Radioimmunoassay for Ovine Luteinizing Hormone. Secretion of Luteinizing Hormone During Estrus and Following Estrogen Administration in the Sheep

Abstract

A solid-phase radioimmunoassay for ovine luteinizing hormone (LH) has been developed, utilizing antibody-coated polystyrene tubes for incubation of the assay and counting of the bound tracer. Tubes were coated with equine antiserum to bovine LH (Snook), and purified ovine LH (Papkoff) was used for iodination with 126 I and assay standards. The procedure is simple and rapid, being completely performed in the assay tubes and giving results after a period of 48 hr. Basal levels of plasma LH were 2.9 ±0.9 ng/ml in the cycling ewe, 1.2 ±0.9 ng/ml in the ram, 12.6 ±5.5 ng/ml in the oophorectomized ewe, and 1.2 ±0.9 ng/ml in the pregnant ewe. These values obtained from jugular venous plasma are 25% higher than those measured in peripheral venous plasma. The sheep does not appear to produce a placental gonadotrophin comparable to human chorionic gonadotrophin in regard to immunological crossreaction with pituitary LH. Plasma levels of LH rose sharply 4-16 hr after the onset of estrus to levels of 80-200 ng/ml, over a total period of only 10 hr. Frequent blood sampling is necessary to delineate the estrous LH release in the sheep, and daily estimations carry a greater than 50% chance of completely missing the LH peak. Administration of estradiol-17/3 to anestrous sheep by intramuscular injection and intravenous infusion was regularly followed by a typical estrous peak of LH secretion. The latent period of the estrogen stimulus was approximately 9 hr, and the dose required to produce an ovulatory LH peak was 6-10 ng, an amount similar to that secreted by the ovary at estrus. It is likely that estrogen secretion by the ovary is a major factor in stimulating the LH release accompanying estrus in the sheep (Endocrinology 85: 133, 1969) T HE STUDY of pituitary gonadotrophin secretion in the sheep was previously dependent upon indirect observations and the use of bioassay techniques. The timing of luteinizing hormone (LH) release has been examined by estimation of pituitary LH content (1) and by measurement of LH in blood obtained from the cavernous sinus (2). These studies have relied upon ovarian ascorbic acid depletion assays (3, 4) for quantitation of LH, a procedure which is adequate for the measurement of pituitary LH content but not generally regarded as satisfactory for the assay of blood LH levels. A decrease in pituitary LH content may indicate release of LH into the circulation, though this is uncertain unless the rate of hormone synthesis is also known, while cavernous sinus

Hypothalamic-Pituitary Function in Normal Ewes and Ewes which Grazed Oestrogenic Subterranean Clover for Several Years

“…All plasma samples which were collected in these experiments were assayed for LH by using the solid-phase radioimmunoassay described by Goding et al (1969), in which a purified LH preparation with a biological potency of 2 x NIH-LH-Sl (Papkoff et al 1965) was used. The antiserum used in the assay was an anti-ovine LH serum which was provided by Dr W. Hansel, Cornell University, New York State.…”

Plasma LH and FSH in Ewes That Were Either Fertile or Infertile After Long-Term Grazing of Oestrogenic Pasture

“…Plasma LH levels in experiments 1-4 were measured by a solid-phase radioimmunoassay (Goding et al 1969) and in experiment 4 by a double antibody method using the same antiserum, raised in a horse against bovine LH (Snook 1968) and used at a final dilution of 1 in 10 6 • The double antibody method used a goat anti-horse serum raised by Dr T. Stelmasiak, Department of Agriculture, Vic., as a source of second antibody (final dilution 1 in 250). Assay procedure was similar to that described by Lee et al (1976).…”

“…For example, Findlay et al (1973) have reported that when infertile ewes which had previously grazed oestrogenic clover were ovariectomized, they were less likely to show elevated plasma LH levels, i.e. positive feedback (Goding et al 1969), in response to oestradiol-17f1 (E 2 ) treatment than were normal ovariectomized ewes. This reduced responsiveness of the hypothalamo-pituitary axis to E2 could be related to changes in the number or affinity of cytoplasmic Ez receptors in the pituitary gland, or both (Tang and Adams 1978).…”

Primordial Follicle Numbers in Ovaries and Levels of LH and FSH in Pituitaries and Plasma of Lambs Selected for and against Multiple Births

“…The peripheral plasma levels and anterior pituitary content of LH and FSH were determined by the radioimmunoassays reported by Goding et al (1969) and Salarnonsen et al (1973). Pituitaries were homogenized in O· 01M phosphate buffer (pH 7·4) and diluted 3 x 10 3 times prior to assay.…”