A double-antibody radioimmunoassay for serum progesterone using progesterone-3-(O-carboxymethyl) oximino-[125I]-iodo-histamine as radioligand.

A reliable, convenient and economical radioimmunoassay (RIA) for serum progesterone has been established and tested. This procedure employs diethyl ether extraction followed by RIA utilizing rabbit anti-11 alpha-hydroxyprogesterone 11-hemisuccinyl-bovine serum albumin (progesterone-11 alpha-BSA) serum, progresterone-3-(O-carboxymethyl) oximino-[125I]-iodohistamine (progesterone-3-[125I]) as radioligand and goat anti-rabbit gamma globulin as second antibody. In conjunction with antiprogesterone-11 alpha-BSA serum, the overall assay specificity of the progesterone-3-[125I] RIA is similar to that of the [3H]-progesterone method using dextran-coated charcoal. The results of serum progesterone measurements during the menstrual cycle obtained by the progesterone-3-[125I] RIA appear comparable to those of [3H]-progesterone assays which employ similar anti-progesterone-11 alpha-BSA sera. The progesterone-3-[125I] double-antibody RIA, however, is more convenient and less expensive than the [3H]-progesterone RIA method.     

Validation of a specific radioimmunoassay to measure plasma cortisol in the fetal sheep.

A procedure utilizing co-chromatography and complementary antiserum comparisons was employed to assess the specificity of a cortisol radioimmunoassay for use in the chronically catheterized fetal sheep preparation. Complementary antiserum comparisons is a technique by which two different cortisol antisera, prepared from conjugates attached at opposite ends of the cortisol molecule, were used to determine cortisol concentrations in the same ovine fetal plasma specimens. Results were not significantly different between the two groups, each measured by a different antiserum. This procedure may be used to assess assay specificity in any species in which steroid radioimmunoassays are being adapted.     

Changes during the menstrual cycle in cytosolic and nuclear concentrations of progestagen receptor in the human Fallopian tube

[3H]R5020 was bound to cytosolic and nuclear samples of human Fallopian tube with high affinity and specificity. The cytoplasmic and nuclear concentrations of progestagen receptor varied, throughout the menstrual cycle, in the ampulla, isthmus and fimbria. Concentrations were higher at the late proliferative stage of the cycle than at the early proliferative and late secretory stages. A positive linear regression was observed between cytosolic and nuclear progestagen receptor concentrations and plasma oestradiol levels. A negative linear relationship was observed between cytosolic progestagen receptor concentration and plasma progesterone levels during the secretory stages of the menstrual cycle.     

Development of a sensitive enzymeimmunoassay (EIA) for FSH determination in bovine plasma.

A highly sensitive enzymeimmunoassay (EIA) procedure for FSH determination in bovine plasma on microtiterplates using the biotin-streptavidin amplification system and the second antibody coating was developed. Biotin was coupled to FSH and used to bridge between streptavidin-peroxidase and the immobilized antiserum in the competitive assay. The EIA was carried out directly in 50 microl of bovine plasma and compared with an established radioimmunoassay (RIA) employing 100 microl plasma. Same FSH standards and FSH specific antiserum were used in both procedures. FSH standards prepared in hormone free plasma were used. The sensitivity of the EIA procedure was 6.25 pg/well FSH which corresponded to 125 pg/ml plasma; the 50% relative binding sensitivity was seen at 200 pg/well. In comparison to RIA, the EIA was at least four times more sensitive besides requiring 6 times less FSH specific antiserum. Plasma volumes for the EIA ranging from 12.5 to 50 microl did not influence the shape of the standard curve even though a slight drop in the OD450 was seen with higher plasma volumes. When both EIA and RIA methods were used to measure FSH in cows, the levels were detectable only by the EIA procedure. The assay detects high and low plasma FSH levels within the physiological variation as well as changes in plasma FSH after stimulation with a GnRH analog. In conclusion, in addition to being non-radioactive and low cost in nature, the method offers several advantages over the conventional FSH RIA procedure; these are (a) higher sensitivity, (b) less labour and time saving, (c) more economical use of precious FSH antiserum and (d) long shelf-life of the biotinyl-FSH label (in contrast to the short half life of iodinated FSH in RIA).     

Determination of progesterone and some of its neuroactive ring A-reduced metabolites in human serum

A method for the separation and assay of some ring A-reduced metabolites of progesterone (pregnanediones and pregnanolones) is described. Serum was extracted with an organic solvent, and the extract chromatographed using high performance liquid chromatography (HPLC). A total of 50 fractions was collected for each sample and split using a stream splitter so that 30% was collected in counting vials for recovery while 70% was collected in test tubes which were assayed by radioimmunoassay. An antiserum raised in our laboratory to progesterone-3-CMO-BSA cross-reacted with five of these compounds (5alpha- and 5beta-dihydroprogesterone, 3alpha- and 3beta-5alpha-tetrahydroprogesterone, and 3beta, 5beta-tetrahydroprogesterone). Since pregnenolone eluted with 5alpha, 3beta-tetrahydroprogesterone, pregnenolone was assayed separately and its effect subtracted. Using this method it was shown that picogram to nanogram/ml amounts of these metabolites are present in all human sera. Levels in men were comparable to those of women in the follicular phase of the menstrual cycle. 5alpha-Dihydroprogesterone and 3alpha,5alpha-tetrahydroprogesterone rose substantially in the luteal phase of the menstrual cycle and all rose considerably during pregnancy.     

Circadian rhythms of the sex steroids of female Papio hamadryas in long-term hypokinesia

Radioimmunoassay was used to study the effect of restricted motor activity on circadian rhythms of blood plasma sexual steroids and cortisol in female Papio hamadryas during different phases of the menstrual cycle. Instact animals manifested clear-cut circadian rhythms of the testosterone and cortisol content during both the phases and those of estradiol and progesterone during the follicular phase of the cycle. Two-week immobilization did not produce any changes in the general pattern of circadian rhythms of cortisol and testosterone but led to a decrease in their mean daily concentrations, with that decrease being more pronounced for testosterone of females immobilized during the luteal phase. The authors established unbalance of sexual steroids with a dramatic fall of the mean daily concentrations of estradiol and progesterone and desynchronism of their circadian rhythms regardless of the cycle phase at the moment of immobilization. The mechanisms responsible for alterations in circadian rhythms of sexual steroids in hypokinetic females are discussed.     

Temporal and endocrine sequelae of aspirating follicular contents in rhesus monkeys

Aspiration of ovarian follicular contents in humans is a well-established procedure used to obtain oocytes for fertilization in vitro (IVF). However, the effects of aspiration on the menstrual cycle and resulting luteal function have been incompletely characterized. The present study was designed to investigate alterations in the temporal and endocrine characteristics of menstrual cycles following aspiration of contents of the dominant preovulatory follicle (DF) on day 10 of the cycle in normal rhesus monkeys. When aspiration was performed prior to the preovulatory surge of luteinizing hormone (LH), cycle length was extended to 38.6 ± 8.6 [15] (x days ± SD, [n]), as compared to 29.5 ± 5.7 [8] days when the surge occurred before the time of aspiration. Mean and maximal amounts of progesterone (P) in the luteal phase and the number of days in which P-values were > 1 ng/ml were significantly greater when aspiration was performed prior to the surge of LH than for aspiration after this event. Laparoscopic observations made in the midluteal phase in animals of the former group demonstrated that the corpus luteum (CL was derived from a follicle other than the original DF which had been aspirated on day 10 of the menstrual cycle; observations in the latter group of animals indicated that the CL was derived from the DF.     

Radioimmunoassay of ovine and bovine serum progesterone without extraction and chromatography

A procedure for the radioimmunoassay of ovine and bovine serum progesterone is described which does not require extraction and chromatography. Serum samples are assayed directly, and a highly specific antiserum that was prepared in rabbits against 11 alpha-hydroxyprogesterone conjugated to bovine serum albumin is used. Interference from serum binding proteins is alleviated by use of a phosphate buffer containing 5% BSA and separation of bound and free tritiated progesterone by a double antibody procedure. Serum samples are assayed in a mini-vial, the bound fraction (double antibody precipitate) is mixed with scintillation solution and the radioactivity is counted in the same vial. The assay procedure is sensitive (10 pg, 100 pg/ml) and has acceptable accuracy and precision. Because there is no extraction or chromatography, serum progesterone is not lost. Most important, the procedure is specific for progesterone and measures serum progesterone concentrations in the ewe and cow which are comparable with those obtained with conventional assay techniques. The progesterone assay described herein provides a rapid, economical procedure that can facilitate the study of ovarian cyclicity and aid in the early diagnosis of pregnancy.     

Circadian rhythms of melatonin release from chicken pineal in vitro: modified melatonin radioimmunoassay

An improved and simplified radioimmunoassay for measuring pineal, serum, and in vitro cultured medium melatonin is described. Using 2-[125I]iodomelatonin as radiolabeled ligand and a polyclonal rabbit antimelatonin antiserum, melatonin concentrations were determined in all three types of samples by a 2-day direct equilibrium double-antibody assay method without prior extraction. Serial dilutions of pineal homogenates, serum, and cultured medium all gave parallel displacement curves. Cross-reactivity of the antisera with other indoles was negligible. Intraassay coefficients of variation (n = 3) were 5.09, 3.32, and 5.05% at 7.81, 62.5, and 500 pg/tube, respectively, and the interassay coefficients of variation (n = 20) were 12.18% at 62.5 pg/tube. A characteristic diurnal rhythm of melatonin was observed using this direct assay for measuring daytime and nighttime chicken pineal and serum samples. An in vitro incubation of chicken pineal glands with a lighting cycle of 12-hr light:12-hr dark showed that the diurnal rhythm of melatonin secretion into the cultured medium was maintained. The direct assay method described in this report for measuring chicken melatonin using 2-[125I]iodomelatonin as radiolabeled ligand coupled with the in vitro cultured chicken pineal gland clearly offers great potential for studying the chicken pineal circadian oscillator and its underlying mechanism.     

Plasma osmolality and the strong ion difference predict respiratory adaptations in pregnant and nonpregnant women

This study tested the hypothesis that plasma osmolality and the strong ion difference ([SID]) predict PaCO2 during rest and during exercise in physically active pregnant (n = 22; gestational age 37.0 +/- 0.2 weeks) and nonpregnant (n = 17) women. Nonpregnant subjects were in varying stages of the menstrual cycle. Arterialized blood gases, hydrogen ion concentration, plasma osmolality, [SID], and circulating levels of progesterone were measured at rest and during upright cycling at work rates corresponding to 70 and 110% of the ventilatory threshold. Pooled data from the two groups at rest revealed significant correlations (P < 0.05) between PaCO2 with plasma osmolality, [SID], and progesterone. Progesterone was also significantly correlated with [SID] and osmolality. Also, changes in PaCO2 with exercise correlated significantly with changes in [SID]. The results support the hypothesis that plasma osmolality and [SID] are important factors in the modulation of respiratory sensitivity in healthy women. Also, the effects of progesterone on PaCO2 may be expressed, at least in part, through progesterone-induced changes in [SID] and osmolality.     

Effect of the variations of female sex hormones during the menstrual cycle upon serum somatomedin and growth-promoting activity

Serum growth-promoting activity measured upon lymphocytes, sulfation activity and radioimmunoassayable somatomedin C (Sm-C) levels were measured in sera from women during the menstrual cycle. The data showed that: estradiol, progesterone, LH or FSH added in vitro do not increase the 3H-thymidine uptake into lymphocytes; the serum thymidine activity decreases during the luteal stage of the cycle, and is negatively correlated with the progesterone levels; the sulfation factor and Sm-C levels do not have significant variations during the menstrual cycle, and the GH maximum values are attained during the luteal stage.     

Determination of plasma progesterone using thin layer chromatography during pregnancy and normal menstrual cycle]

Plasma progesterone levels were measured during pregnancy and the normal human menstrual cycle by thin-layer densitometry of a specific fluorescence reaction for delta4-3-ketosteroids. 94 determinations were carried out from the 7th week of pregnancy until term. 84 plasma progesterone concentrations were estimated between day 16 and day 26 and other 10 during the preovulatory phase of the normal menstrual cycle.     

Changes in electrical impedance of the vaginal medium during the menstrual cycle of female rhesus monkeys (Macaca mulatta)

Changes in electrical impedance of the vaginal medium during the menstrual cycle were recorded in female Rhesus monkeys using electrical probe and were correlated with estradiol-17 beta and progesterone plasma concentrations. A gradual decrease in impedance was observed during the follicular phase, the lowest values being observed between days 12-17 of the cycle. Impedance increased again during the first third of the luteal phase until day 21. The reversal of the impedance gradient's sign was nearly concomitant with the appearance of a detectable plasma progesterone concentration. These results support the use of vaginal impedance measurements as a help for the diagnosis of the periovulatory time in the female Rhesus monkey.     

Reproductive and hormonal patterns in the African green monkey (Cercopithecus aethiops).

The results of breeding Cercopithecus aethiops under time-mated laboratory conditions and analysis of total estrogen, progesterone, and LH concentrations in plasma during the menstrual cycle and plasma estrogen and progesterone concentrations during pregnancy indicate that this species is a suitable alternative for the rhesus monkey as a model for investigations of reproductive function in man.     

Estrogen-induced luteolysis in the rhesus monkey: Reversal with indomethacin

Normally cycling Rhesus monkeys were treated with diethylstilbestrol (25 mg/day) alone or in combination with indomethacin (25 mg/day) for five consecutive days beginning in the early luteal and mid-luteal phase of the menstrual cycle. Blood specimens were obtained daily to monitor corpus luteum function (progesterone), and the length of each menstrual cycle was recorded. Diethylstilbestrol alone cause premature luteolysis as indicated by decreasing plasma progesterone and shortened menstrual cycle, and indomethacin effectively blocked the luteolytic action of diethylstilbestrol. These results suggest that the probable mechanism of diethylstilbestrol action in causing luteolysis is mediated via the prostaglandins.     

Raised salivary testosterone in women is associated with increased attraction to masculine faces

Women's preferences for masculinity in men's faces, voices and behavioral displays change during the menstrual cycle and are strongest around ovulation. While previous findings suggest that change in progesterone level is an important hormonal mechanism for such variation, it is likely that changes in the levels of other hormones will also contribute to cyclic variation in masculinity preferences. Here we compared women's preferences for masculine faces at two points in the menstrual cycle where women differed in salivary testosterone, but not in salivary progesterone or estrogen. Preferences for masculinity were strongest when women's testosterone levels were relatively high. Our findings complement those from previous studies that show systematic variation in masculinity preferences during the menstrual cycle and suggest that change in testosterone level may play an important role in cyclic shifts in women's preferences for masculine traits.     

A new assay and solubilization procedure for steroid 17,20-lyase from rat testes

We report an assay for testicular 17, 20-lyase which depends on the use of [21?¹⁴C]progesterone as a substrate. The method is made possible by a simplified procedure for the synthesis of [21?¹⁴C]progesterone. A chromatographic separation of the unreacted substrate and the 2-carbon by-product on mini silica gel colums permitted a quantitative assay of the lyase activity.The lyase complex from rat testes has been solubilized by treatment with Triton CF-54 detergent. The solubilized enzyme complex catalyzes the formation of androstenedione (4-androstene-3,17-dione) from progesterone without equilibrium with added 17-hydroxyprogesterone and the solubilized enzyme complex responds to the presence of cytosol activator. Both of these characteristics are similar to the properties of the intact microsomes. Thus, solubilization with this detergent preserves the special properties of the microsome bound enzyme complex.     

Phase of menstrual cycle affects lysine requirement in healthy women

The aim of this study was to investigate whether the phases of the menstrual cycle affect lysine requirement in healthy adult females, as determined by the indicator amino acid oxidation (IAAO) method. Five healthy females with regular menstrual cycles were studied at seven graded levels of lysine intake, in random order, with an oral [13C]phenylalanine tracer protocol in both the follicular and luteal phases. A total of 14 studies were conducted for each subject. Breath and plasma samples were collected according to the standard IAAO protocol. Serum 17beta-estradiol and progesterone concentrations were measured on each IAAO study day. The rate of release of 13CO2 from [13C]phenylalanine oxidation (F13CO2) was measured, and a two-phase linear regression crossover model was applied to determine lysine requirement. F13CO2 was higher during the luteal phase (P < 0.001) and was positively associated with serum concentrations of 17beta-estradiol and progesterone. The F13CO2 data were adjusted for subjects and sex hormones and used to define breakpoints for lysine requirements. The lysine requirement of healthy females in the luteal phase was 37.7 mg.kg(-1).day(-1) and higher (P = 0.025) than that of females in the follicular phase (35.0 mg.kg(-1).day(-1)). At all lysine intake levels, plasma amino acids were lower and phenylalanine oxidation was higher in the luteal relative to the follicular phase. Therefore, we reason that the higher lysine requirement observed in the luteal phase is probably due to higher amino acid catabolism.     

A competitive radioligand assay for danazol (17 alpha-pregn-4-en-20-yno(2,3-d)isoxazol-17-ol) using pregnant guinea pig plasma

A method is described for the radioligand assay of circulating levels of a novel, pituitary gonadotrophin inhibiting agent, danazol (17α-Pregn-4-en-20-yno[2,3-d]isoxazol-17-ol). The assay described is based upon the ability of danazol to bind avidly to pregnant guinea pig plasma proteins, a binding system hitherto considered highly specific for progesterone. Danazol is separated from progesterone and other possible interfering substances by paper chromatography. The method was used to measure danazol in a series of normal human subjects given the drug at 800 mg/day.     

Peripheral inhibin levels during estrous cycle in buffalo (Bubalus bubalis)

A sensitive, specific RIA was validated and used for measurement of peripheral plasma immunoreactive inhibin (irinhibin) levels during the estrous cycle in Murrah buffalo. The RIA employed an 125-I iodinated inhibin as tracer and an antiserum against dimeric inhibin. The procedure had a sensitivity of 16 pg/tube, and the nonspecific effects of buffalo plasma were compensated for by including 200 ul bullock plasma in the standards. Separation of free and bound inhibin was affected by the use of a second antibody and precipitation with polyethylene glycol. Blood samples were collected once daily for 30 d from Murrah buffalo (n = 6) during the hot month of July. Cyclic activity and estrus were confirmed by plasma progesterone determination. Peripheral plasma concentrations of ir-inhibin fluctuated between 0.40 +/- 0.07 and 0.67 +/- 0.13 ng/ml during the estrous cycle in buffalo. During the same period, plasma progesterone levels increased from 0.21 +/- 0.01 ng/ml at Day 0 to a peak of 3.30 +/- 0.72 ng/ml on Day 13, declining sharply by Day -5. Ir-inhibin levels exhibited an increase during the follicular phase, with the maximum concentration of 0.65 +/- 0.01 ng/ml occuring on the day of estrus, a decline thereafter, and no pattern during the luteal phase. The differences, however, were not statistically significant throughout the estrous cycle.