Radioimmunoassay of cortisol in peripheral blood plasma of buffaloes peripartum

Changes in the concentration of cortisol were observed in the jugular venous plasma of pregnant buffaloes on days 30, 15, 5, 2 and 1 prepartum, at partum, at regular 6-hr intervals up to 72 hr postpartum and on days 4, 6, 10, 18, 34 and 50 postpartum. A radioimmunoassay (RIA) procedure for cortisol standardized in the laboratory was used. Mean plasma cortisol levels showed little fluctuation (P<0.05) between days 30 and 2 prepartum with the values ranging from 1.28 +/- 0.23 to 1.46 +/- 0.13ng/ml. A small (but nonsignificant) rise in the hormone level was observed one day prepartum followed by a sharp increase to a high mean value of 3.78+/-0.36 ng/ml (P<0.05) at parturition. A sharp decline (P<0.05) to a low mean value was recorded within 6 hr postpartum followed by marked fluctuations in the hormone level up to 72 hr postpartum. The hormone levels subsequently varied narrowly between 1.74+/-0.39 and 2.01+/-0.27 ng/ml up to 50 days postpartum.     

Radioimmunoassay of estradiol-17beta in bovine peripheral plasma with and without chromatography.

An assay of estradiol-17beta (E217beta) in bovine peripheral plasma is described. The plasma is incubated with an antiserum to E217beta-BSA and the gamma-globulin fraction precipitated with ammonium sulphate. After extraction with diethyl ether E217beta in the precipitate is estimated by radioimmunoassay using a specific antiserum against E217beta-6-BSA. Plasma concentrations of E217beta during the normal estrous cycle determined by this method and by a method involving Sephadex LH-20 chromatography range from 4 to 23 pg/ml.     

Radioimmunoassay of progesterone, 17-hydroxyprogesterone, estradiol-17beta and prostaglandin F in human corpus luteum.

Radioimmunoassay procedures have been adapted for the assay of progesterone, 17-hydroxyprogesterone, estradiol-17beta, and prostaglandin F in human corpus luteum. The method utilises a single homogenisation and extraction of the tissue followed by fractionation of the steroids on alumina, and separation of the prostaglandins of the F series from the E and A series on silica gel, prior to radioimmunoassay. An attempt has been made to validate the method for the progestins by comparison with results after fractionation of the progestins on Sephadex LH-20, for estradiol-17beta by comparison with values obtained with competitive protein-binding, and for prostaglandin F by comparison with values after additional purification. The results showed that peak concentrations of the three steroids in corpora lutea from women during the luteal phase of the menstrual cycle were comparable to those found in corpora lutea from women in early pregnancy. However, in six out of fourteen corpora lutea from non-pregnant women, prostaglandin F levels were higher than those found in corpora lutea from seven women in early pregnancy, i.e. 13-46 ng/g compared with 1-7 ng/g. Of the above six corpora lutea, four were on days 23-25 of the cycle, at a time when luteolysis would be commencing. The results in this paper support the conclusion that the corpus luteum is a major site of synthesis of the three steroids examined, although the site of synthesis of prostaglandin F is still equivocal.     

Contrasting effects of estradiol-17 beta and 17 alpha-ethinyl estradiol-17 beta on cultured whole embryos

Estradiol-17 beta (E2) and 17 alpha-ethinyl estradiol-17 beta (EE) were compared in terms of their relative capacities to alter growth and developmental patterns of cultured whole embryos during the early stages of organogenesis. Embryos exhibited a notable differential susceptibility to the embryotoxic effects of parents E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically significant effects whereas EE elicited marked embryotoxicity. Inclusion of a P-450-dependent biotransformation system in the culture media resulted in a significant attenuation of the embryotoxic effects of parent E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically embryotoxicity by hepatic S9. The divergent results produced by the two steroids could not be attributed to differences in rates of catecholestrogen generation in the culture medium or by the conceptuses. The results demonstrate definitive dissimilarities between the effects of two steroidal estrogens on developmental parameters and document marked differences in the effects of biotransformation on their embryotoxic potential. The data strongly suggest that the embryotoxicity of these steroids is not mediated via interactions with estrogen receptors. Additionally, the data show that the differential capacity of these two steroids to produce embryotoxic effects is diametrically opposite to earlier reported patterns of their carcinogenic potential in the Syrian hamster kidney.     

Radioimmunoassay of 17-hydroxyprogesterone

A method is described for the determination of 17-hydroxyprogesterone in peripheral venous plasma (0.1–1.0 ml) from men and women using an antiserum to 17-hydroxyprogesterone-3-carboxymethyl oxime bovine serum albumin (BSA).

The coefficients of variation on replicate analyses ranged from 7–16%. The louest level of 17-hydroxy-progesterone uhich may be determined is 5 ng/100 ml plasma. The concentration (mean ± S.D.; ng/100 ml plasma) in a group of healthy men (aged 20–40 yrs) uas 123 ± 65. From women during days 1–10 of the menstrual cycle the value uas 40 ± 15, during days 18–32 of the cycle 134 ± 57 and during pregnancy (12th week to term) 622 ± 262. Progesterone was determined in the same samples using an antiserum to 11-hydroxyprogesterone-11-hemisuccinate-BSA.     

Competitive protein-binding assay of estrone, estradiol-17 or estradiol-17 in human or ruminant plasma

A procedure for the assay of estrone, estradiol-17β or estradiol-17α in plasma is described. The technique also appears applicable to the assay of estriol in plasma. The procedure uses a semi-automatic extraction of plasma, rapid micro-alumina column chromatography and competitive binding of the estrogens to stable proteins of sheep uterine cytosol. The use of alumina column chromatography results in consistently low blanks. The assay has been evaluated for the measurement of estradiol-17β and estrone in human and sheep plasma, and for estradiol-17α and estrone in goat plasma. The change in binding affinity of estradiol-17α relative to estradiol-17β when incubated in sheep uterine cytosol at two different temperatures (25°C and 4°C), makes it possible to differentiate the two epimers of estradiol. Measurement of estradiol-17β down to 10 pg and of estrone and estradiol-17α to 25 pg are maintained in routine analyses. The specificity of the procedure was thoroughly checked by various methods, including comparison with spectrophotofluorimetric analysis.     

Uterine growth responses of the mature castrate rat to estradiol-17B

To examine estrogen-stimulated uterine growth we have monitored changes in uterine DNA synthesis, ornithine decarboxylase (ODC) activity and protein content as well as luminal epithelial (LE) cell mitotic index and ultrastructural changes. We have utilized this model to examine castrate mature rat uterine growth as a function of time between 18 and 40 hours following a single injection of 25.0 ug of estradiol-17B. LE cell mitotic index and protein content increases were maximally elevated as early as 18 hours postinjection while uterine ODC activity was maximal at 28 hours; uterine DNA synthesis increases continued throughout the experiment. In addition, the infusion of either 1 or 2 ug E2 plus progesterone over a 24 hour period, stimulated elevated ODC activity under both treatment regimens and LE cell mitotic index which was inversely related to E2 dose.     

Comparison of the methylation of estradiol-17 beta and of estradiol-17 alpha by dimethyl sulfate

E₂-17β and E₂-17α give substantially similar yields of 3-methyl ether and dimethyl ether when methylated by Brown's procedure.     

Sulfotransferase 2A1 forms estradiol-17-sulfate and celecoxib switches the dominant product from estradiol-3-sulfate to estradiol-17-sulfate

Using recombinant sulfotransferases (SULTs) expressed in E. coli, β-estradiol (E2) sulfonation was examined to determine which SULT enzyme is responsible for producing E2-17-sulfate (E2-17-S). SULTs 1A1*1, 1A1*2, 1A3, 1E1 and 2A1 all sulfated E2 to varying extents. No activity was observed with SULT1B1. Among the SULTs studied, SULT2A1 produced primarily E2-3-sulfate (E2-3-S), but also some E2-17-S and trace amounts of E2 disulfate. SULT2A1 had a K_m value of 1.52 μM for formation of E2-3-S and 2.95 μM for formation of E2-17-S. SULT2A1 had the highest V_max of 493 pmol/min/mg protein for formation of E2-3-S, which was 8.8- and 47-fold higher than the maximal rates of formation of E2-17-S and E2 disulfate, respectively. SULT2A1 formed E2-3-S more efficiently. However, when celecoxib (0–160 μM) was included in the incubation with either SULT2A1 or human liver cytosol, sulfonation switched from E2-3-S to E2-17-S in a concentration-dependent manner. The ratio of E2-17-S/E2-3-S went up to 15 with SULT2A1, and was saturated at 1 with human liver cytosol. In both cases, more E2-17-S was formed, with the unreacted E2 remained unchanged, suggesting celecoxib probably bound to a separate effector site to cause a conformational change in SULT2A1, which favored production of E2-17-S. The ability of celecoxib to alter the position of sulfonation of E2 may in part explain its success in the experimental prevention and treatment of breast cancer.     

Prevalence of aerobic and anaerobic uterine bacteria during peripartum period in normal and dystocia-affected buffaloes

Parturition complications predispose establishment of uterine infections, which in turn affect subsequent fertility. The aim of present study was to characterize and compare the type of bacterial flora prevalent within the uterine lumen of dystocia-affected buffaloes and compare them with the normally calving buffaloes. The study was conducted on 40 buffaloes; of which 10 calved normally (Group I) and 30 were treated for dystocia (Group II). Bacteriological examination was performed using uterine swabs, which were collected before delivery, immediately after delivery and day's 24-60 postpartum. A total of 30 uterine swabs from Group I and 79 swabs from Group II were collected, of which 19 (63.3%) and 71 (89.9%) yielded significant bacterial growth, respectively. A total of 205 isolates belonging to 10 different genera of bacteria were identified, 8 facultative anaerobes and 2 obligate anaerobes. In Group II, 91.6% of the bacteria positive swabs (n = 71) yielded mixed cultures, whereas the remainder being pure cultures. In contrast, 89.5% of the bacteria positive swabs of Group I (n = 19) yielded pure cultures. Mixed infections comprised mostly Arcanobacter (Actinomyces) pyogenes together with obligate anaerobes, Fusobacterium spp. and Bacteroides spp. In Group II, the frequency of incidental and coliform group bacteria was highest at the time of parturition, i.e., before and immediately after delivery, and decreased to nil during the 24-60-day postpartum period. However, in Group I, the incidental and coliform group of bacteria present at the time of parturition apparently persisted beyond the period when uterine involution is complete. The frequency of obligate anaerobes and A. pyogenes at the time of parturition was nil in the Group I while they predominated in dystocia-affected buffaloes (Group II). During the postpartum period of 24-60 days, the frequency of both obligate anaerobes and A. pyogenes increased significantly in Group II, whereas in Group I, only occasional isolates were obtained. To conclude, at the time of calving the prevalence of obligate anaerobes and A. pyogenes occurring in combination was highest in dystocia-affected buffaloes, and further increased in the postpartum period suggesting that these infections act synergistically.     

Plasma estradiol-17beta level in the domestic fowl

Using healthy white Leghorn chickens the estradiol-17β level in plasma were determined by radioimmunoassay and the dependency on age and sex examined. In one and two year old laying hens average values between 48.0 and 54.4 pg/ml were found. The estradiol level in cocks of the same age were between 6.0 and 7.3 pg/ml. Significant difference with respect to the estradiol level were already noticeable in sexually immature 4 month old chickens ♀: 8.1 pg/ml, ♂: 1.0 pg/ml). The significance of the estradiol-17β level with respect to plasma lipids and electrophoretic mobility of serum lipoprotein is shown.