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In a recent report (Watson, 1971) a method for the bioassay of luteinizing hormone (LH) depending on progesterone synthesis by superovulated rat ovarian tissue in vitro was described. The method, although specific and precise is, however, only useful for the determination of physiological levels of LH in small volumes of body fluids towards its lower limit of sensitivity. A method of improving the sensitivity five times by pretreatment of the experimental animals with the pituitary hormone prolactin is reported here. It has been known for some time that prolactin has the effect of inhibiting the appearance of the enzyme 20α-hydroxysteroid dehydrogenase in the rat (Wiest, Kidwell & Balogh, 1968). Thus, in incubated ovaries of prolactintreated rats, progesterone, and not its reduced form 20α-hydroxy-pregn-4-en-3-one, is the principle steroid accumulated (Armstrong, Miller & Knudsen, 1969).
The experimental procedures used were those previously described (Watson, 1971) except that in addition to priming with
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SUMMARY
Plasma levels of luteinizing hormone (LH) in normally cyclic women during the menstrual cycle and in rats during the oestrous cycle were measured by bioassay. With the human subjects, it was possible to establish a mid-cycle peak of LH and correlate it with basal body temperature, while with the rats a peak of LH secretion was noted between 15.00 and 19.00 h on the day of pro-oestrus. The levels of LH in both groups of subjects were of the same order as those measured by other assay techniques.
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SUMMARY
A bioassay for luteinizing hormone (LH) activity is described. The method depends on progesterone synthesis by pooled, sliced rat ovarian tissue in vitro, the ovaries being obtained from immature rats pretreated with pregnant mare serum gonadotrophin.
The main advantage of the method is its increased sensitivity compared with the commonly used ovarian ascorbic acid depletion assay, its high degree of precision, and the fact that it allows the use of large numbers of well randomized samples from a small group of animals.
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Prostaglandin F2α (PGF2α) secreted by the reproductive tissues of the pig in vitro was measured and it was found that the levels secreted by the corpus luteum and endometrium of early pregnant sows were significantly lower than those secreted by tissues during the late stage of the oestrous cycle. They were, however, comparable to levels secreted by tissues from the mid-stage of the oestrous cycle. Embryos also secreted significant amounts of PGF2α. Secretion of progesterone and oestradiol by the corpora lutea of both cyclic and pregnant pigs fell within accepted limits but embryos were also found to secrete significant amounts of oestradiol. The results suggest that luteal maintenance in the early pregnant pig is unlikely to be directly due to reduced synthesis of PGF2α.
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SUMMARY
A method for the continuous superfusion of porcine corpus luteum tissue is described which readily allows both the introduction of regulatory factors to the incubating tissue, and sampling of the tissue. Oestrogen (principally oestradiol) and progestin (principally progesterone) can be measured for up to 24 h in the superfusate from corpora lutea of all ages, and the secretion of both steroids is stimulated by the addition of luteinizing hormone. The pattern of response of both steroids to a pulse of gonadotrophin was similar in that a rapid transient increase in secretion occurred followed some time later by a secondary and more prolonged response. A second pulse of gonadotrophin introduced 6 h after the first also stimulated steroid secretion, indicating that during superfusion in vitro the porcine corpus luteum does not become refractory to the steroidogenic effect of gonadotrophin.
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ABSTRACT
Determination of the presence and characterization of oestrogen receptors (ERs) in subcutaneous and internal fat depots were performed and compared with ERs in the uterus using ligand binding and immunological techniques. Successful and consistent measurement of ERs in ovine adipose tissue could only be accomplished in animals depleted of endogenous sex steroids by combined ovariectomy and adrenalectomy. Scatchard, sucrose gradient and Western blot analyses all confirmed the presence of ERs in the cytosolic fractions of various adipose and uterine tissues from ovariectomized-adrenalectomized ewes. The approximate K d values of 0·1–0·4 nmol/l for oestradiol binding in cytosolic fractions of gluteal, omental and perirenal adipose tissues were similar to the expected high affinity binding of K d 0·35 nmol/l observed in uterine tissue. The binding was specific for oestrogens, as unlabelled diethylstilboestrol and oestradiol effectively competed with labelled hormone for receptor sites and progesterone, R5020, testosterone and dexamethasone all failed to compete. Mean (± s.e.m.) concentrations of ERs, expressed as fmol specific binding sites per mg protein, were much lower (P<0·05) in adipose tissues than in uterine tissue (975 ± 33). However, the content of ERs was greater (P<0·05) in subcutaneous gluteal fat (11·5 ± 0·8) than in the internal omental or perirenal fat (5 ± 0·6) depots. ERs from adipose and uterine tissues both migrated as moieties of 8S on 5–20% sucrose gradients. Western blot analysis of ERs from uterine and adipose tissues in the presence of protease inhibitors demonstrated an immunostaining band with a molecular mass of 67 kDa. In the absence of protease inhibitors, the adipose ER was degraded into two smaller bands with molecular masses of 45 kDa and 36 kDa.
In conclusion, these results support the presence of specific ERs in adipose tissues of sheep with characteristics similar to those of the ERs of the uterus. Furthermore, their presence is supportive of sex steroid-dependent effects on adipose accretion and metabolism.
Journal of Endocrinology (1993) 139, 107–115
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SUMMARY
Plasma levels of oestradiol-17β, progesterone and luteinizing hormone (LH) and pituitary levels of LH have been measured during the first 6 days of pregnancy, in normal rats and in rats receiving two doses of Tamoxifen (trans-1-(p-β dimethylamino-ethoxyphenyl)-1-2 diphenylbut-1-ene) on day 2 of pregnancy.
In normal rats oestradiol rose strongly from early on day 3 to reach a peak concentration between 22.00 h on day 3 and 08.00 h on day 4. Progesterone concentrations rose from day 2 to reach peak values on day 3–4. In animals in which implantation was delayed 20–24 h by administration of Tamoxifen (0·1 mg/kg) orally on day 2 the increased level of plasma oestrogen was also delayed by 20 h. A higher dose of Tamoxifen (0·2 mg/kg) on day 2, which prevented implantation, completely eliminated the increase in plasma oestradiol. Neither dose of Tamoxifen affected the levels of progesterone.
In both normal rats and rats treated with 0·1 mg Tamoxifen/kg, plasma LH levels declined by day 3 while pituitary levels rose steadily. There was no detectable change in either plasma or pituitary LH levels, accompanying the increase in plasma oestradiol in the normal rats. In animals receiving Tamoxifen (0·2 mg/kg), plasma LH increased to a maximum by day 4 while levels of pituitary LH decreased.
The results show that the oestrogen ' surge' of early pregnancy, occurs normally about midnight on day 3 and not late on day 4 as previously thought. It is considered that the plasma oestradiol peak in early pregnancy results from an increased release of FSH rather than an increased release of LH. Tamoxifen may owe part of its antifertility action to a capacity to inhibit the synthesis of oestradiol from progesterone.
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Abstract
In order to identify the molecular weight forms of bioactive and immunoactive inhibin in human plasma, plasma/serum was sequentially fractionated by immunoaffinity chromatography (using immobilised inhibin α subunit antiserum), reversed phase HPLC and preparative SDS-PAGE. The electroeluted gel fractions were assayed for inhibin in vitro bioactivity and immunoactivity, the latter by RIA. Initial experiments examined human follicular fluid as an inhibin-rich source. Bioactive and immunoactive fractions of 30, 35, 53, 65 and ∼120 kDa were identified in addition to bio-inactive, immunoactive fractions of 26 kDa and 32 kDa. These molecular weights correspond to those of known inhibin forms and are attributed to differing degrees of glycosylation of the inhibin α subunit and variable processing of the α and β inhibin subunits.
Fractionation of male plasma pools revealed the presence of higher molecular weight immunoactive forms (55–120 kDa) as well as 28–31 kDa forms although the molecular weight distribution of activity between pools varied. To assess if the molecular weight pattern was modified by storage and/or subsequent fractionation, protease inhibitors were added initially to plasma and fractionated as above. The molecular weight distribution of immunoactivity was largely unaffected by the treatment, indicating that minimal processing had occurred. Postmenopausal serum itself showed low to undetectable activity. The addition of recombinant human 31 kDa inhibin to postmenopausal serum resulted in a molecular weight profile of inhibin immunoactivity consistent with the presence of 31 kDa inhibin. Fractionation of a serum pool from women undergoing gonadotrophin stimulation, in which inhibin levels were elevated, showed a range of bioactive and immunoactive inhibin forms over the 30–120 kDa range. A good correspondence between activities was observed.
It is concluded that: 1. inhibin exists in plasma/serum as a range (28–120 kDa) of molecular weight forms. 2. In female serum, the majority of inhibin isoforms appear to be bioactive. 3. This fractionation procedure provides a basis for investigating the forms of inhibin in plasma and provides a means of assessing the specificity of new assay methods.
Journal of Endocrinology (1995) 144, 261–269
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ABSTRACT
The function of blood and uterine luminal neutrophils from ovariectomized mares treated with ovarian steroids was investigated 18 h after intrauterine infusion of 1 × 109 Streptococcus zooepidemicus. Random migration of blood neutrophils under agarose was reduced by treatment with progesterone compared with that of neutrophils from oestradiol-treated and control mares. In-vitro addition of progesterone to blood neutrophils from acyclic ponies also reduced migration. Uterine neutrophils did not migrate under agarose which was probably an effect of bacterial phagocytosis. Hormone treatment had little effect on phagocytosis of yeast blastospores by blood neutrophils. Phagocytosis by uterine neutrophils from oestradiol-treated and control mares was significantly better than that by blood neutrophils. In progesterone-treated mares, however, phagocytosis by uterine neutrophils was significantly lower than that in the other two treatment groups and was similar to that measured in blood neutrophils.
The results indicate a marked effect of progesterone in reducing both migration of blood neutrophils and phagocytosis by uterine neutrophils.
J. Endocr. (1987) 112, 443–448
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Despite the unquestioned galactopoiesis evoked in cows by thyroxine or dried thyroid gland [see Folley, 1940, for review] there is little unanimity about the effect of thyroidectomy on lactation [see Folley, 1938]. As regards the rat in particular there is a clear divergence of opinion on this point. Nelson & Tobin [1937] found that their rats weaned normal litters after thyroidectomy performed during pregnancy. Folley [1938], on the other hand, showed that thyroidectomy carried out early in lactation resulted in a prompt reduction in the growth of the pups, only a small proportion of which lived to be weaned and then only because the death of the majority of the young reduced the demands on the depleted milk supply. Nelson [1939] subsequently reaffirmed his original findings, though he now reported a slight retardation of the growth of litters of rats thyroidectomized during the current lactation. Results which may be considered
