Simultaneous determination of plasma estrogens, androgens, and progesterone during the human menstrual cycle

A method is described for the purification and quantitation of estradiol-17β (E₂), estrone (E₁), testosterone (T), androstenedione (A) and progesterone (P) from a single plasma sample extraction. A combination of Sephadex LH-20 column chromatography and thin layer chromatography is used to separate the phenolic and neutral steroids; quantitation of the purified steroids is by radioimmunoassay (E₂, E₁) and competitive protein binding (T,A,P) techniques. The precision and accuracy of this method is comparable with that reported by others. A preliminary investigation of the temporal relationship between estrogens androgens, progesterone and serum LH during five normal and one short luteal phase menstrual cycle is presented.     

Characterization and properties of steroid binding protein in Bufo arenarum serum

Serum steroid binding properties of mature Bufo arenarum females were studied. Binding data obtained using charcoal adsorption assay and equilibrium dialysis methods indicates a single protein, named Bufo arenarum sex binding protein (Ba SBP), which binds 5 α-dihydrotestosterone (DHT), testosterone (T), and estradiol-17β (E₂) with high affinity (10⁷ M^?1 – 10⁸ M^?1) and fair capacity (10^?6 M). Scatchard plot analysis demonstrated the coexistence of two binding sites. Ba SBP has a sedimentation coefficient of 5.2 S in sucrose gradient centrifugation in low salt and under steady-state conditions. The specificity of this protein, determined by competitive binding experiments, is comparable to human SBP. DHT and T bind with higher affinity than E₂. Estriol and estrone competed poorly, while diethylstilbestrol and C₂₁ steroids did not compete. The binding capacity of this protein is under estrogenic control. © 1994 Wiley-Liss, Inc.     

Sex-specific effects of 17β-estradiol and dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERβ and require palmitoylation to translocate the receptors to the plasma membrane

Rat costochondral cartilage growth plate chondrocytes exhibit cell sex-specific responses to 17β-estradiol (E₂), testosterone, and dihydrotestosterone (DHT). Mechanistically, E₂ and DHT stimulate proliferation and extracellular matrix synthesis in chondrocytes from female and male rats, respectively, by signaling through protein kinase C (PKC) and phospholipase C (PLC). Estrogen receptors (ERα; ERβ) and androgen receptors (ARs) are present in both male and female cells, but it is not known whether they interact to elicit sex-specific signaling. We used specific agonists and antagonists of these receptors to examine the relative contributions of ERs and ARs in membrane-mediated E₂ signaling in female chondrocytes and DHT signaling in male chondrocytes. PKC activity in female chondrocytes was stimulated by agonists of ERα and ERβ and required intact caveolae; PKC activity was inhibited by the E₂ enantiomer and by an inhibitor of ERβ. Western blots of cell lysates co-immunoprecipitated for ERα suggested the formation of a complex containing both ERα and ERß with E₂ treatment. DHT and DHT agonists activated PKC in male cells, while AR inhibition blocked the stimulatory effect of DHT on PKC. Inhibition of ERα and ERβ also blocked PKC activation by DHT. Western blots of whole-cell lysates, plasma membranes, and caveolae indicated the translocation of AR to the plasma membrane and specifically to caveolae with DHT treatment. These results suggest that E₂ and DHT promote chondrocyte differentiation via the ability of ARs and ERs to form a complex. The results also indicate that intact caveolae and palmitoylation of the membrane receptor(s) or membrane receptor complex containing ERα and ERβ is required for E₂ and DHT membrane-associated PKC activity in costochondral cartilage cells.     

The pituitary-gonadal axis before puberty: Effects of various estrogenic steroids in the ovariectomized rat

A series of experiments were conducted to determine the effects of several estrogens upon FSH and LH secretion in immature ovariectomized rats. Groups of animals were castrated at 26 days of age and treated for 5 days post-operatively with various dosages of one of the following steroids: estrad1ol-17β(E₂), estradiol benzoate (EB), estrone (E₁), equilenin (EQ), 17α-ethinyl estradiol (EE), or mestranol (ME). Uterine weights were recorded and blood taken for radioimmunoassay.Estradiol was able to suppress both FSH and LH within a “physiologic dosage range” (PDR), wherein both gonadotropins were suppressed to intact control levels by a dose which did not stimulate uterine weight higher than intact control weight. EB and ME suppressed LH but not FSH at the PDR; the other steroids suppressed at higher than PDR doses. LH was preferentially suppressed, as compared to FSH, by all 6 steroids. By biological potency the order of activity was E₂ = EE ? EB ? ME ? E₁ ? EQ. For relative ability to suppress FSH (compared to LH), the order was E₁ or E₂, ? ME ?EB ? EE ? EQ. At higher doses (near maximum uterine stimulation), e₁, E₂ and EE produced higher FSH and LH than suppressed levels seen at lower doses; a pharmacologie dosage of E₂ caused re-suppression of both gonadotropins.Results indicate that a feedback system is present before puberty and this system is sensitive to very low levels of estrogens. Likewise, there is a potential for positive feedback present for higher estrogen levels, and a complete suppression occurs at pharmacologie levels. There appears to be a significant discrepancy between the biologic activity (by uterine weight) of estrogens and their ability to affect gonadotropin     

Comparisons of affinity constants of PGEs-receptor interaction with concentrations of PGEs needed for half maximal stimulation of adenylate cyclase

The specific binding of ³H-prostaglandin E₁ (³H-PGE₁) to bovine corpus luteum cell membranes was not affected by cholesterol or various progestins at concentrations of up to 9.0 × 10⁻⁶M. At concentrations above 2.5 × 10⁻⁶M; estrone, 17β-estradiol (but not 17α-estradiol or 17β-estradiol glucuronide), estriol, equilin, D-equilenin, 17-ethynyl estradiol, diethylstilbestrol, cortisol, corticosterone, deoxycorticosterone and aldosterone inhibited specific binding of ³H-PGE₁. On the other hand, testosterone and dihydrotestosterone (DHT) (but not androstenedione) significantly enhanced ³H-PGE₁ binding. These findings permitted the following correlations between steroid structure and modulation of ³H-PGE₁ binding: steroids with a free phenolic ring and a 17β-hydroxyl or 17-keto group or C-21 steroids with a C-20 ketone and a C-21 hydroxy group decrease, whereas C-19 steroids with a C-17 hydroxy group enhance specific binding of ³H-PGE₁. PGE receptors are heterogeneous with respect to affinity for ³H-PGE₁. The steroids that decreased ³H-PGE₁ binding caused a lowering to a complete loss of low affinity PGE receptors. Steroids that increased ³H-PGE₁ binding caused appearance of new low affinity PGE receptors. Association rate constants for ³H-PGE₁ binding were decreased by 17β-estradiol (61%) and increased by DHT (59%).     

Steroid hormone modulation of 3H-prostaglandin E1 binding to bovine corpus luteum cell membranes

The specific binding of ³H-prostaglandin E₁ (³H-PGE₁) to bovine corpus luteum cell membranes was not affected by cholesterol or various progestins at concentrations of up to 9.0 × 10⁻⁶M. At concentrations above 2.5 × 10⁻⁶M; estrone, 17β-estradiol (but not 17α-estradiol or 17β-estradiol glucuronide), estriol, equilin, D-equilenin, 17-ethynyl estradiol, diethylstilbestrol, cortisol, corticosterone, deoxycorticosterone and aldosterone inhibited specific binding of ³H-PGE₁. On the other hand, testosterone and dihydrotestosterone (DHT) (but not androstenedione) significantly enhanced ³H-PGE₁ binding. These findings permitted the following correlations between steroid structure and modulation of ³H-PGE₁ binding: steroids with a free phenolic ring and a 17β-hydroxyl or 17-keto group or C-21 steroids with a C-20 ketone and a C-21 hydroxy group decrease, whereas C-19 steroids with a C-17 hydroxy group enhance specific binding of ³H-PGE₁. PGE receptors are heterogeneous with respect to affinity for ³H-PGE₁. The steroids that decreased ³H-PGE₁ binding caused a lowering to a complete loss of low affinity PGE receptors. Steroids that increased ³H-PGE₁ binding caused appearance of new low affinity PGE receptors. Association rate constants for ³H-PGE₁ binding were decreased by 17β-estradiol (61%) and increased by DHT (59%).     

Search for a substance which selectively inhibits FSH--effects of steroids and prostaglandins on serum FSH and LH levels

In an attempt to find a preferential inhibitor of serum FSH, a number of C₁₈, C₁₉ and C₂₁ steroids were evaluated for suppressive effects on LH and FSH and stimulatory effects upon ventral prostate weight in the acutely castrate adult male rat. C₂₁ steroids had no effect. The C₁₉ steroids were listed in order of potency with regard to suppressing LH, FSH and stimulating ventral prostate weight. All C₁₉ steroids that had an inhibitory effect on gonadotropins also stimulated ventral prostate weight and all demonstrated preferential inhibition of LH over FSH. Two C₁₈ steroids were tested, estradiol-17β and estrone; estradiol-17β suppressed LH and FSH in a parallel fashion. Four prostaglandins were evaluated with regard to effect on gonadotropins and ventral prostate weight; none demonstrated effect.     

A single injection of 17 beta-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E₂) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E₂, as was the combination of E₂ and DHT. The relative efficacy of a single dose of T, DHT, and E₂ plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E₂B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E₂, and E₂B suggests that an appreciable, but not necessarily sustained, elevation of E₂ levels in the brain may be critical in the facilitation of male copulatory behavior in mice.     

A single injection of 17β-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E₂) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E₂, as was the combination of E₂ and DHT. The relative efficacy of a single dose of T, DHT, and E₂ plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E₂B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E₂, and E₂B suggests that an appreciable, but not necessarily sustained, elevation of E₂ levels in the brain may be critical in the facilitation of male copulatory behavior in mice.     

New Aromatic Esters of Progesterone as Antiandrogens

The in vivo and in vitro antiandrogenic activity of four new progesterone derivatives: 4-bromo-17α-(p-fluorobenzoyloxy)-4-pregnene-3,20-dione 1,4-bromo-17α-(p-chlorobenzoyloxy)-4-pregnene-3,20-dione 2, 4-bromo-17α-(p-bromobenzoyloxy)-4-pregnene-3,20-dione 3 and 4-bromo-17α-(p-toluoyloxy)-4-pregnene-3, 20-dione 4 was determined. These compounds were evaluated as antiandrogens on gonadectomized hamster prostate and reduced the weight of the prostate glands in gonadectomized hamsters treated with testosterone 5 (T) or dihydrotestosterone 6 (DHT) in a similar manner to that of commercially available finasteride, thus indicating a potent in vivo effect. The in vitro studies showed that steroids 1–4 have a weak inhibitory activity on 5α-reductase with IC50 values of: 280 (1), 2.6 (2), 1.6 (3) and 114?μM (4). The presence of Cl and Br atoms in the C-17 benzoyloxy group tends to increase the inhibitory potency of the compounds.

The binding efficiency of the synthesized steroids 1–4 to the androgen receptor of the prostate gland is also evaluated. All compounds form a complex with the receptor and this explains the weight reduction of the seminal vesicles in the animals treated with DHT plus steroids 1–4.     

The site of estradiol sensitization of the gonadotrope is prior to calcium mobilization

In primary pituitary cell cultures prepared from ovariectomized rats, estradiol-17B (E₂) sensitizes gonadotropes to stimulation of luteinizing hormone (LH) release by gonadotropin releasing hormone (GnRH). The calcium ionophore A23187, which stimulates LH release from the cells by Ca²⁺ mobilization at a post-receptor locus, and veratridine, which stimulates LH release by activation of endogenous ion channels, were used to localize the site of E₂ action. Cells cultured in medium which was charcoal stripped (to remove steroids) or which contained 10^?8 M added E₂ responded equally well to the ionophore and equally well to veratridine, indicating that the molecular locus of E₂ action precedes Ca²⁺ mobilization. This type of analysis can be used to locate the site of action of compounds which alter the responsiveness of the pituitary to GnRH.     

Decrease of Hormone Binding Capacity of Estrogen Receptor by Calcium

Abstract

We have adressed the question as to whether calcium may modify the [³H]estradiol ([³H]E₂) binding properties of the estrogen receptor (ER). A human recombinant full length ER (yER) expressed in yeast was used to limit the potential interference of ER-associated proteins and proteases present in the target tissues.

Ca⁺⁺ (0.1–10 mM) always produced an important loss of [³H]E₂ binding capacity without any effect on the hormone binding affinity of residual receptors. This loss was reflected in a decrease of immunoreactivity for monoclonal antibodies raised against the hormone binding domain. An ER recombinant expressing solely this domain confirmed that the ion operated at this level. Binding of [¹²⁵I]Z-17 α-(2-iodovinyl)-11β-chloromethyl estradiol-17β (an compound with very high selectivity for ER) as well as [¹²⁵I]tamoxifen aziridine were similarly affected. Size-exclusion chromatography failed to reveal the emergence of any ER isoforms of low molecular weight rejecting the hypothesis of a Ca⁺⁺- induced proteolysis. In agreement with this conclusion, EDTA reversed the loss of [³H]E₂ binding capacity. Phosphoamino acids (PY, PT and PS) partly antagonized the effect of Ca⁺⁺ suggesting its interaction with phosphoamino acid residues. Worthy of note, the effect of Ca⁺⁺ appeared more marked when assessed by DCC than HAP assay. The phosphocalcic nature of the HAP matrix may explain this phenomenon which was observed with cytosolic ER from various origins.     

Molecular characterization of gonadotropin subunits and gonadotropin receptors in black porgy,Acanthopagrus schlegeli: Effects of estradiol-17β on mRNA expression profiles

The cDNAs of three gonadotropin (GTH) subunits (GTHα, FSHβ, and LHβ) and two GTH receptors (FSHR and LHR) from pituitary and gonads of black porgy were cloned. The nucleotide sequences of the GTHα, FSHβ, and LHβ cDNA were 354, 363, and 414 base pairs (bps) in length with open reading frames (ORF) encoding peptides of 117, 120, and 137 amino acids, respectively. The FSHR and LHR cDNA was 2118 and 2076 bps in length with ORFs encoding peptides of 705 and 691 amino acids, respectively. To study the mechanism of the estradiol-17β (E₂) action, we examined the expression pattern of GTH subunit mRNAs in pituitary and GTH-receptor mRNAs in gonads, and the changes of plasma E₂ level when E₂ treatment was applied to immature black porgy. E₂ treatment increased mRNA expression levels of the genes and plasma E₂ levels, indicating that E₂ stimulated the increases in GTH subunit and GTH-receptor mRNAs. These data indicate that E₂ plays an important regulatory role in the brain–pituitary–gonad axis of immature black porgy. We provide the molecular characterization and expression of the GTH subunits and GTH receptors during sex change in the protandrous black porgy.     

Perioestrous patterns of circulating LH,FSH, prolactin and oestradiol-17β in the gilt

Concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) were measured in jugular blood and those of oestradiol-17β (E₂17β) in utero-ovarian blood. Samples were taken from five intact gilts every 15 min for 108 h starting between day 15 and day 18 of the oestrous cycle. In the late luteal/early follicular phase, high pulsatile LH secretion, close to one pulse per hour, was observed. This could be the stimulus necessary for the final maturation of the ovarian follicles.Thereafter, frequency and amplitude of pulses, and the baseline value, decreased and were low at least between 36 and 12 h before the preovulatory LH surge. PRL and FSH concentrations also declined. This was probably due to the increase of oestrogen secretion. As E₂17β concentrations were still high, the surge of LH which was accompanied by increase in FSH and PRL, occurred for approximately 13 to 20 h. While LH and PRL mean levels decreased, FSH concentrations continued to increase. Peaks of PRL were observed during the late luteal/early follicular phase and during the LH discharge. During the period of estrus, each exposure to the boar was immediately followed by one of these peaks, which could play a role in the sexual behavior of the gilt.     

Metabolic and proliferative responses to estrogen by hepatocytes selected for plasma membrane binding-sites specific for estradiol-17beta.

Hepatocytes were isolated by established procedures from freshly-excised livers of ovariectomized rats. Integrity of the cells was verified by DNA, protein, and calcium contents, and by dye exclusion. The cells also showed progressive increments in oxidation to ¹⁴CO₂ of [26-¹⁴C]cholesterol during one to five hours' incubation. Analysis was undertaken of cellular reactivities toward estrogen and the hepatocarcinogen dibutylnitrosamine (DBN). Binding and retention of [³H]estradiol-17β (E₂β) by isolated liver cells was specific for E₂β, saturable, temperature-dependent, and maximal after 30-minute incubation. The apparent dissociation constant for the binding process at 22°C is 2 × 10^?9 M, and the total number of binding-sites at saturation corresponds to approximately 3,400 E₂β molecules per liver cell. To probe for steroid binding-sites at their external surfaces, cells were incubated 30 minutes with mounted 17β-estradiol-17-hemisuccinyl:albumin:nylon fibers. The covalentlyimmobilized estrogen (1 ng/mg albumin) was accessible for interaction with antiserum directed against 17β-estradiol-17-hemisuccinyl:albumin. Significant numbers of isolated liver cells were retained by estrogen-derivatized fibers at 22°C after extensive washes. Binding was markedly reduced by incubation at 4°C and by prior exposure to free E₂β (× 10^?8 M), but not to the relatively inert estradiol-17α (E₂α). Fiber-bound cells could be dislodged by brief incubation in 150 mOsM saline with 2 × 10^?7 M E₂β or diethylstilbestrol, but not E₂α, cortisol, progesterone, or testosterone, and recovered intact. Cells that had been retained by the fibers and those that were not adherent were collected and washed under identical conditions, then plated in serum-free, chemically-defined medium at 37°C. After 72 hours, specific binding of E₂β by the fiber-binding cells during 30 minutes' incubation was 2.5-fold that of cells which had not bound the immobilized steroid. Similarly, stimulation of the oxidation to ¹⁴CO₂ of [26-¹⁴C]cholesterol by E₂β was greater in fiber-binding than in non-binding liver cells after three hours' incubation. In the absence of added mitogen, thymidine incorporation into macromolecular form (20 hours), and cell proliferation (48 hours) were significantly greater in fiber-binding cells as compared to non-binding hepatocytes. Moreover, in parallel experiments, when cells were exposed to 1 × 10^?9 M estrogens or to 1 × 10^?4 M nitrosamines to assess the capacities of these substances to increase basal thymidine incorporation, total DNA, and cell numbers, only those cells with estrogen-binding sites at their surfaces showed significant E₂β- and DBN-induced increments in these parameters as compared with paired controls that had been treated with E₂α or the noncarcinogen diphenylnitrosamine. These data indicate that the accessibility of hormone-binding components at the plasma membrane may contribute to the capacity of a given liver cell to respond to E₂β, as well as to other known hepatocarcinogens.     

Bone loss in ovariectomized rats: dominant role for estrogen but apparently not for FSH

Estrogen deficiency as the sole factor underlying post‐menopausal osteoporosis was challenged, in light of reports that both follicular stimulation hormone (FSH) receptor and FSHβ knockout mice were resistant to bone loss, suggesting a detrimental role for FSH. We assessed whether lowering FSH levels by gonadotropin realizing (GnRH) analog decapeptyl in ovariectomized female rats (OVX) affects bone. Wistar‐derived 25 days old OVX female rats were injected for 10 weeks with estradiol‐17β (E₂), with GnRH analog (decapeptyl) or with both. FSH and luteinizing hormone (LH) serum levels were markedly increased in OVX rats, with smaller growth plates with disrupted architecture; heavy infiltration of bone marrow with numerous adipocytes and reduced thickness of cortical bone. In OVX rats treated with E₂, FSH, and LH levels were intermediate, the tibia was similar to that of intact rats, but there was reduced thickness of cortical bone. In decapeptyl treated OVX rats, FSH and LH levels were suppressed, the organization of growth plate and the trabecular bone were disrupted, and there were fewer proliferative and chondroblastic cells and a large adipocytes population in bone marrow, but an increased trabecular bone volume (TBV). In the E₂ + decapeptyl treatment, FSH and LH levels were suppressed, with partially restored growth plate architecture and improved TBV. In conclusion, E₂ deficiency is the dominant factor impairing bone loss in OVX and concomitant changes in FSH/LH levels achieved by decapeptyl have some modulating, though complex role in this setting. The role of high FSH levels in post‐menopausal bone loss requires further investigation using combined sub‐optimal doses of the different hormones. J. Cell. Biochem. 112: 128–137, 2011. © 2010 Wiley‐Liss, Inc.     

Dihydrotestosterone and estradiol-17β mutually neutralize their inhibitory effects on human vascular smooth muscle cell growth in vitro

We reported previously that high concentrations of either estradiol-17β (E₂) or dihydrotestosterone (DHT) inhibit growth of human cultured vascular smooth muscle cells (VSMC), mediated by cell membrane receptors and MAP-kinase–kinase activity (MEK). We now tested whether the presence of the opposite gender's dominant sex hormone modifies these effects. We incubated VSMC with various concentrations of E₂ and DHT or protein bound hormones (E₂–BSA or T–BSA), alone or in various combinations. High concentration of E₂ or E₂–BSA inhibited VSMC growth and stimulated MEK. In the presence of 3 nM DHT, high concentration of E₂ no longer inhibited ³[H] thymidine incorporation or increased MEK. Moreover, when high DHT concentration (300 nM) was added to VSMC exposed to high E₂, VSMC growth actually increased without change in MEK. DHT at 300 nM suppressed VSMC growth and increased MEK while 0.3 nM E₂ had only marginal effect on this interaction, and 30 nM E₂ reversed the inhibitory effect of DHT on cell growth. The inhibitory effects of both E₂ and DHT on VSMC cell growth and the stimulation of MEK was apparently mediated by cell membrane receptors, as it persisted when bovine serum albumin (BSA)-bound hormones were used. Further, inhibition of VSMC growth induced by E₂–BSA was reversed in the presence of T–BSA and vice versa. These results suggest that while female and male sex hormones affect VSMC growth similarly, they interfere in a dose-, hormone- and MEK-dependent manner with each other's effect.     

Serum and urinary levels of reproductive hormones associated with the estrous cycle in white-tailed deer (Odocoileus virginianus)

Concentrations of estradiol-17β (E₂), progesterone (P_o), and luteinizing hormone (LH) in serum and estrone glucuronide (E₁G) and pregnanediol glucuronide (PdG) in urine were determined by direct radioimmunoassay in five yearling and three adult female white-tailed deer (Odocoileus virginianus) from 14 October to 20 December 1985. Elevated levels of LH were recorded before the first estrus of the breeding season and immediately preceding or during behavioral estrus. Urinary E₁G and PdG concentrations were poorly correlated with serum levels of E₂ and P_o, respectively. Serum P_o levels prior to the first estrus of the breeding season indicate that pubertal yearlings experience silent ovulations and suggest that silent ovulations may be important in the transition from adolescence to cycling adult in white-tailed deer. © 1992 Wiley-Liss, Inc.     

“Cleavable” hapten-biotin conjugates: Preparation and use for the generation of anti-steroid single-domain antibody fragments

Antibody engineering technology has the potential to provide artificial antibodies with higher performance than conventional antibodies. Filamentous phage particles are often used to express a vast diversity of mutated antibody fragments from which clones displaying improved fragments can be isolated. We recently showed that hapten-biotin conjugates, combined via a linker involving a reductively cleavable disulfide bond, are useful for isolating phage clones displaying high-affinity anti-hapten antibody fragments. Here we prepare cleavable hapten-biotin conjugates and use them to isolate anti-hapten antibody fragments with relatively low affinities. Three diagnostically important steroids (estradiol-17β [E₂], cortisol, and 17α-hydroxyprogesterone) were each coupled with a biotin derivative containing a disulfide bond. These conjugates could be bound simultaneously by their relevant anti-steroid antibody and NeutrAvidin, and their linkers were easily cleaved by dithiothreitol (DTT) treatment. The E₂-biotin conjugate was used to generate anti-E₂ single-domain antibody fragments (sdAbs). Random point mutations were introduced by error-prone PCR into the gene fragment encoding the V_H domain of a mouse anti-E₂ antibody, and these products were expressed as phagemid particles that were reacted with the E₂-biotin conjugates that had already been immobilized on a solid-phase via NeutrAvidin. Thorough washing off of nonspecific phages and subsequent DTT treatment provided a phagemid clone that displayed a mutated sdAb with improved binding properties.     

Effect of thyroid state on estradiol-17β metabolism in the rabbit

It is well known that the metabolic clearance rate (MCR) of a hormone is Influenced highly by the level of its specific binding protein. It was interesting, therefore, to study the metabolism of estradiol-17β (E₂) in an animal model such as the rabbit where there is a lack for a highly specific binding protein for the steroid. The kinetics of the hormone was studied in relation to the thyroid state, namely in rabbits receiving thyroxin or propylthiouracil.In the absence of any significant decrease of the level of the rabbit androgen binding protein (R-ABP), the accelerated MCR^E2 and the elevated conversion ratio of estradiol to estrone (CR ^E₂→E₁) observed in hyperthyroid rabbits were attributed to the important role of metabolizing enzymes in the liver and/or extrahepatic tissues. In hypothyroid rabbits, while the CR ^E₂→E₁ decreased significantly the MCR^E2 was not altered.