G. D. Niswender

Radioimmunoassay for Rat Luteinizing Hormone with Antiovine LH Serum and Ovine LH-131I

Summary A highly sensitive and specific radioimmunoassay for rat LH has been developed utilizing antiovine LH serum and ovine LH for iodination. Estimates of the LH potency of pituitary preparations with widely varying LH to FSH and LH to TSH potencies obtained with this assay agree with estimates obtained with bioassay and with those obtained with a radioimmunoassay utilizing antirat LH serum and rat LH for iodination. With this technique it has been possible to measure the LH in serum obtained from females throughout the estrus cycle and in serum from normal and castrated males.

Influence of the site of conjugation on the specificity of antibodies to progesterone

Abstract In order to determine how the site on the molecule used for conjugation influences the specificty of the resulting antiserum, progesterone was conjugated to bovine serum albumin (BSA) through substituents on the A(C3), B(C6), C(C11), and D(C20) rings for use as a hapten to elicit antibody formation in rabbits. Specificty of the antisera was determined by testing the ability of 24 representative steroids to displace radioactive progesterone in a radioimmunoassay procedure. Progesterone-tyrosine methyl ester (TME) conjugates were radioiodinated and used as the radioactive form of the hormone and radioactivity bound to antibody was separated from free radioactivity by a double antibody procedure. Immunization with progesterone conjugated at C20 resulted in the formation of antibodies which could not distinguish between progesterone and other Δ4-3-ketosteroids with structures similar in the A, B, and C ring (namely 17-hydroxyprogesterone, 20α and 20β-hydroxy-4-pregnen-20-one, deoxycorticosterne and testosterone). Immunization with progesterone-3-BSA resulted in the formation of antisera which were fairly specific for progesterone while immunization with progesterone conjugated at the 11 or 6 positions resulted in antisera which were very specific for progesterone. It was concluded that steroid hormones should be conjugated to protein at sites on the B or C ring of the molecule for the production of specific antisera.

Regulation of Luteal Function in Domestic Ruminants: New Concepts

Publisher Summary This chapter discusses the control of luteal function in domestic ruminant animals. It focuses on data regarding the regulation of receptors for luteinizing hormone (LH) and the characterization of two distinct types of steroidogenic luteal cells. The function of the corpus luteum in ruminants is regulated by a complex interaction of the secretions from at least two other endocrine organs. The adenohypophysis secretes LH, generally accepted as the primary luteotropic hormone in these species. The chapter suggests that the number of luteal receptors for LH—but not circulating concentrations of LH—is correlated with serum levels of progesterone throughout the estrous cycle of ewes. Down-regulation of LH receptors depends upon occupancy of the receptor, based on correlation between number of receptors occupied and number lost after administration of 1 mg of LH in vivo. The major pathway for loss of human chorionic gonadotropin (hCG) or LH bound to the luteal receptor for LH is via internalization and degradation of the hormone. Over 80% of 125I-labeled hCG bound to luteal cells in culture is internalized and degraded. A lesser amount of 125I-labeled LH is internalized and degraded because more of this hormone dissociates from the receptor. A major portion of the internalized population of receptors is recycled to the plasma membrane.

Computer program sequence for analysis and summary of radioimmunoassay data

Abstract A sequence of three programs is described for efficient design and analysis of radio-immunoassays. The first program designs the assay; the second program analyzes all available data according to the organizational base provided by the first program; and the third program summarizes the results of several assays in either tabular or graphical form. The analysis program uses a logit response-log dose transformation to obtain a linear inhibition curve for all preparations assayed at multiple levels. The curves are subjected to a weithted, least squares, regression analysis. All curves are tested for linearity and parallelism with the curve chosen as standard, and a weighted mean potency estimate with error limits is computed for all preparations run at single or multiple dose levels with any degree of replication.

Levels of Luteinizing Hormone, Estradiol and Progesterone in Serum During the Estrous Cycle and Pregnancy in the Beagle Bitch

Summary Levels of luteinizing hormone (LH), estradiol-17/3 and progesterone were determined by specific radioimmunoassays in sera obtained from Beagle bitches during proestrus, estrus and diestrus. Concentrations of LH (expressed as NIH-LH-S1 equivalents) were: 2.8 ± 0.1 ng/ml in proestrus, 35.5 ± 10.0 ng/ml during early estrus and 2.2 ±0.1 ng/ml in early diestrus. Peak levels of estradiol-17/3 (68.9 ±11.0 ng/ml) were detected 24 hr prior to the LH peak, declined rapidly and reached basal levels (17.8 ± 6.3 ng/ml) by five days following the LH peak. Levels of progesterone were 1.7 ± 0.3 ng/ml during proestrus, 3.5 ± 0.3 ng/ml during early estrus and 23.3 ±2.8 ng/ml on day 5 after the LH peak. Progesterone levels remained elevated through day 28 of diestrus and pregnancy. A significant decrease (P < 0.05) in levels of progesterone occurred between day 28 of pregnancy and one day prior to whelping (3.3 ± 1.2 ng/ml), with a further decrease on the day of whelping (1.1 ± 0.2 ng/ml). Levels of estradiol-17β and LH did not change significantly (P > 0.05) during diestrus or pregnancy.

Nuclear changes in ovine luteal cells in response to PGF2α.

Percentages of normal and apoptotic parenchymal cells, fibroblasts and endothelial cells in ovine corpora lutea at 12, 24 and 36 hr following administration of a luteolytic dose of PGF2 alpha were determined and compared to percentages for identical cell types in corpora lutea removed from control ewes on days 10 (n = 5) and 12 (n = 6) postestrus. In corpora lutea obtained from control ewes greater than or equal to 95% of nuclei examined were scored normal for each of the respective cell types with no difference (P greater than .05) observed between luteal tissue obtained on days 10 and 12 postestrus. Following treatment with PGF2 alpha there were significant (P less than .05) reductions in the percentages of nuclei scored normal. Compared to controls the percentage of endothelial cell nuclei scored normal was reduced at 12 hr following PGF2 alpha-treatment; however significant reductions in percentages of parenchymal and fibroblast nuclei scored normal were not evident until 24 and 36 hr, respectively. Consistent with the concept of apoptosis, nuclear condensation and/or margination indicative of apoptosis did not occur synchronously within a given cell type: i.e., irrespective of the time point examined some cells appeared normal, whereas others had undergone nuclear condensation and/or margination. A sequence of events to explain structural and functional changes that occur during luteolysis following the interaction of PGF2 alpha with specific receptors in large steroidogenic luteal cells is discussed.

A specific, non-chromatographic radioimmunoassay for human plasma cortisol

A radioimmunoassay technique has been developed for the measurement of cortisol in a single methylene chloride extract of human plasma without chromatography. The antiserum, obtained by immunizing rabbits with cortisol-3-carboxymethyl-oxime conjugated to bovine serum albumin, had a high affinity (KA = 1.8 X 10(9) 1/mole) and capacity (2.3 X 10(-6) moles/L undiluted serum) for cortisol. The minimum detectable amount determined at the lower 95% confidence limit of the buffer control tubes was 8.3 +/- 4.7 pg/tube and a log dose - logit response standard curve was linear between 20 pg and 20 ng/tube. The antiserum was highly specific for cortisol with only corticosterone, cortisone, 11-deoxycortisol and 21-deoxycortisol showing significant cross-reaction (12.4, 6.6, 3.8 and 3.7%, respectively). The cross-reaction for the other tested naturally occurring and synthetic steroids did not exceed 1%. Regression analysis of cortisol concentration estimates obtained on 20 samples before and after Sephadex LH-20 column chromatography gave a coefficient of correlation (r) of 0.995 and a regression coefficient (b) of 1.04. Recovery of cortisol added to plasma samples was quantitative. The intra-assay error was 8.5% and the inter-assay error averaged 5.7%. The method is simple requiring a single solvent extraction of plasma, therefore permitting large numbers of samples to be handled efficiently by a single technician.

Uterine Vein Infusion of Interferon Tau (IFNT) Extends Luteal Life Span in Ewes

Interferon tau (IFNT) from the ovine conceptus has paracrine actions on the endometrium that alter release of prostaglandin F2alpha (PGF) and protect the corpus luteum (CL). Antiviral activity in uterine vein blood and expression of interferon-stimulated genes (ISGs) in CL is greater in pregnant than in nonpregnant ewes. We hypothesized that IFNT contributes to antiviral activity in uterine vein blood and has endocrine actions on the CL. Preadsorption of IFNT with antiserum against recombinant ovine (ro) IFNT revealed that antiviral activity in uterine vein blood from pregnant ewes was mediated by IFNT. Endocrine actions of IFNT were examined after infusing either roIFNT or bovine serum albumin (BSA; 200 μg/24 h; mini-osmotic pump) into the uterine vein of nonpregnant ewes from Day 10 to Day 11 postestrus. The abundance of ISG15 mRNA and protein was greater in CL (P < 0.05) from ewes receiving 24-h roIFNT infusion compared to that from ewes receiving 24-h BSA infusion. Injection of PGF at 12 h following insertion of mini-osmotic pumps resulted in a decline in serum progesterone concentrations 6 through 12 h later in BSA-infused ewes; however, in roIFNT-infused ewes, a similar decline in progesterone concentrations at 6 h was followed by recovery to control values at 12 h. Ewes then received infusions (200 μg/day) of either roIFNT or BSA for 7 days beginning on Day 10 of the estrous cycle. All BSA-infused ewes returned to estrus by Day 19, whereas 80% of roIFNT-infused ewes maintained luteal-phase concentrations of progesterone through Day 32. In conclusion, IFNT is released from the uterus into the uterine vein and acts through an endocrine mechanism to induce ISGs in the CL and delay luteolysis.

Prostaglandin Metabolism in the Ovine Corpus Luteum: Catabolism of Prostaglandin F2α (PGF2α) Coincides with Resistance of the Corpus Luteum to PGF2α

Abstract To examine possible mechanisms involved in resistance of the ovine corpus luteum to the luteolytic activity of prostaglandin (PG)F2α, the enzymatic activity of 15-hydroxyprostaglandin dehydrogenase (PGDH) and the quantity of mRNA encoding PGDH and cyclooxygenase (COX-2) were determined in ovine corpora lutea on Days 4 and 13 of the estrous cycle and Day 13 of pregnancy. The corpus luteum is resistant to the action of PGF2α on Days 4 of the estrous cycle and 13 of pregnancy while on Day 13 of the estrous cycle the corpus luteum is sensitive to the actions PGF2α. Enzymatic activity of PGDH, measured by rate of conversion of PGF2α to PGFM, was greater in corpora lutea on Day 4 of the estrous cycle (P < 0.05) and Day 13 of pregnancy (P < 0.05) than on Day 13 of the estrous cycle. Levels of mRNA encoding PGDH were also greater in corpora lutea on Day 4 of the estrous cycle (P < 0.01) and Day 13 of pregnancy (P < 0.01) than on Day 13 of the estrous cycle. Thus, during the early estrous cycle and early pregnancy, the corpus luteum has a greater capacity to catabolize PGF, which may play a role in the resistance of the corpus luteum to the actions of this hormone. Levels of mRNA encoding COX-2 were undetectable in corpora lutea collected on Day 13 of the estrous cycle but were 11 ± 4 and 44 ± 28 amol/μg poly(A)+ RNA in corpora lutea collected on Day 4 of the estrous cycle and Day 13 of pregnancy, respectively. These data suggest that there is a greater capacity to synthesize PGF2α, early in the estrous cycle and early in pregnancy than on Day 13 of the estrous cycle. In conclusion, enzymatic activity of PGDH may play an important role in the mechanism involved in luteal resistance to the luteolytic effects of PGF2α.

Steroidogenic enzyme activity after acute activation of protein kinase (PK) A and PKC in ovine small and large luteal cells.

Intracellular effector systems which utilize PKA and PKC can be pharmacologically activated by forskolin and phorbol 12-myristate 13-acetate (PMA) and appear to be important for regulation of steroidogenesis by cells of the corpus luteum. In this study the effect of pharmacologic activation of PKA (forskolin) or PKC (PMA) on the activity of adenylate cyclase, cholesterol esterase, P450 cholesterol side chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5, delta 4 isomerase (3 beta HSD) was determined. Basal adenylate cyclase activity (as measured by intracellular and secreted cAMP) was extremely low in both large and small luteal cells. Forskolin stimulated adenylate cyclase activity in both large and small luteal cells but progesterone production was increased only in small cells. PMA inhibited progesterone production by large and forskolin-stimulated small cells without altering adenylate cyclase activity. Basal cholesterol esterase activity was greater in small than in large cells and was stimulated by forskolin only in small cells. PMA did not significantly alter cholesterol esterase activity in either cell type. Activity of P450scc or 3 beta HSD was measured by conversion of hydroxylated cholesterol derivatives (P450scc) or pregnenolone (3 beta HSD) to progesterone. Although basal progesterone production was 47 times greater in large than small cells, there was only 5.1 (P450scc) and 6.4 (3 beta HSD) times greater enzyme activity in large than in small luteal cells. Activation of PKA and/or PKC did not alter the activity of P450scc or 3 beta HSD in either cell type.(ABSTRACT TRUNCATED AT 250 WORDS)