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SUMMARY
The concentration of cortisol in peripheral plasma (μg./100 ml.) was 0·6 ± 0·1 (s.e.) in penned sheep accustomed to handling and 1·1 ± 0·2 in untrained grazing sheep, or about 1/10 of the basal level in man or the guinea-pig. Plasma cortisol in the goat (mean 1·2) and the ox (2·6 ± 0·3) was also low.
Freely diffusible cortisol was distinguished from protein-bound hormone by gel filtration after equilibration of the plasma with 3H- or 14C-labelled steroid. The binding capacity (μg./100 ml.) of sheep plasma for cortisol at room temp. (1·7 ± 0·2) was only slightly above that of 5% ovine albumin solutions. It compared with 3·5 (2·2–4·6) in the goat, 6·1 ± 1·0 in the ox, 14–21 in the guinea-pig, 17–23 in normal human and 25–59 in human pregnancy plasma. These are minimal estimates, since partial dissociation of the transcortin-cortisol complex occurred under the test conditions. Sheep plasma showed no significant elevation in cortisol concentration or binding power during pregnancy, nor in response to oestrogen administration.
At physiological cortisol levels and room temperature, 39 ± 5% of the cortisol in sheep plasma was protein bound, but values above 40% were associated with low plasma cortisol levels and hence confined to samples from trained or adrenalectomized sheep. ACTH administration or addition of very small amounts of cortisol to ovine plasma in vitro caused a sharp fall in this percentage. Moreover, at body temperature only 9 ± 2% of the plasma cortisol in the sheep was non-diffusible. This finding may account for the great sensitivity of target tissues to the changes of blood cortisol level in this species. In human pregnancy plasma 86–95% of plasma cortisol (> 14μg./100 ml.) was bound at room temperature and 60–74% at 37°.
Published data and those now presented suggest that plasma level and biological half-life of cortisol in different animal species are directly related to binding capacity.
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SUMMARY
Testosterone and androstenedione were determined chemically in testes of bulls ranging in age from 28 days to 17½ years. The earliest age at which it was possible to detect both steroids was 39 days. Their combined contents in the testes varied from 4·7 to 61 μg at 39–90 days, from 31 to 475 μg at 3½–5½ months, and from 155 to 3450 μg at 10½ months-17½ years of age.
The ratio androstenedione/testosterone decreased with age. In immature bull calves, less than 4 months old, it exceeded 1:1, in animals above 9 months of age it was less than 1:10.
The appearance of chemically demonstrable amounts of testosterone and androstenedione in the testes of young bull calves coincided with the onset of an active process of fructose and citric-acid production by the seminal vesicles. Between the 2nd and 6th month of life, the weight, fructose level and citric-acid level of the seminal vesicles increased at an exponential rate, and this sharp increase in the activity of the seminal vesicles was accompanied by a similarly sharp increase in the testosterone content of the testes. In adult bulls, differing widely in age and breed, the testicular content of testosterone was significantly correlated with the weight, fructose content and citric-acid content of the seminal vesicles.
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SUMMARY
Dispersed human endometrial and myometrial cells grown for up to 4 months as monolayer cultures responded to addition of oestradiol-17β (1 × 10−9 mol/l) to the medium by increased [3H]thymidine incorporation into DNA, and an increased rate of cell division. Cultures derived from uterine leiomyomata showed a less consistent mitotic response to the hormone.
Normal endometrial and myometrial cells grown in the oestradiolenriched medium showed a significantly higher efficacy of colony formation (increases of about 60 and 90%, respectively) than cells grown in control medium throughout the experimental period. Exposure to the hormone for 3–4 days was sufficient to induce this effect, which suggests that it does not depend on selection of hormone-sensitive cells. The mitogenic effect of oestradiol, as expressed by increased cloning efficacy, persisted for several days after transfer of the cells to a hormone-free medium. Cultures of foetal rat skeletal muscle failed to respond to oestradiol-17β in the cloning test, and oestradiol-17α was without effect on uterine cells.
It is concluded that oestradiol-17β exerts a direct mitogenic action on some cellular components of the human endometrium and myometrium in vitro.
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Changes in the peripheral plasma level of oestradiol during the bovine oestrous cycle were determined by a competitive protein-binding assay, following chromatographic purification of the steroid. Oestradiol concentration increased during the 3 days preceding oestrus and attained its peak value [17 ± 1·9 (s.e.m.) ng/100 ml] 4 h before oestrous behaviour could be detected. The level declined steeply during the day of oestrus to reach a nadir (0·8 ± 0·11) before the time of ovulation. A minor rise was observed on day 4 and a more sustained increase on days 10–13, with a peak on day 11 (8·1 ± 3·6). Comparison with published data on luteinizing hormone (LH) levels suggests that the pre-oestrous surge of oestradiol precedes the pre-ovulatory surge of LH in the cow.
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The concentrations of testosterone, progesterone and 20α-hydroxypregn-4-en-3-one (20α-OHP) were measured in the ovaries of immature rats in which ovulation was induced by treatment with pregnant mare serum gonadotrophin (PMSG) and, 48 h later, with human chorionic gonadotrophin (HCG). The concentration of testosterone in the tissue increased significantly 48 h after treatment with PMSG, reached a peak 4 h after the administration of HCG and declined to the basal level 4 h later. Increases in the levels of progesterone and 20α-OHP were observed 4 h after the administration of HCG. Whereas the level of 20α-OHP continued to rise during the subsequent 30 h, progesterone levels declined near the presumed time of ovulation (12 h after administration of HCG). It is concluded that 20α-hydroxysteroid dehydrogenase activity is present in the immature rat ovary before ovulation and that an increase in the production of testosterone in the ovaries of rats treated with PMSG and HCG precedes increased production of progesterone and 20α-OHP in these ovaries.
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A sensitive and specific method is described for the determination of oestradiol-17β and oestrone in blood by gas-liquid chromatography with the use of an electron capture detector. The rate of secretion of these steroids into the ovarian venous blood of rats was determined on the day of pro-oestrus (six animals) and at various times during the first 8 days of pregnancy (240 animals). Oestradiol output (pg/ovary/30 min, mean ± s.e.m.) was low during day 1 (the day sperm were present in the morning vaginal smear) and until 11.00 h on day 2 (59 ± 13), but rose rapidly and significantly on the afternoon of day 2 (299 ± 46). From day 3 to day 8 an increased secretion rate was maintained (451 ± 26), though this was well below the level found on the day of pro-oestrus (mean 8·8 ng oestradiol and 1·2 ng oestrone/ovary/30 min). Oestradiol secretion during early pregnancy tended to be higher in the afternoon than in the morning.
Oestrone output roughly paralleled that of oestradiol, but was only about one third as high. No distinct peak in the rate of secretion of either steroid was demonstrable on day 4. The results strengthen the view that ovarian oestrogen secretion has an essential role in ovum implantation in the rat, but are incompatible with the hypothesis that the time of uterine receptivity to the blastocyst and of uterine sensitivity to atraumatic deciduoma induction is determined by a discrete discharge of oestradiol or oestrone from the ovary on the afternoon of the 4th day post coitum — the so-called 'oestrogen surge' theory.
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To examine whether lysosomal enzymes play a part in the involution of corpora lutea, cathepsin D was assayed in both the lysosomal fraction ('bound' cathepsin D) and the postlysosomal supernatant fluid ('free' enzyme), by measuring the increment in absorbance at 280 nm caused by acid-soluble material released from haemoglobin. From these data the release ratio (free: bound specific activity) was calculated. In corpora lutea on days 5 and 15 of pregnancy, the values for lysosome-bound specific activity (units of E 280/h/mg protein) were 4·76 ± 0·51 and 5·00 ± 0·29 (s.e.m.), and the release ratios were 0·12 ± 0·02 and 0·11 ± 0·01 respectively. Similar values were obtained in newly formed corpora lutea of dioestrous and pro-oestrous rats, but on the day of oestrus the level of cathepsin D bound to lysosomes decreased to 2·66 ± 0·36 and the release ratio increased to 0·36± 0·03. On day 5 of prolactin-induced pseudopregnancy, the activities of cathepsin D in both cellular fractions resembled those of pregnancy. An even higher level of lysosome-bound cathepsin D (7·11 ± 0·47) with a relatively low level of free activity (release ratio 0·18 ± 0·02) was observed in lactating rats (day 7 of lactation), in the persistent corpora lutea of pregnancy. Administration of prostaglandin F2α (PGF2α) on days 4, 5 and 6 of lactation significantly decreased the level of lysosome-bound cathepsin D measured on day 7 in the persistent corpora lutea of pregnancy (3·81 ± 0·24) and increased the release ratio (0·40 ± 0·05). The intracellular distribution of acid phosphatase did not show a consistent relationship with the reproductive state. It appears that a decrease in the amount of lysosome-bound cathepsin D is associated with incipient luteolysis, that prolactin inhibits release of cathepsin D from the lysosomes and that PGF2α counteracts this action of prolactin.
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SUMMARY
An antigen containing the phyto-oestrogen genistein (5,7,4′-trihydroxyisoflavone) was prepared by coupling genistein-2-carboxylic acid to a copolymer of tyrosine, glutamic acid and lysine. Immunization of rabbits with this antigen resulted in the production of antibodies to the carrier and to the hapten. Genistein-specific antibodies were isolated by means of an immunoadsorbent containing genistein linked to a heterologous carrier; these reacted with genistein-containing antigens in the passive cutaneous anaphylaxis, complement fixation and 125I-labelled antigen binding tests. The antibodies failed to cross-react with oestradiol-17β coupled to ovalbumin.
The reaction of the genistein—polypeptide conjugate with the purified antibodies was inhibited by genistein and by the closely related oestrogenic isoflavone biochanin-A. No such inhibition was produced by oestradiol-17β, nor were the anti-genistein antibodies capable of binding 14C-labelled oestradiol.
The potential use of immunization with isoflavone conjugates for the protection of livestock against phyto-oestrogens is discussed.
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The aim of this study was to search for direct biochemical effects of highly purified FSH on isolated ovarian follicular theca in vitro. Granulosa cells (GC; approximately 1 × 105 cells per follicle) were flushed from isolated follicles of pro-oestrous rats. The remaining theca layer and the isolated GC were incubated with highly purified ovine FSH. Prostaglandin E (PGE) accumulation was measured by radioimmunoassay. Follicle-stimulating hormone induced a 15-fold increase in PGE accumulation over the basal level in the follicular theca, the stimulated rate exceeding threefold that observed in the GC fraction derived from the same follicle. Follicle-stimulating hormone caused no significant increase in cyclic AMP level or steroidogenesis in the theca layer, but was active on these parameters in the GC. In contrast, LH increased the accumulation of cyclic AMP, progesterone and testosterone, as well as of PGE, in follicular theca. Exogenous 8-bromo cyclic AMP or cyclic GMP also stimulated PGE production in follicular theca or GC, but FSH was without any effect on the level of endogenous cyclic GMP in GC or follicular theca. Antibodies to FSH prevented the effect of FSH (but not that of LH) on PGE formation by follicular theca and GC, while antibodies to the β-subunit of LH blocked the effect of LH but not of FSH. We conclude that highly purified FSH has a stimulatory effect on PGE formation by the follicular theca.
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Follicle-stimulating hormone (FSH) preparations from various species stimulate the rate of formation of cyclic AMP by ovarian tissue, but there has been doubt whether this effect is due to an inherent property of FSH or to contamination of the FSH preparations with luteinizing hormone (LH) (Marsh, Mills & Lemaire, 1972). To answer this question we have made use of an antiserum directed against the β-subunit of ovine LH. Such antisera neutralize LH, but unlike antisera raised with intact LH do not cross-react with FSH and thyroid-stimulating hormone (TSH) which have a similar α-subunit (Pierce, Liao, Howard, Shome & Cornell, 1971).
Ovine LH (NIH-LH-S18) purified on Sephadex G-100 was dissociated and separated into its α- and β-subunits as described by Reichert, Rasco, Ward, Niswender & Midgley (1969). The concentrated β-subunit fraction was emulsified with Freund's complete adjuvant and used for immunizing rabbits (twice 200 μg antigen/rabbit by multiple intradermal injections, 2
