The boar testis: the most versatile steroid producing organ known.

A review of the remarkable production of steroids by the testes of the boar is presented, with the principal aims of highlighting the achievements of the Leydig cells and, at the same time, pointing to the considerable deficiencies in our understanding of its biological relevance. The onset of gonadal steroidogenesis at an early stage of sex differentiation and the pattern of pre- and postnatal secretion of steroids are outlined. This is followed by a list of steroids identified in extracts of the boar testis, with emphasis on those that can reasonably be assumed to be secretory products of the Leydig cells. For example, the high concentrations of 16-unsaturated C19 and sulphoconjugated compounds are noted. Next, an impressive list of steroids found in venous blood from the boar testis is given; among them are the 16-unsaturated steroids, the oestrogens and dehydroepiandrosterone, all mainly in the form of sulphates. However, the list also includes some less likely members, such as 11-OH and 19-OH androgens as well as 5alpha-reduced steroids. Lastly, the high concentrations of steroids reported in testicular lymph, especially sulphates, are mentioned. Although roles for testosterone are uncontested, and even for the pheromone-like C19 steroids, there is little that can be said with assurance about the other compounds listed. Some speculations are made on their possible contributions to the reproductive physiology of the boar. This is done to provoke interest and, perhaps, even action towards reaching a more complete understanding of the biological significance of the steroidogenic powers of porcine Leydig cells.

Evidence for 15alpha- and 7alpha-hydroxylase activity in gonadal tissue of the early-life stages of sea lampreys, Petromyzon marinus.

Gonads of premetamorphosing larval (PML), transforming (TL) and newly metamorphosed (juvenile) sea lampreys (JL) (Petromyzon marinus) were incubated in vitro with tritiated pregnenolone ([(3)H]P(5)), progesterone ([(3)H]P(4)), and androstenedione ([(3)H]A(4)) to identify the major products of steroidogenesis in early developmental stages. Reverse-phase high-performance liquid chromatography, using two mobile phase gradients, was used to separate the radioactive steroid metabolites. 7alpha-Hydroxylase activity was evident, based on the loss of radioactivity from [(3)H]P(5) labelled at position 7, appearing as tritiated water, and on the appearance of radiolabelled 7alpha-hydroxypregnenolone in the incubation medium. In addition, there was evidence of the synthesis of 15alpha-hydroxylated steroids from the three steroid precursors used. For the progestogen precursors, one of the major 15alpha -hydroxylated metabolites synthesized by both testis and ovarian tissue co-eluted with authentic 15alpha-hydroxyprogesterone, and for [(3)H]A(4), the product was predominantly [(3)H]15alpha-hydroxyandrostenedione. Additional polar steroids were produced, some of which co-eluted with authentic 15alpha-hydroxytestosterone and 15alpha-hydroxyestradiol, whereas others could not be correlated with the authentic 15alpha- or 15beta-hydroxylated steroids available. Ovarian tissues from PML and TL developmental stages synthesized several very non-polar compounds, some of which were present as unconjugated compounds, and others only in the conjugated fraction. These molecules had retention times consistent with pregnanes, and their presence in the incubation medium was therefore indicative of the presence of 5alpha-reductase. These metabolites were not present in the incubation medium from testis, or the JL ovary, suggesting that there is no expression of 5alpha-reductase activity in these tissues. Traces of 17beta-estradiol were found in the incubation medium from ovarian tissue incubated with P(5), but not following incubation with P(4) or A(4). Testosterone was not present in the incubation medium from either ovarian or testis fragments incubated with any of the substrates used.

The role of CYP 1A1 in the in vitro metabolism of pregnenolone by the liver of rainbow trout embryos.

The in vitro metabolism of pregnenolone (P5) was investigated using whole liver preparations taken from rainbow trout (Oncorhynchus mykiss) embryos sampled between 55 and 61 days post-fertilization. The intent of the study was to use HPLC techniques to separate and identify the metabolites of hepatic P5 metabolism and identify the enzyme(s) involved. The major metabolite of [3H]P5 catabolism was [3H]7alpha-hydroxypregnenolone ([3H]7alphaOHP5), and the enzyme involved was hypothesized to be a cytochrome P450 (CYP) isozyme. To test that hypothesis, whole liver preparations from embryos were pre-treated with selected CYP inhibitors prior to incubation with [3H]P5 and post-mitochondrial supernatant (PMS) fractions of embryo livers were pre-treated with specific antibodies raised against rainbow trout CYP 1A1 prior to incubation with radiolabelled steroid precursor. Three of the four inhibitors used (Miconazole, Clotimazole, Ketokonazole) and the CYP 1A1 antibodies totally blocked the conversion of [3H]P(5) to [3H]7alphaOHP5, and the fourth, Metyrapone, partially blocked the conversion. These results suggest that CYP 1A1 is the major enzyme involved in hepatic catabolism of P5 by rainbow trout embryos.

In vitro metabolism of pregnenolone to 7alpha-hydroxypregnenolone by rainbow trout embryos.

Tissues taken from rainbow trout embryos at several developmental stages, were incubated in the presence of radioactively-labelled pregnenolone in order to determine the capability of salmonid embryos to metabolize steroids, such as pregnenolone, that are incorporated into the oocyte during gonadal growth and maturation. High performance liquid chromatography was used to separate the steroid products, and gas chromatography-mass spectrometry was applied for the chemical identification of the product. 7alpha-Hydroxypregnenolone, previously known to be produced only by ovarian tissues, was found to be the sole metabolite of pregnenolone metabolism by rainbow trout embryos. Sulfate and glucuronide conjugated forms of 7alpha-hydroxypregnenolone were also produced. We hypothesize that this metabolite provides a pathway for excretion of pregnenolone, enabling the embryo to maintain its own steroid milieu, although the possibility of 7alpha-hydroxypregnenolone also playing a physiological role cannot be excluded.

Estradiol-17beta sulfotransferase activity in canine osteosarcoma D17 cells.

Estrogen sulfatase and sulfotransferase (EST) activities are present in breast cancer tissues but there are no reports on EST in cancerous bone cells. We incubated [(3)H]estradiol-17beta with cells from a canine osteosarcoma D17 line for periods up to 24 h. Radioactive steroids were recovered from the media and separated into unconjugated and conjugated fractions using Sep-Pak C18 cartridges. The conjugate fraction was solvolyzed and the resulting free steroids were obtained from a second C18 cartridge. Little metabolism was apparent in 4 h of incubation, but by 24 h as much as one half of the radioactivity was seen in the conjugate fraction. Most of the conjugates were recovered as sulfates in all three experiments. HPLC profiles showed a limited metabolism of estradiol to other compounds except for estrone, which was clearly present in both free and sulfate fractions. These results suggest that EST may have a role in the local metabolism of estrogens in bone.

Androgen and estrogen metabolism in the reproductive tract and accessory sex glands of the domestic boar (Sus scrofa).

Steroid metabolism in target tissues has relevance in assessing biological response. We have investigated the metabolism of testosterone and estrogens in the reproductive tract and accessory sex glands in the boar. Seminal vesicles were taken from four 6-mo-old animals; and seminal vesicles, prostate, vas deferens, and regions of the epididymis were taken from two mature boars (10 and 24 mo old). Tissues were incubated in 5 ml medium (TC-199) at 34 degrees C under 5% CO(2) and 95% air for 2 h with (3)H-labeled testosterone, estrone, and estradiol-17beta. Aliquots of spent media were taken to measure radioactivity before separation of unconjugated and conjugated steroids on Waters C(18) Sep-Pak cartridges. Sulfoconjugated steroids and glucuronidates were recovered in series from C(18) cartridges after solvolysis and enzyme hydrolysis, respectively. Profiles of metabolites for free and hydrolyzed fractions were obtained from gradient HPLC with acetonitrile:water on a reversed-phase C(18) column. No clear evidence of conjugation was seen for testosterone metabolites. 5alpha-Dihydrotestosterone was the principal metabolite, but the amounts formed depended on the source, with little from the epididymal tissues and seminal vesicles, but greater quantities from the vas deferens (>25%) and prostate (>30%). The most noteworthy feature of estrogen metabolism was the extent of conjugation by all tissues. Almost all radioactivity in the conjugate fractions for the epididymis and vas was present as sulfates. Glucuronidates were seen for the prostate and were the dominant form of conjugation (about 60%) for the seminal vesicles. A striking parallel existed for the profiles of estrogen metabolites from all tissues for unconjugated and hydrolyzed fractions. Only in quantitative terms were some distinctions noted. These overall findings underscore a need to consider local metabolism of steroid hormones in target tissues of the male reproductive system.

Metabolism of oestrone and oestradiol-17beta to conjugated steroids by the accessory sex glands of the male pig.

Oestrogens are secreted in large amounts by boar testes and are known to have a synergistic effect with testosterone on the production of large volumes of seminal plasma. Thus, oestrogens play a role in regulating the large accessory sex glands in the boar. Since testosterone metabolites (e.g. 5alpha-dihydrotestosterone) account for much of its action in target tissues we have looked at the metabolism of oestrogens in the accessory sex glands of the male pig. Tissues from seminal vesicles and bulbourethral glands of 6-week-old castrate and intact males, and 12-week-old castrate animals, were incubated with (3)H-labelled oestrone and oestradiol-17beta. Aliquots of spent culture medium and of methanolic tissue extracts were taken to measure radioactivity, prior to separation of unconjugated and conjugated steroids on Waters C(18) Sep-Pak cartridges. About one-third of the radioactivity appeared as conjugates in the media from both glands with each oestrogen. Subsequently, sulphoconjugated steroids and glucuronidates were recovered in series from C(18) cartridges after solvolysis and enzyme hydrolysis respectively. Furthermore, about one-third of the conjugated fraction in each case remained unhydrolysed after these treatments. In conclusion, it is clear that a study of the actions of oestrogens on these glands must consider the dynamics of metabolism of the oestrogens presented to them by the testes and would include conjugation of steroids by the glands themselves.

Metabolism of estrogens and androgens by embryonic tissues of Arctic charr, Salvelinus alpinus.

This study examines the ability of Arctic charr (Salvelinus alpinus) embryos to metabolize tritiated androstenedione (A4), testosterone (T), 17 beta-estradiol (E2), and estrone (E1) in vitro; the metabolic products were separated by HPLC. A4 was poorly metabolized, with 48 to 64% of the substrate remaining even after 24 hr of incubation. The major metabolites of A4 metabolism are E1 and some other unidentified metabolites. T was mostly converted to A4, along with some reduced steroids, but E2 was a minor metabolite. Further, while E2 was almost exclusively transformed into E1, when E1 was used as the precursor, there was little metabolism; the products of E1 metabolism were small amounts of E1 sulfate, glucuronide, E2, and an unknown metabolite which cochromatographed with reference steroid androstenetrione (also called 11-ketoandrostenedione). It is concluded that in Arctic charr embryos there is preferential expression of a form of 17 beta-hydroxysteroid dehydrogenase resembling the type 2 isozyme of mammals that converts T and E2 to A4 and E1, respectively.

Expression and localization of relaxin in the ovary of the mare.

Immunoreactive, chromatographic and molecular techniques were used to study the expression of relaxin in mare ovaries at different stages of the oestrous cycle. Relaxin in follicular fluid ranged from 1.6 to 2.5, from 1.4 to 5.2, from 1.2 to 6.7 and from 1.0 to 3.5 ng ml-1 in small (< or = 2 cm), medium (> 2 < or = 3 cm), medium-large (> 3 < or = 4 cm) and large (> 4 cm) follicles, respectively, and total content of fluid relaxin per follicle increased (P < 0.05) with follicular size. When subjected to reverse phase HPLC analysis, follicular fluid yielded absorbance profiles corresponding closely to those of purified relaxin, and immunoreactive peaks in follicular fluid fractions measured by radioimmunoassay matched peaks of the relaxin standard. While relaxin was localized immunocytochemically to granulosa and theca cells of preovulatory follicles, northern blot and reverse transcriptase-PCR followed by Southern blot analysis failed to detect a relaxin transcript in these tissues. A single relaxin transcript (428 bp) corresponding to mRNA encoding relaxin was identified in early, mid- and late stage corpora lutea but not in corpora haemorrhagica or albicantia. Northern blot analysis revealed a weakly expressed 1 kb transcript in total cellular RNA from mature corpora lutea. In situ hybridization studies localized the mRNA to the large luteal cells of mature corpora lutea and relaxin protein was detected by immunocytochemistry in the same tissue. This is the first report demonstrating relaxin in the equine ovary and its expression by luteal cells, thereby suggesting a role for relaxin in follicular or corpus luteum function in cyclic mares.

Steroid metabolism by embryonic tissues of Arctic charr, Salvelinus alpinus.

High-performance liquid chromatography analysis of extracts of Arctic charr (salvelinus alpinus) eggs revealed the presence of several steroids, predominantly progestogens together with testosterone. Yolk sac embryos were incubated with tritiated progesterone ([3H]P4) or 17-hydroxyprogesterone ([3H]17OHP) to examine the ability of the embryos to metabolize progestogens in vitro; both progestogen precursors were converted to various free and conjugated steroids metabolites (sulfates and glucuronides). [3H]P4 was completely metabolized to form steroids that coeluted with standard 11-oxygenated androgens, corticosteroids, progestogens, and some other unidentified metabolites. This report also describes the biosynthesis of 17,20 beta-dihydroxy-4-pregnen-3-one and 17,20 beta-dihydroxy-4-pregnen-3-one 20-sulfate by embryos of Arctic charr that were incubated with 17OHP.

Postnatal decline in gonadal secretion of dehydroepiandrosterone and 3 beta-hydroxyandrosta-5,7-dien-17-one in the newborn foal.

Dehydroepiandrosterone (DHEA) and 3 beta-hydroxyandrosta-5,7-dien-17-one (7-dehydro-DHEA) are secreted in large quantities by the remarkably hypertrophied fetal gonads of both sexes in the pregnant mare. Their secretion serves as the fetal component of a feto-placental unit for oestrogen production in equine pregnancies. They are secreted in large amounts but show a decline in late pregnancy when the fetal gonads regress and levels of oestrogens in the mare fall as a consequence. We have examined the levels of these precursor steroids in the newborn foal in the first days after birth. DHEA and 7-dehydro-DHEA were measured in peripheral plasma in a direct RIA with a DHEA antibody which cross-reacts with 7-dehydro DHEA (> 150%). Subsequent studies were performed with solid-phase extraction, separation of unconjugated from conjugated steroids, and HPLC fractionation followed by RIA. Detection on HPLC at 254 and 280 nm was compared with results from RIA. It was concluded that DHEA is the major steroid produced by the gonads at birth. The concentrations are highly variable in the first day of postnatal life (70.45 +/- 63.06 ng/ml, n = 52) and decline rapidly to < 2 ng/ml (n = 6) at 96 h after birth. At this time the sulphate form is also seen, with an increasing ratio of DHEAS/DHEA as the value for total DHEA falls. The mechanism and significance of the apparent abrupt decline in gonadal steroidogenesis in the newborn foal remain unknown.

Secretion of 19-hydroxyandrostenedione and 19-hydroxytestosterone by porcine Leydig cells in vitro and in vivo.

19-Hydroxytestosterone and 19-hydroxyandrostenedione have been identified as secretory products of the testes in the mature male domestic pig. Their isolation and identification were made by reverse-phase high-performance liquid chromatography and capillary gas chromatography-mass spectrometry (CGC-MS) of extracts from testicular vein blood and media of incubations with Leydig cells. Blood was collected from veins on the surface of the testes of anaesthetized boars. Collagenase-dispersed Percoll-purified cells (> 90% pure) were incubated (20 x 10(6) cells/flask) with androstenedione (8.75 mumol/l) or [3H]androstenedione (5 x 10(6) c.p.m.) for < 60 min. Steroids were recovered from plasma or media by solid-phase extraction and the unconjugated fractions chromatographed isocratically in two solvent systems (acetonitrile:water, 37:63 (v/v) and methanol:water, 70:30 (v/v)) before CGC-MS analysis. 19-Hydroxytestosterone was present in greater quantities than 19-hydroxyandrostenedione in testicular vein blood; it was also seen as a quantitatively significant metabolite of unlabelled and radioactive androstenedione in the incubation studies. The demonstration of the secretion of 19-hydroxyandrogens from porcine testes thus raises questions concerning the physiological significance of a testicular, rather than an adrenal, secretion of these compounds.

Hydroxylation of 19-norandrogens by porcine Leydig cells.

The metabolism of 19-norandrogens by porcine Leydig cells was investigated. Non-radioactive 19-norandrostenedione (19-Nor A) and [3H]19-nortestosterone (19-Nor T) were used as substrates in incubations with cell preparations from mature male pigs. Steroid products were separated by reversed-phase HPLC and material in selected peaks was rechromatographed before attempts to identify them by GC-MS. Both 11 beta- and 6 beta-hydroxylated derivatives of 19-Nor A were found and a third product (11-oxo-19-Nor A) was tentatively identified. The profile of radioactive metabolites from [3H]19-Nor T also favours the view of a capacity for hydroxylation of 19-norandrogens by porcine Leydig cells. The significance of these findings together with our earlier report of direct 11 beta-hydroxylation of C19 steroids by such cells remains to be examined.

Identification of 5 alpha-androstane-3 beta,17 beta-diol and 3 beta-hydroxy-5 alpha-androstan-17-one sulfates as quantitatively significant secretory products of porcine Leydig cells and their presence in testicular venous blood.

By means of high performance liquid chromatography and gas chromatography-mass spectrometry it has been found that 5 alpha-androstane-3 beta,17 beta-diol sulfate and 3 beta-hydroxy-5 alpha-androstan-17-one sulfate (epiandrosterone) are major secretory steroids of the mature boar testes. These same compounds were similarly identified in culture media when porcine Leydig cells were incubated with androstenedione as substrate. In addition, they were seen as the principal secretory products when [3H]androstenedione and [3H]testosterone were used as substrates; and their presence was greatly reduced by an inhibitor of 5 alpha-reductase (N,N-diethyl,4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide). Greater quantities of 5 alpha-androstanediol than epiandrosterone were noted in all instances. These findings provide further evidence of the versatile activity of the boar testes in steroidogenesis.

Effect of a 5 alpha-reductase inhibitor on the metabolism of 19-norandrogens by porcine Leydig cells.

The metabolism of 19-norandrostenedione and [3H] 19-nortestosterone was examined in porcine Leydig cell preparations in the absence, or presence, of a 4-azasteroid inhibitor of 5 alpha-reductase. Evidence for a major production of 5 alpha-estrane-3 beta, 17 beta-diol and 3 beta-hydroxy-5 alpha-estran-17-one, as sulfo-conjugated steroids, was obtained by HPLC and gas-chromatography/mass spectrometry. The 4-aza-steroid clearly reduced the formation of both of the above 5 alpha-reduced products from the 19-norandrogens. From the HPLC profiles of the radioactive metabolites, it was also concluded that estrogen secretion was increased significantly by exposure of the cells to the 5 alpha-reductase inhibitor.

Formation of C19 11-hydroxysteroids by porcine Leydig cells.

In a previous study, we reported the presence of 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone in testicular vein blood from mature male pigs. Since C19 steroids with an oxygen function at C11 have not been recorded as products of steroid biosynthesis in normal mammalian testes, we have examined their possible production in purified preparations of porcine Leydig cells. Both androstenedione and cortisol were added as substrates in studies using cell incubations of Leydig cells from mature boars (greater than 8 months old). Steroids were recovered from media by solid-phase extraction and separated by reversed-phase high performance liquid chromatography. Peaks corresponding to retention times of authentic standard steroids were seen for both 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone from each substrate. Generally, lesser amounts of C19 11-oxosteroids were noted also. Definitive confirmation was made by gas chromatography - mass spectrometry for 11 beta-hydroxyandrostenedione in the media.

Isolation of 11 beta-hydroxylated androgens from testicular vein blood of the mature boar.

The isolation of 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone from testicular vein blood of the mature male domestic pig is described. Blood was collected from veins and arteries on the surface of the testes of mature boars. Steroids were extracted from plasma with SEP-PAK C18 cartridges and recovered with acetonitrile. A separation of steroids was made by high performance liquid chromatography (HPLC) using acetonitrile/water (37/63; v/v), and fractions were collected manually with detection at 254 nm. Preliminary identification was based on comparison with the HPLC retention time of an authentic steroid standard. Final characterization was achieved by means of capillary gas chromatography-mass spectrometry (GC-MS). The findings record the first evidence for the secretion of C19-11-hydroxylated steroids by normal testes in a mammalian species.

Aromatization of 19-norandrogens by porcine Leydig cells.

The aromatization of norandrogens was investigated with highly purified preparations of Leydig cells from mature male pigs. Cell incubations with norandrostenedione and nortestosterone gave rise to large amounts of estrone sulfate in the medium as determined directly by a specific radioimmunoassay (RIA). Estrogen production was at least equal to that seen with androstenedione and testosterone as substrates. Similar findings were made with cells in culture for 5 days before addition of the androgen substrates in a 4h-test of aromatase activity. Stimulation of estrogen formation was noted when cells were exposed for 48 h to either hCG (0.5 i.u.) or FGF-beta (10 ng) daily, as a pretreatment, before adding androstenedione for the test of aromatase activity. Little or no increase was seen with norandrostenedione or nortestosterone as substrate. Further evidence for estrogen production was obtained from HPLC separations of metabolites of cell incubations with norandrostenedione and [14C]nortestosterone monitored by RIA and radioactivity, respectively. It is suggested that norandrogens could serve as important substrates for aromatization in the boar testes.

Differences in aromatase activity between Leydig cells from the scrotal and abdominal testis in the naturally unilateral-cryptorchid boar.

In an earlier study, estrogen production was much lower in Leydig cells from the abdominal than from the scrotal testis in naturally occurring unilateral cryptorchidism in the boar. A more direct assessment of aromatase activity was made in thirty-two mature male pigs to examine this observation further, using nonradioactive androstenedione (delta 4A 1.0 x 10(-6) M - 1.5 x 10(-5) M) and [1 beta, 2 beta-3H] delta 4A as substrates. Purified Leydig cells were prepared from normal boars and from unilaterally and bilaterally cryptorchid animals. Combined estrone sulfate (E1S) and estrone (E1) formation from delta 4A were measured by radioimmunoassay. Little or no estrogen secretion was seen with cells from the abdominal testis in unilaterally cryptorchid boars (n = 7), and E1S formation from delta 4A was 6- to 14-fold higher for scrotal cells (n = 6). Aromatase activity as reflected in percent conversion of substrate to [3H]-labeled water was clearly lower in cells from the abdominal testis (1.10 +/- 0.08 and 11.22 +/- 0.7%, respectively, p less than 0.01, n = 6). No marked reduction was noted for unilaterally cryptorchid boars with an inguinally located testis (10.18 +/- 0.27 and 13.09 +/- 0.58% for inguinal and scrotal testes, respectively, n = 3). Concentrations of E1S in testicular arterial and venous blood (n = 9) gave additional evidence of lower estrogen production by the undescended testis of the cryptorchid boar. It was concluded that lower aromatase activity is present in Leydig cells of the abdominal testis.

Identification of 3 beta-hydroxy-5,7-androstadien-17-one as a secretory product of the fetal horse gonad in vivo and in vitro.

Isolation of 3 beta-hydroxy-5,7-androstadien-17-one, as a major component of steroids extracted from vein blood of the fetal gonads of the horse, supports the proposed role for the compound as a precursor for equilin formation in the placenta of the mare. The 5,7-diene was extracted from blood collected from gonadal veins of fetal ovaries and testes in situ, and from a fetal testis connected to an artery in the neck region of the mare. Perfusion of fetal gonads in the laboratory was carried out to allow longer periods of collection. In addition, isolated cell preparations from a fetal testis were incubated for 4-8 h in tissue culture to investigate steroid secretion in vitro. Final purification of neutral steroids in the extracts was carried out by high performance liquid chromatography, and identification was made by u.v. and mass spectrometry. The presence of 3 beta-hydroxy-5,7-androstadien-17-one in extracts from all sources provided evidence for its secretion in vivo and in vitro. Other 5,7-dienes, which were less polar than the C19 compound, were noted in extracts of media but not identified. These data support the view that a 5,7-diene pathway is involved in the biosynthesis of 3 beta-hydroxy-5,7-androstadien-17-one in the fetal horse gonad.