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.

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.

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.

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.

Blood steroid profile and in vitro steroidogenesis by ovarian follicles and testis fragments of adult sea lamprey, Petromyzon marinus.

The main purpose of the study was to identify the principal gonadal steroids synthesized by male and female sea lampreys, Petromyzon marinus. To achieve this, we used high performance liquid chromatography to separate the steroids in the serum of sexually mature animals, and to separate the steroids produced by gonadal tissue incubated in the presence of radiolabelled precursor steroids, as a means of identifying the major steroidogenic pathways. We were unable to detect evidence of the 'classical' steroids, such as 17beta-estradiol (E(2)) or testosterone (T) in the serum of either male or female lampreys. Instead, the principal chromatographic peaks contained very polar compounds that had elution times consistent with 15alpha-hydroxylated estrogens and androgens, and there were sex-specific differences in the chemical nature and the quantity of these compounds. Testis fragments or ovarian follicles co-incubated with tritium-labelled pregnenolone ([3H]P(5)), 17-hydroxyprogesterone ([3H]17OHP(4)), or androstenedione ([3H]A(4)), provided additional confirmation that the gonads synthesize a range of very polar steroids, and the metabolites found were consistent with the presence of a 15alpha-hydroxylated (15alphaOH) metabolic pathway common to testis and ovary. For ovarian tissue, the major 'end product' metabolites from all three precursors were 15alphaOH-estrogens, and for testis tissue 15alpha-hydroxyprogesterone (15alphaOHP(4)) and 15alpha-hydroxytestosterone (15alphaOHT) and small amounts of 15alphaOH estrogen. Small amounts of E(2) were also produced by both ovarian (all substrates) and testicular tissue (some substrates). Although it was assumed that the E(2) was synthesized via the aromatization of T, [3H]T was not found as an intermediate metabolite. The study suggests that the principal gonadal steroids in sea lamprey are 15alpha-OH compounds, and that only small amounts of E(2) or T are synthesized by the gonads at this stage of reproductive development. There was no direct evidence of progesterone (P(4)) synthesis from [3H]P(5), although the metabolites synthesized by both testis and ovary were indicative of a metabolic pathway that involved P(4) as an intermediate.

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.

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.

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.

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.

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.

Ontogenesis of estrogen secretion by porcine fetal testes.

High levels of estrogen secretion is a characteristic of steroidogenesis in the pig testis in both the adult and newborn male. We have now examined the ability of fetal gonads to secrete estrogens, and compared it with testosterone secretion during prenatal development. Fetuses were recovered from sows (N = 33) at 27-114 (term) days of gestation. Gonads were removed for organ culture in TC-199 medium, or used as minced tissues or free cell preparations when taken later in development. Organ cultures were maintained for 96 h with luteinizing hormone added for the last 72 h for one gonad of each pair. Estrone, estradiol-17 beta and testosterone were measured by radioimmunoassay in media samples. Trace amounts of estrone were detected almost as early as testosterone secretion commenced, but quantities sufficient for confirmation by radioimmunoassay after chromatography were not seen until day 35 of gestation. Estrogen production increased to > 0.37 nmol.gonad-1.4 h-1 at term. Testosterone secretion in organ culture was increased by luteinizing hormone but no effect was seen on estrone levels for the first half of pregnancy. Thus, estrogen secretion is a feature of steroidogenesis in the porcine testes even in the early stages of fetal development.

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.

Plasma levels of several androgens and estrogens from birth to puberty in male domestic pigs.

Plasma concentrations for several androgens and estrogens were determined in male domestic pigs from birth to eight months of age. Samples (N = 6) of blood were collected from Yorkshire males at weekly intervals from birth to four weeks, and thereafter at monthly intervals to eight months. Radioimmunoassays were done without extraction from plasma for dehydroepiandrosterone sulphate, androstenedione and estrone sulphate. Other steroids were measured after solid-phase extraction, separate elution of unconjugated and conjugated fractions, and solvolysis of sulpho-conjugated steroids (testosterone, 5 alpha-androstane-3 beta, 17 beta-diol, epiandrosterone, 19-nortestosterone and estradiol-17 beta). All steroids showed a peak in plasma levels at 2-4 weeks after birth. Concentrations remained low from 2-5 months and rose markedly thereafter. Most steroids were present in much greater quantities as sulpho-conjugated compounds. Concentrations of testosterone sulphate and testosterone were similar (9.4 mumol/l) at three weeks but the sulphated form predominated after six months of age. This study shows that during postnatal development the testes of the domestic pig are remarkably active in steroidogenesis with a peak at 2-4 weeks after birth. Also, the range of steroid products seen at this stage is comparable to that shown by the mature boar.

Measurements of faecal oestradiol and progesterone in non-pregnant and pregnant domestic and exotic cats.

Faecal samples collected for variable periods from 12 animals and five species of cats were assayed for progesterone and oestradiol content by application of standard radioimmunoassays to aliquots (50 microliters) of methanol extracts (4 ml) of a mixture of 0.5 g sample, 0.5 ml water and 1 g aluminium oxide, following partitioning of the total extract with petroleum ether (3 ml), further dilution of assay aliquots and drying. Recoveries averaged 100 and 72% for oestradiol and progesterone, respectively. Results included increases in progesterone during luteal phases or pregnancies to 7688 ng g-1 (tiger, Panthera tigris), 2594 ng g-1 (lion, P. leo), 3000 ng g-1 (cheetah, Acinonyx jubatus) and 4915 ng g-1 (caracal, Felis caracal). Faecal oestradiol peaks near oestrus included 246 ng g-1 (tiger), 175 ng g-1 (lion) 190 ng g-1 (cheetah), 23 ng g-1 (caracal) and 190 ng g-1 (domestic cat, F. catus).