Effect of testosterone, estrone sulphate and androstenone on 3β-hydroxysteroid dehydrogenase protein expression in primary cultured hepatocytes

Androstenone (5-androst-16-en-3-one) is a steroid pheromone, excessive accumulation of which in pig adipose tissue contributes to the phenomenon of “boar taint”. The incidence of boar taint increases with age and weight of entire male pigs. One of the reasons for the excessive accumulation of androstenone in adipose tissue of some pigs is a low rate of androstenone degradation in pig liver, which is related to defective expression of the androstenone-metabolising enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD). The mechanism regulating 3β-HSD expression in pig liver remains unclear. The present study investigated the effects of the sex steroids testosterone, estrone sulphate and androstenone on the expression of 3β-HSD protein using primary cultured hepatocytes as a model system. The study was performed on hepatocytes from pigs of two weight/age groups: “lighter weight” (average carcass weight 70 kg, age 21 weeks) and “heavier weight” (average carcass weight 92 kg, age 26 weeks). Testosterone at the range of concentrations from 5 to 100 nM induced 3β-HSD expression in “lighter weight” pigs but not in “heavier weight” animals. In contrast, estrone sulphate (5 to 100 nM) and androstenone (5 to 500 nM) induced 3β-HSD expression only in “heavier weight” but not “lighter weight” pigs. Induction of 3β-HSD expression by the sex steroids was protein-synthesis-dependent. Overall this study demonstrates for the first time that sex steroids are involved in regulation of 3β-HSD protein expression in primary cultured pig hepatocytes and that the effect of sex steroids depends on age and weight of animals. The results of this work might contribute to understanding the mechanism controlling androstenone deposition in pigs.     

Relationship between metabolism of androstenone and skatole in intact male pigs

The relationship between the metabolism of androsterone and skatole, the major compounds responsible for boar taint, was investigated in F4 Swedish Yorkshire x European Wild Pig intact males. The metabolism of androstenone and skatole were studied in liver microsomes, and the testicular steroid production was measured in testes microsomes. Including androstenone in the assays of skatole metabolism reduced the formation of 6-hydroxyskatole (pro-MII), and three other skatole metabolites (P<.05). The formation of three additional metabolites was not affected. Liver microsomal incubations of androstenone produced two metabolites, I and II. The rate of the formation of metabolite I and the rate of androstenone metabolism were correlated with the rate of skatole metabolism. Liver metabolism of androstenone was not related to levels of androstenone in fat. Testicular synthesis of 16-androstene steroids was correlated with combined synthesis of estrogens and androgens, plasma levels of androstenone, levels of skatole in fat, and skatole metabolism in the liver (P<.05). Plasma levels of estrone sulfate were correlated with levels of skatole in fat and with androstenone levels in fat and plasma and were negatively correlated with synthesis of skatole metabolite F-1 and pro-MII sulfation. These results indicate that the liver metabolism of androstenone and skatole are related. However, it is likely that the relationship between levels of androstenone and skatole in fat is due more to a link between the testicular synthesis of androstenone rather than to the metabolism of androstenone and skatole in the liver. Sex steroids may affect this relationship because of their biosynthesis along with androstenone and possible inhibition of skatole metabolism in the liver.     

Castration and testosterone effects on endogenous and somatocrinin-induced growth hormone release in intact and castrated male pigs

Growth hormone (GH) release is influenced mainly by two hypothalamic factors, growth hormone-releasing factor (GRF) and somatostatin and is modulated by other hormones such as gonadal steroids. The objective of this study was to determine if castration (CA) and exogenous testosterone (TE) affect endogenous and GRF-induced GH release. Purebred Yorkshire male pigs (n = 32) were assigned to one of the following treatments: T1:CA; T2:CA +/- TE; T3: intact (IN); T4: IN +/- TE, in a 2 x 2 factorial design. Piglets were castrated at 3 days of age. Testosterone propionate (1.0 mg/kg) in sesame oil (2 ml) or sesame oil alone was injected sc SID during a 10-day period before each sampling day at 9, 15 and 21 weeks of age. Jugular blood samples were collected for a 6-hr period preceding and following iv injection of hGRF (1-29)NH2 (10 micrograms/kg). These procedures were repeated at 9, 15 and 21 weeks of age. The overall mean GH levels and the area under the GH peaks before and after GRF stimulation were lower (P less than .05) in castrated animals than in intact animals. Testosterone treatment increased (P less than .05) circulating TE levels and increased the amplitude of the endogenous GH peaks but did not affect (P greater than .05) the GRF-induced GH release. Increasing age produced a marked reduction of the amplitude of the GH peaks, the area under the GH peaks, the baseline mean and the overall mean GH levels during the 6-hr period preceding GRF injection. The present data support the hypothesis that castration in pigs reduces circulating and GRF-induced GH release. Exogenous testosterone for 10 days did not stimulate endogenous or GRF-induced GH release with the exception of the amplitude of the endogenous GH peaks.     

Effect of live weight and dietary supplement of raw potato starch on the levels of skatole, androstenone, testosterone and oestrone sulphate in entire male pigs

This study evaluated the effect of supplement of raw potato starch (RPS) on the levels of skatole, androstenone, testosterone and oestrone sulphate in plasma from entire male pigs. The study also evaluated relationships between plasma levels of skatole and testicular steroids at three different live weights (LW) of approximately 90, 100 and 115 kg. A total of 111 entire male pigs of a crossbred (Yorkshire dams×Swedish Landrace sires) were used. Animals were raised either in mixed pens, with females and males, or single-sex pens. Each pen contained seven or nine pigs. The most fast-growing three pigs from the pens with nine pigs were slaughtered when they reached 90 kg LW, and the remaining pigs were slaughtered at 115 kg LW. All pigs were fed the same commercial diet until the average pen weight reached 100 kg. Then, 33 out of 80 remaining pigs received RPS, 0.6 kg per pig and day, for 2 weeks prior to slaughter. Blood samples were taken from the pigs at three occasions: first, the day prior to first slaughter occasion, low-weight group; second, the day prior to change in diet, middle-weight group; and third, the day prior to second slaughter occasion, high-weight group. Plasma was analysed for the levels of skatole, androstenone, testosterone and oestrone sulphate. Fat samples were taken at slaughter and analysed for the levels of skatole and androstenone. The levels of skatole and testicular steroids in plasma were significantly higher in entire male pigs from the high-weight group fed no RPS compared to those from low- and middle-weight groups. The levels of the investigated compounds did not differ between low- and middle-weight groups (P>0.1). The diet with RPS induced a decline in skatole levels in plasma and fat (P<0.001), but not plasma levels of testicular steroids and fat levels of androstenone (P>0.05). Skatole levels were positively correlated to testosterone and oestrone sulphate levels in the middle- and high-weight pigs fed no RPS as well as to testosterone in the low-weight group. In the high-weight group fed RPS, skatole levels were not correlated to any of the analysed compounds. Approximately 26% of the entire male pigs (11 out of 43) from the high-weight group fed no RPS produced skatole levels in fat above 0.20 μg/g, whereas the pigs from the low- and high-weight group fed RPS did not produce skatole levels above 0.20 μg/g in fat. Androstenone levels in fat were high in all groups. In total 47% (52 out of 111) pigs expressed androstenone levels above the rejection levels in fat of 1.0 μg/g and 88% (98 out of 111) had androstenone levels above 0.5 μg/g. It was concluded that a lower slaughter weight and the supplement of raw potato starch to the diet could be used to reduce skatole levels in entire male pigs. Androstenone levels in fat, however, could not be reduced by either a lower weight at slaughter or dietary manipulation.     

Effect of active immunization against GnRH on androstenone concentration, growth performance and carcass quality in intact male pigs

The objective of this study was to investigate the efficacy of active immunization against gonadotropin releasing hormone (GnRH) in male pigs and to compare it with surgical castration. Piglets were randomly assigned to two groups, one of 263 animals for surgical castration (SC) and one of 270 animals for immunocastration (IC). Surgery was done at 14 days of age and vaccination (Improvac®, CSL, Australia) performed twice at an interval of 4-5 weeks with the second injection given 4-7 weeks before slaughter. At slaughter, testes were weighed and fat samples collected for androstenone analysis. Androstenone was tested olfactorially by heating salivary glands in a microwave oven. Daily growth rate and meat quality were assessed in all animals. Regarding mean androstenone concentrations in backfat, no significant difference was found between SC and IC (0.042 (0.041; 0.044) μg/g vs. 0.058 (0.044; 0.071) μg/g; mean (95% confidence interval)). Testes weight was significantly (P<0.001) smaller in immunized (230.8 g (218.23; 243.52)) as compared to normal boars (761.8 g (722.77; 801.01)) of the same age, as a reference. In this study, androstenone production was suppressed in all vaccinated animals and the carcasses given free for consumption. Mean daily growth gain was not significantly different between SC (0.817 kg (0.804; 0.830)) and IC (0.827 kg (0.814; 0.840)), although there was a trend for better performance in the latter group. The yield of lean meat was significantly (P<0.001) improved in IC (54.50% (54.26; 54.73)) when compared to SC (53.76% (53.53; 54.00)). From our results, we conclude that vaccination against GnRH is a practical and effective method to suppress androstenone synthesis. Pain and stress associated with surgical castration can thus be avoided.     

Expression of 3beta-hydroxysteroid dehydrogenase, cytochrome P450-c17, and sulfotransferase 2B1 proteins in liver and testis of pigs of two breeds: relationship with adipose tissue androstenone concentration

An excessive accumulation of androstenone in pig adipose tissue is a major contributor to the phenomenon of boar taint. Androstenone deposition is dependent on the rate of androstenone biosynthesis in testis and androstenone degradation in liver. The aim of the current study was to examine the possibility of the existence of breed-specific mechanisms controlling androstenone accumulation in pig adipose tissue. The specific objective was to investigate the expression of some of the key enzymes involved in testicular and hepatic androstenone metabolism in pigs of 2 breeds by using animals with high and low androstenone concentrations within each breed. The study was conducted with Norwegian Landrace (N. Landrace) and Duroc boars. The mean androstenone values for the low- and high-androstenone groups were 0.1 +/- 0.01 microg/g and 7.58 +/- 0.68 microg/g for N. Landrace boars, and 0.22 +/- 0.04 microg/g and 13.55 +/- 1.14 microg/g for Duroc boars. The enzymes investigated were 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450-c17, and sulfotransferase 2B1 (SULT2B1). Expression of cytochrome P450-c17 in liver and testis did not differ between animals with high and low androstenone concentrations in either the N. Landrace or Duroc breed. Expression of hepatic 3beta-HSD, which catalyzes the first stage of androstenone degradation, was decreased in high-androstenone N. Landrace boars (P < 0.01), but not in high-androstenone Duroc boars. In contrast, the expression of hepatic SULT2B1, which catalyzes the second stage of steroid catabolism, was decreased in high-androstenone Duroc animals (P < 0.05), but not in high-androstenone N. Landrace animals. Sulfotransferase 2B1 was also inhibited in testis of high-androstenone pigs of both breeds compared with low-androstenone animals. We report breed differences in expression of the androstenone-metabolizing enzymes 3beta-HSD and SULT2B1 in the liver of high- and low-androstenone pigs. It is suggested that accumulation of androstenone in adipose tissue of N. Landrace boars might be related to a low rate of hepatic androstenone degradation in metabolic stage I, whereas the high androstenone concentration in Duroc boars might be related to a low rate of androstenone metabolism in metabolic stage II.     

Effect of bovine growth hormone gene expression, sex and age on plasma gonadotropins, estrone and testosterone in prepuberal pigs.

Chronic supraphysiological blood levels of growth hormone (GH) may retard sexual maturation in swine. Pigs used in this study included four founder transgenic pigs (two gilts and two boars) expressing a mouse transferrin (TF) promoter fused to a bovine (b) GH structural gene, 13 second- or third- generation transgenic pigs (seven gilts and six boars) expressing a mouse metallothionein (MT) promoter fused to a bGH structural gene and 16 control littermates (eight gilts and eight boars). Blood plasma levels of LH, FSH, estrone and testosterone were measured to determine whether expression of bGH genes altered secretion of hormones between 80 and 180 days of age. Presence of a bGH gene was detected by hybridization of DNA in dot blots of tail biopsies. Expression of a bGH gene was detected by radioimmunoassay of plasma bGH. In four TFbGH founder transgenic pigs bGH ranged from 164 to 1948 ng/ml; in one MTbGH transgenic boar of line 3104 bGH was 1211 ng/ml; and in 12 pigs of line 3706 bGH ranged from 25 to 190 ng/ml. Expression of bGH in transgenic pigs lowered (P = .0192) plasma LH with no significant differences between sexes, had no significant effect on plasma FSH and lowered plasma estrone (P = .0001) and testosterone (P = .0269) in boars (but not gilts). Plasma estrone and testosterone were higher (P = .0001) in boars than in gilts. Plasma FSH was higher (P = .0001) in gilts than boars and decreased (P = .0001) with advancing age in gilts but not in boars.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetics of sulfamethazine in male, female and castrated male swine

The concentration of sulfamethazine in plasma and sulfamethazine and its metabolites in urine were compared in male, female and castrated male swine. A surgical technique for placement of catheters in the urinary bladder was used to facilitate the collection of urine in males and castrated males. The elimination rate of sulfamethazine from plasma and the excretion of parent drug and metabolites into urine did not differ significantly among females, males and castrated male swine.     

Plasma concentrations of testosterone and nandrolone in racing and nonracing intact male horses

Pennsylvania (PA) State Racing Commissions regulate the endogenous androgenic steroid, testosterone (TES), in racing intact males (RIM) by quantification of TES in post-race samples. Post-race plasma samples (2209) collected between March 2008 and November 2010 were analyzed for TES, nandrolone (NAN), and other anabolic steroids (ABS). Of the 2209 plasma samples, 2098 had quantifiable TES ≥ 25 pg/mL. Plasma (mean ± SD) concentrations of TES and NAN in RIM were 329.2 ± 266.4 and 96.0 ± 67.8 pg/mL, respectively. Only 64.6% of RIM had quantifiable concentration of NAN, and there was no relationship between TES and NAN. Plasma TES concentrations were significantly (P < 0.0001) higher during the months of April, May, June, July, and August. A significantly higher (P < 0.006) plasma TES was observed in Thoroughbred (TB) (347.6 ± 288.5 pg/mL) vs. that in Standardbred (STB) (315.4 ± 247.7 pg/mL). Plasma concentrations of TES from breeding stallions (BS) were 601.6 ± 356.5 pg/mL. Statistically significant (P < 0.0001) lower plasma concentrations of the two steroids were observed in RIM horses. Based on quantile distribution of TES in the RIM and BS populations, 99.5% were at or below 1546.1 and 1778.0 pg/mL, respectively. Based on this population of RIM, the suggested upper threshold plasma concentration of endogenous TES in horses competing in PA should remain at 2000 pg/mL.     

Immunolocalization of 3beta-hydroxysteroid dehydrogenase in normal and hyperplastic ram prostates

The enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is essential in the synthesis of all steroids by cleaving dehydroepiandrosterone to androstenedione. In the present study, 3beta-HSD immunoreactivity was investigated in the prostate of Akkaraman breed rams aged older than 3 years. Five normal and five hyperplastic ram prostates were processed for immunohistochemistry. Prostate hyperplasia was determined by histopathological evaluation of 375 ram prostate and confirmed with significantly (P<0.01) increased number of cells expressing proliferating cell nuclear antigen (PCNA) immunoreactivity in the glandular epithelia. The 3beta-HSD immunoreactivity with a variable intensity and pattern of distribution was present in the glandular epithelia and endothelia of blood vessels in normal and hyperplastic ram prostates. While immunoreactivity was focally present in some glands, some sections had a homogenous distribution. The presence of 3beta-HSD immunoreactivity indicates that steroids are locally synthesized in the ram prostate. No differences in the distribution pattern of 3beta-HSD immunoreactivity and the percentage of immunoreactive cells were observed between normal and hyperplastic prostates (P>0.05), suggesting that locally produced steroids have little or no effect on the pathogenesis of the ram prostate hyperplasia which affects a very small proportion of the ram population (5 out of 375).     

Maintenance of physiologic concentrations of plasma testosterone in the castrated male dog, using testosterone-filled polydimethylsiloxane capsules.

Effects of various numbers of polydimethylsiloxane (PDS) capsules filled with testosterone (PDS-T) on plasma testosterone (PT) in castrated male dogs were studied. Dogs were implanted with 1 empty PDS capsule or 1, 3, or 5 PDS-T capsules. Blood samples were collected prior to and after implantation, after castration with capsules in situ, and after capsule removal. The PT was determined in these samples by radioimmunoassay. One empty capsule had no effect on PT concentration; after castration, PT values fell to nondetectable amounts. One PDS-T capsule maintained PT at concentrations above nondetectable amounts after castration, but these concentrations were significantly (P less than 0.05) lower than were preimplantation values. Three or five PDS-T capsules were capable of maintaining PT concentrations in the castrated male dog similar to those concentrations seen in the intact dog.     

Induction of parturition in swine and epostane, a competitive inhibitor of 3 beta-hydroxysteroid dehydrogenase

The ability of epostane, a competitive inhibitor of the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) enzyme system, to decrease the peripheral levels of progesterone and induce sows to farrow was examined. Twenty-five sows were randomly divided into five groups. On d 109 of pregnancy, each sow received one of the following treatments: no epostane (Group C); one oral dose of 5 mg (Group O5) or 10 mg (Group O10) of epostane/kg of body weight; or a sc injection of 1 mg (Group I1) or 5 mg (Group I5) of epostane/kg of body weight. During the 24 h after treatment with epostane, the levels of progesterone were approximately one-half of pre-treatment levels. Progesterone was influenced (P less than .01) by treatments and time. The treatment X time interaction (P less than .05) appeared to be due mostly to the difference in response between the sows that received epostane or vehicle. Similar results were observed for estrogen. The interval from treatment to the birth of the first piglet for Groups C, O5, O10, I1 and I5 was 112, 31, 33, 77 and 32 h, respectively, and the intervals differed (P less than .01). Three gifts in Group I1, with an interval of 53, 124 and 133 h, were not considered to have been induced. The route of administration of epostane or dose did not influence (P greater than .05) the interval to the onset of farrowing. The interval from the birth of the first to the last piglet in a litter, the proportion of piglets born live and weaned, and the birth and weaning weights for the five groups were similar (P greater than .05). These results suggest that inhibitors of the 3 beta-HSD can be used to induce farrowing without adversely affecting either the sow or its litter.     

Insulin-like growth factor-I concentrations in plasma of intact and castrated male and female cattle at four ages

The influence of age, sex and castration on plasma concentrations of insulin-like growth factor-I (IGF-I) and other metabolic hormones related to growth was studied in cattle. Plasma was sampled from bulls, steers, heifers, and ovariectomized heifers at 20-min intervals for 12 hr at 5, 8, 12, and 15 mo of age. Plasma samples from each animal taken during each 12-hr period were composited for analysis of IGF-I, testosterone, total estrogens, thyroxine, triiodothyronine, insulin, and glucose. The mean plasma IGF-I concentration in all cattle increased from 61.6 to 158.6 ng/ml as the animals aged (p less than .01). Over all ages, bulls had greater concentrations of IGF-I than steers, heifers, or ovariectomized heifers (P less than .01). Bulls also had higher concentrations of testosterone (P less than .01) and total estrogens (P less than .01). Triiodothyronine concentration was greater in ovariectomized heifers than in bulls (P less than .01) or steers (P less than .05). Females had higher concentrations of thyroxine than males (P less than .01). Concentrations of triiodothyronine in the cattle were greater (P less than .01) during the winter and early spring as compared with the summer. Concentrations of insulin and glucose were not influenced by sex or castration; however, insulin increased in all cattle with age (P less than .01). The mean increase in IGF-I concentration with age within each of the four groups was associated with an increase in concentration of plasma insulin but the differences due to sex were not related to differences in insulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long non‐coding RNA analysis of muscular responses to testosterone deficiency in Huainan male pigs

Long non‐coding RNAs (lncRNAs) participated in growth and development of skeletal muscle; however, little is known about their response to testosterone deficiency in porcine skeletal muscle. We compared lean mass related carcass traits and lncRNAs expression files in Longissimus dorsi (LD) muscle between intact and castrated Huainan male pigs. The results showed that castration significantly reduced eye muscle area and lean meat percentage (P < 0.05), but increased the fat mass weight (P < 0.05). Meanwhile, 8946 lncRNAs, including 6743 intergenic lncRNAs (lincRNAs), 498 anti‐sense lncRNAs, and 1705 intronic lncRNAs, were identified in porcine LD, among which, 385 lncRNAs were considered as the differentially expressed candidates between intact groups and castrated groups (q‐value < 0.05). Functional analysis indicated that these differently expressed lncRNAs and their target genes were involved in the estrogen receptor signaling pathway and skeletal and muscular system development and function. We first detect porcine muscular lncRNA response to castration, and the results suggested that lncRNAs and their target genes participated in the regulation of testosterone deficiency‐related skeletal muscle growth.     

Immunohistochemical detection of inhibin-alpha, -betaB, and -betaA chains and 3beta-hydroxysteroid dehydrogenase in canine testicular tumors and normal testes.

Immunohistochemical detection of inhibin-alpha, -betaA and -betaB chains and 3beta-hydroxysteroid dehydrogenase (HSD) was carried out on primary testicular tumors from 15 dogs and normal testes from three adult dogs. Histopathologically, the tumors were composed of three types: Leydig cell tumors in five dogs, Sertoli cell tumors in five dogs, and seminoma in five dogs. In normal testes, immunostaining against inhibin-alpha, -betaA, and -betaB chains and 3beta-HSD revealed positive reactivity in the cytoplasm of Leydig cells. In testicular tumors, immunoreactive cells against inhibin-alpha, -betaA, and -betaB chains and 3beta-HSD were localized in all Leydig cell tumors but not in any Sertoli cell tumors or seminomas. The results of radioimmunoassay for plasma inhibin in dogs with Leydig cell tumors showed higher concentrations than those in dogs with Sertoli cell tumors and seminomas and those in normal dogs. The concentration of inhibin in the plasma was markedly decreased by the surgical removal of the Leydig cell tumor in one dog. Our findings suggest that inhibin is synthesized by normal and neoplastic Leydig cells in the canine testis, and the secreted inhibin may be inhibin A and inhibin B.     

Immunohistochemical localization of 3beta-hydroxysteroid dehydrogenase in the granulosa and theca interna layers of bovine cystic follicles

The aim of the present study was to determine whether the alteration of population of cells containing 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is responsible for the formation of cystic follicles. Paraffin sections of healthy (2 to 5 mm in diameter), atretic (2 to 5 mm) and cystic follicles (more than 25 mm) were immunohistochemically stained with rabbit polyclonal antibody to bovine 3beta-HSD. The 3beta-HSD-positive cells were counted in 4 different regions of the follicles from the apical to the basal side. The frequencies of 3beta-HSD-positive granulosa cells in cystic follicles were significantly higher than those in the healthy follicles (P<0.05), although the number of 3beta-HSD-positive granulosa cells in the cystic follicle were fewer than half the cells (30 to 40%) and was much smaller than that in preovulatory follicles (Conley et al., 1995). The frequencies of 3beta-HSD-positive cells were higher in the granulosa layer and lower in the theca interna layer of the cystic follicles than the atretic follicles. These results suggest that the differentiation of granulosa cells to express 3beta-HSD might be insufficient in cystic follicles and accordingly they fail to ovulate. The differences of frequencies of 3beta-HSD-positive cells in the granulosa and theca interna layers between cystic and atretic follicles may be one of the reasons why regression is delayed in cystic follicles.