lyase (P450c17). The re-establishment of steroidogenesis was initiated by the removal of the GnRH-agonist implant (18.5 mg azagly nafarelin, Gonazon) at 5 months after treatment. Testes were removed at 3-week intervals (weeks 0–24) and four groups were formed according to the stage of spermatogenesis as revealed by the most developed germ cells observed (developmental group [DG] spermatocytes to DG elongated spermatids). Five dogs served as untreated controls. Positive immunostaining for StAR, P450scc and P450c17 was restricted to Leydig cells. Western blot indicated the specifity of the respective antibodies with hints of a expression of canine-specific P450scc and P450c17 proteins. A significant effect of group was observed for a percentage of the immunopositive area (PIA) as an indicator of active Leydig cells for StAR (P<0.05), P450scc (P<0.001) and P450c17 (P<0.001), with PIA being lowest for the DG spermatocytes. With regard to the strength of the immunopositive signal, a significant effect of group was found for P450scc (P<0.01) and P450c17 (P<0.05), with the lowest intensity being observed in DG spermatocytes. At the mRNA level, the upregulation from DG spermatocytes to DG round spermatids was clearly evident but was only significant for P450scc (P<0.05). Thus, downregulation affects the whole cascade of steroidogenesis, whereas withdrawal of inhibition results in a rapid restart, in part indicating a rebound phenomenon.