Diaryl dimers of estradiol and of estrone may be formed as major metabolites by mouse mammary glands.

The formation of a novel estrogen metabolite by mammary tissues was investigated. Polar and nonpolar metabolites of endogenous estrogens are formed in liver and other tissues. Polar products such as the catechol estrogens are implicated in tumorigenesis in breast tissue, whereas a nonpolar metabolite, 2-methoxyestradiol, may be protective. Diaryl ether dimers, as a novel form, have been reported as nonpolar products from liver microsomes. We have noted major amounts of nonpolar metabolites in other tissues that were neither 2-methoxyestrogens nor estrogen fatty acid esters. The possible formation of such novel metabolites by breast tissues from adult nulliparous mice with [(3)H]-labeled estrogens as substrates was considered. Steroids were recovered from media by solid-phase extraction and profiles were obtained from HPLC (acetonitrile:water). Saponification was done with an internal standard of estradiol stearate. Major amounts of nonpolar metabolites were formed in all instances, with one or two principal peaks. Alkaline hydrolysis had no effect on the nonpolar product(s) but released estradiol from its stearate. Strong acid treatment also had no effect as shown by HPLC. Thus, it is suggested that diaryl dimers of estrogens may be formed as major metabolites by mouse mammary glands.

The presence of 19-norandrostenedione and its sulphate form in yolk-sac fluid of the early equine conceptus.

C(18) neutral steroid formation by cytochrome P450 aromatase has been recorded for several equine and porcine tissues. High activity of P450 aromatase is reflected in the quantities of estrogens in yolk-sac (y-s) fluid of early equine conceptuses. In a previous study of y-s fluid we detected large amounts of androgens by radioimmunoassay (RIA), using an antiserum for androstenedione (A(4)). Here, we report that RIA, following chromatography, gave tentative identification of the major peak as norandrostenedione (19-norA) not as A(4). Furthermore, even greater quantities of 19-norA seemed to be present in y-s fluid as a sulphoconjugate, as noted from extraction, solvolysis, HPLC, followed by RIA. Confirmation of these unusual findings was attained after further purification with two HPLC systems and definitive identification by LC-MS with an authentic standard of 19-norA. Initial extraction of the steroid sulphate as a methylene-blue complex also yielded 19-norA suggesting that the 3-enol form had enabled sulphoconjugation. The biological significance of retention mainly as a sulphate is not known; however, the large amounts of 19-norA found in the fluid accords well with reports on the catalytic activity shown in vitro by the blastocyst isozyme of P450 aromatase in the pig and horse.

Synthesis of free and sulphoconjugated 16-androstene steroids by the Leydig cells of the mature domestic boar.

This study examined the involvement of sulphoconjugation in the biosynthesis of the 16-androstene steroids in Leydig cells of the mature boar, since the formation of steroid sulphoconjugates can reduce the levels of these steroids that accumulate in fatty tissue. Leydig cells were purified from testes of mature male pigs and incubated with pregnenolone, or various individual 16-androstene steroids for 10 min, 1, 4 and 8h. Sulphoconjugated steroids were recovered by solid-phase extraction followed by solvolysis. Profiles of unconjugated and sulphoconjugated steroids were analysed by HPLC. Steroids present in the sulphoconjugated fractions were purified, derivatised as O-methoxime/trimethylsilyl ethers (MO-TMS), and subsequently identified using gas chromatography-mass spectrometry (GC-MS). The principal metabolite produced from incubations with pregnenolone, androstadienol, androstadienone and 5alpha-androstenone was 3beta-androstenol. 16-Androstene steroids that were sulphoconjugated included 5alpha-androstenone, 3beta-androstenol and 3alpha-androstenol. Approximately 70% of the total amount of each 16-androstene steroid was in its sulphoconjugated form after incubations for 4h or more. The finding that sulphoconjugated 5alpha-androstenone was present in large amounts suggests that this steroid may be converted from a 3-keto to a 3-enol form which is subsequently sulphoconjugated. These findings emphasise the need to consider the impact of sulphoconjugation of the 16-androstene steroids and their role in contributing to boar taint.

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.

Increase in plasma testosterone concentrations after injection of adrenocorticotrophin into the boar.

Injection of a 'rapid-acting' preparation of porcine adrenocorticotrophic hormone (ACTH) into three boars resulted in a rapid rise in plasma testosterone levels which accompanied the expected rise in plasma corticosteroids. Urinary dehydroepiandrosterone (DHA) levels were measured in one boar and were found to be raised also. The results suggest that the effect involved enhaced testicular steroid activity and was related to the dosage of ACTH employed. This action of ACTH is thought to be mediated through the adrenal cortex since injection of cortisol elecited a rise in testosterone similar to that observed after injection of ACTH. ACTH had no effect on testosterone levels in a castrated boar. When a 'long-acting' preparation of ACTH was administered to two boars twice daily for 5 days, testosterone levels were depressed. It was concluded that ACTH may bring about an increase or a decrease in plasma testosterone levels in the boar depending upon the length of time increased levels of ACTH are present in the circulation.

Effect of cortisol on the response to gonadotrophin releasing hormone in the boar.

The effect of intracarotid perfusion of 40 mg cortisol for 1 h on the hormonal response to three different doses of an intramuscular injection of synthetic gonadotrophin releasing hormone (GnRH) was compared to that of GnRH injected during intracarotid perfusion with 0.9% (w/v) NaCl solution in five boars. The increase in production of LH, above basal values, in response to injection of 0.25 microgram GnRH midway through perfusion was only slightly greater (P greater than 0.05) in boars receiving cortisol compared to that when the same boars received saline. When 0.5 microgram GnRH was injected midway through perfusion, a significantly greater (P less than 0.05) increase in production of LH above basal levels occurred during cortisol administration than occurred when saline was given. Injection of 1.0 microgram GnRH in boars during cortisol perfusion resulted in significantly greater (P less than 0.01) production of LH, above basal levels, compared to the increase above basal levels that resulted when this dose of GnRH was given during intracarotid saline treatment. Increases in plasma values of testosterone reflected the increases in levels of LH. The results suggest that acute elevations in plasma cortisol may, under some circumstances, enhance the increased production of LH in the boar by increasing the responsiveness of the anterior pituitary gland to GnRH.

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.

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.

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.

Effects of gonadotrophin-releasing hormone on testosterone secretion by fetal and neonatal rat testes in vitro.

Testosterone production by fetal (20.5 days) and neonatal (1-day-old) rat testes was measured to study the possible direct effect of gonadotrophin-releasing hormone (GnRH) on steroidogenesis in early development. Single gonads were incubated in the presence and absence of GnRH (10(-10) to 10(-6) M) or agonistic analogues (10(-11) to 10(-7) M). For comparison, some testes were incubated with ovine luteinizing hormone (0.001-0.01 microgram oLH1 NIH-LH-S20/ml of medium TC199. No clear evidence of stimulation by an agonist (10(-7) M) was seen with fetal testes, but the lowest concentration (10(-11) M) gave results suggestive of an inhibitory action. Similar experiments with neonatal testes showed stimulation. With the highest concentration of GnRH or agonist the amounts of testosterone produced were about 2-3-fold greater than from control testes. Greater quantities of testosterone were found with testes exposed to LH. Hourly sampling in one experiment showed that significant stimulation of testosterone secretion had occurred within the first hour with GnRH (10(-6) M). It was concluded that fetal-neonatal rat Leydig cells are responsive to GnRH.

Effects of castration on early postnatal development of male accessory sex glands in the domestic pig.

In the neonatal pig there is a remarkable production of steroids by the testes for the first few weeks after birth. Several androgens and estrogens reach a peak at about one month of age. In order to gain an understanding of the significance of this early steroid secretion we examined the effect on accessory sex glands of removal of the testes before the peak in these compounds would have occurred. Pigs were castrated (n = 38) at 2-3 weeks of age, with littermates serving as intact controls (n = 33). Animals were killed at ages ranging from 4-12 weeks. Blood samples were taken and both bulbourethral (BU) and vesicular glands (VG) were removed, as well as the testes of intact males. Organ pairs were weighted and samples fixed for histological examination. Plasma samples were stored at -20 degrees C until assayed, without extraction, for testosterone, dehydroepiandrosterone sulfate (DHEAS) and estrone sulfate (E1S) by radioimmunoassay. Of the hormones measured, plasma DHEAS concentrations were highest, but variable over the time period (304.2 and 75.6 nmol/l; 87.7 and 21.8 ng/ml at 5 and 12 weeks respectively). E1S declined steadily from 76.6 to 5.8 nmol/l (20.7 to 1.56 ng/ml). Testosterone levels were lowest but rose from 2.67 to 9.54 nmol/l (0.77 to 2.75 ng/ml). No steroids were clearly detectable in samples from castrated males. Testes weights (wt) increased fourfold, as did body wt for both intact and castrate males. Both BU and VG showed absolute increase in wt (3.5x and 5x respectively) in intact males, and each was about 2.8x greater than in castrates (mg/kg body wt). Histological sections were markedly distinctive for both BU and VG between intact and castrate animals, and a lack of developmental changes in both glands was noted in the castrates. Our findings provide clear evidence of an influence of the testes on accessory sex glands in the early postnatal life of the pig.

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.

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.

Impaired estrogen production by Leydig cells of the naturally retained testis in unilaterally cryptorchid boars and stallions.

Estrogen production in vitro was compared for Leydig cells from cryptorchid and scrotal testes in boars and stallions. Animals with natural and experimental cryptorchidism were used. Purified Leydig cells were prepared from testes of mature animals by collagenase treatment and Percoll density gradients. After incubation for 3 hours (1 X 10(6) cells), estrone sulfate and estrone in the media were measured by direct radioimmunoassay. Androstenedione and testosterone in media extracts also were determined. Cells from the abdominal testis of unilateral cryptorchid boars and stallions showed impaired estrogen production compared with that of the contralateral scrotal testis. Surgical translocation of the scrotal testis to the abdominal cavity in four unilaterally cryptorchid, prepubertal boars did not result in a reduced capacity for estrogen secretion by Leydig cells examined after puberty. Cells from the naturally retained testis in each of these four animals produced practically no estrogen. In a naturally bilateral cryptorchid stallion, there was a high rate of estrogen secretion by both testes. It was concluded that the scrotal testis of a unilaterally cryptorchid animal exerts a suppressive influence on estrogen formation by the abdominal testis.

Estrogen concentrations in semen of the stallion.

Large amounts of estrogens are secreted by the tests of the mature stallion. In a recent study by Claus et al. [Claus, Dimmick, T., Gimenez, T., Hudson, L.W., 1992. Estrogens and prostaglandin F2 alpha in the semen and blood plasma of stallions. Theriogenology 38, 687-693.], it was stated that high levels of estrogens were also present in semen. As a preliminary step to study possible implications for fertility in the stallion, we have measured estrone sulphate (E1S), the principal estrogen in blood, in both seminal plasma and spermatozoa. Semen was collected from four Standardbred stallions at each of two stud farms in Southern Ontario during the breeding season (March-May) in 1994 and 1995, respectively, and from five stallions at the second farm in 1996. Blood samples from the jugular vein were also taken at the time of semen collection in 1996. Gel-free semen samples (n = 98, 1994-1995, n = 12, 1996) and blood were stored at -20 degrees C until analysis. Sperm was removed from thawed samples (1 ml) by centrifugation, washed 5 x with saline solution and extracted with 80% methanol. Seminal plasma (200 microliters) was diluted with absolute methanol (800 microliters), vortexed and centrifuged before aliquots were taken for radioimmunoassay (RIA). Blood plasma was diluted with RIA buffer (1:10), and all aliquots were assayed using an antiserum for direct measurement of E1S. Concentrations of E1S (ng ml-1) ranged from 0.73-8.15 (n = 110) in seminal plasma and from 64.7-153.5 (n = 12) in blood plasma. E1S concentration in the sperm pellet from 1 ml of semen had a mean value of 1.3 ng and a range of 0.54-2.48 ng (n = 53 from four animals). The amounts of E1S in total gel-free ejaculates (n = 22) from four stallions range 26-121 ng. It was concluded that the high concentrations of E1S in peripheral blood of the stallion are reflected in lesser, but high levels of the steroid in the ejaculate. E1S concentrations were quite variable in seminal plasma among stallions but less so for collections from an individual animal. The presence of E1S in washed spermatozoa requires further study.

Plasma concentrations of steroid hormones in sows infected experimentally with Leptospira pomona or porcine enterovirus strain T1 in late gestation.

Plasma levels of progesterone, corticosteroids and oestrone were determined during the late stages of pregnancy in four sows experimentally infected with Leptospira pomona and in a group of three sows in which fetuses were inoculated in utero with a strain of porcine enterovirus. Only one of the seven infected sows farrowed at full term. All fetuses were dead and delivery was prolonged in litters infected with the virus. In the sows with leptospirosis almost all piglets were expelled dead or in a weak condition. The amounts of progesterone in plasma were within the normal range and showed a decrease shortly before abortion similar to that observed before parturition in normal animals. The elevation in plasma corticosteroids at normal parturtion was not seen at abortion. An irregular pattern with rising levels of plasma oestrone was found in most sows. Peak levels of oestrone were usually reached close to the time of delivery, but occured earlier in most sows which aborted well before term. In conclusion differences were noted between the endocrine patterns in normal parturition and in abortion caused by infectious agents in sows.

Early postnatal plasma concentrations of testicular steroid hormones, pubertal development, and carcass leanness as potential indicators of boar taint in market weight intact male pigs.

Testicular steroid hormone concentrations in plasma of early postnatal male pigs were compared with plasma steroid hormone concentrations and androstenone concentrations in the fat of pigs at market weight. Positive correlations were found between the concentrations of fat androstenone at market weight and the concentrations of plasma androstenone (r = 0.46; P < 0.01), estrone sulfate (r = 0.42; P < 0.01), and testosterone (r = 0.26; P < 0.05) at market weight. These correlations were not found in animals that had reached an advanced state of pubertal development as judged by high estrone sulfate concentrations in plasma. Significant correlations were observed between plasma testosterone concentrations at market weight and plasma concentrations of androstenone (r = 0.57; P < 0.05), and estrone sulfate (r = 0.49; P < 0.05) in early postnatal animals. However, concentrations of androstenone in the fat of market weight animals were not correlated with plasma concentrations of estrone sulfate, androstenone, or testosterone in early postnatal animals. Plasma concentrations of steroid hormones in early postnatal animals cannot, therefore, be used to predict the potential for boar taint in the same animals at market weight. In market weight animals, there was a negative correlation (r = -0.57; P < 0.01) between backfat thickness and concentrations of androstenone in fat. Animals were subsequently sorted according to backfat thickness into lean and fat groups of animals. There was a strong, negative correlation between back-fat thickness and androstenone concentrations in fat (r = -0.80; P < 0.01), as well as a positive correlation between plasma androstenone and concentrations of androstenone in fat (r = 0.42; P < 0.05) among the lean group of animals. This was not seen in the fat group of animals. This suggests that the accumulation of androstenone from plasma into fat may be affected by the leanness of the pig.

The effect of early postnatal treatment with a gonadotropin-releasing hormone agonist on the developmental profiles of testicular steroid hormones in the intact male pig.

Three studies examined the effects of early postnatal treatment with a GnRH agonist on plasma concentrations of testosterone, dehydroepian-drosterone sulfate, 16-androstene steroids in fat and salivary glands, androstenone in fat and plasma, and testicular development of intact male pigs. The first study involved 45 7-d-old pigs assigned to three treatment groups: 1) boars administered 100 microg/kg of Lupron depot, 2) boars administered 200 microg/kg of Lupron depot, and 3) control boars receiving a saline carrier. The second study involved 20 7-d-old pigs assigned to two treatments: daily injection of 200 microL of 0.5 mg/mL Lupron from d 7 to 35 and controls treated with saline. The third study involved a total of 100 animals assigned to 10 groups of 10 based on their age at slaughter. These groups were subdivided into one of two treatments: 1) boars injected with 200 microL of 0.5 mg/mL of Lupron from d 3 to 35 and 2) control boars injected with saline. Testicular steroid hormone concentrations in plasma decreased (P < 0.01) within 7 d of GnRH agonist treatment. Following cessation of treatment, steroid levels increased to control levels and remained constant until the final rise at 5 mo. Plasma testosterone levels in the 100 microg/kg depot treatment group were higher (P < 0.05) than that of the 200 microg/kg and control group at 164 d of age. There were no differences between treatments (P > 0.05) in testicular steroid hormone levels at the end of study 2 or 3. There were no differences (P > 0.05) in concentrations of 16-androstene steroids in salivary glands between any of the treatment groups at market weight in studies 1 and 2. Fat androstenone levels measured in the third study ranged between 0.6 microg/g and 4.2 microg/g at 7 to 28 d of age. Treatment with GnRH agonist decreased plasma steroid levels and testicular development; however, by d 60 testicular size and weight were at control levels and remained similar until 180 d of age. The results of these studies indicate that daily administration of a GnRH agonist significantly decreased testicular development and steroidogenesis only during treatment, but testis growth and steroidogenesis had returned to control levels by 60 d of age in male pigs. Suppression of the early postnatal rise in testicular steroid hormones did not affect growth performance or steroid hormone levels at 5 to 6 mo of age.