Changes in the 17 beta-hydroxysteroid dehydrogenase activity of mouse blastocysts during delayed implantation

The rate of estrone (E1)----estradiol-17 beta (E2) or E2----E1 conversion catalyzed by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity was determined for each mouse embryo in modified F-10 medium containing 0.95 microM 3H-E1 or 3H-E2. During delayed implantation, the E1----E2 conversion rate was decreased (p less than 0.005) from 5.69 +/- 0.34 fmol/h/blastocyst on Day 5 to 3.50 +/- 0.46 fmol/h/blastocyst on Day 9, whereas E2----E1 was increased (p less than 0.005) from 7.44 +/- 1.08 to 18.60 +/- 2.04 fmol/h/blastocyst. After estrogen injection, the Day 9 implanting blastocyst showed an increase (p less than 0.005) in E1----E2 conversion to 9.05 +/- 0.64 fmol/h/blastocyst but a slight, insignificant decrease in E2----E1 conversion to 14.2 +/- 1.82 fmol/h/blastocyst. A similar trend was also observed in Day 5 implanting blastocysts when compared to Day 5 delayed blastocysts. Thus, 17 beta-HSD activity in delayed blastocysts favors E2----E1 over E1----E2 conversion in a ratio of 5:1. After estrogen induction of implantation, the E1----E2 conversion rate is stimulated and the ratio of E2----E1 to E1----E2 rate is decreased to 1.5:1. The results suggest that 17 beta-HSD activity may be involved in blastocyst implantation.     

A novel HPLC-RIA method for the simultaneous detection of estrone, estradiol and estrone sulphate levels in breast cancer tissue

Estrogen deprivation is an effective approach for treatment of hormone sensitive breast cancer. While much is known about plasma estrogen levels with respect to castration in premenopausal women and use of aromatase inhibitors in postmenopausal women, currently there is increasing interest in intra-tumour estrogen production. However, knowledge about alterations in intra-tumour estrogen levels is limited, mainly due to methodological problems with measurements of estrogen fractions in tissue samples. Here we describe a new method for simultaneous measurement of the three main estrogen fractions, estrone (E(1)), estradiol (E(2)) and estrone sulphate (E(1)S) in breast tumour tissue. Following incubation with -labelled estrogen standards, crude fractions were separated by ether extraction. The E(1)S fraction was hydrolysed with sulphatase followed by eluation on a Sephadex column. High pressure liquid chromatography (HPLC) was used to purify the individual estrogen fractions prior to RIA analysis. Estrone and E(1)S were converted into E(2), and all three estrogen fractions were finally measured by the same highly sensitive and specific radioimmunoassay using estradiol-6-(O-carboxymethyl)-oximino-2-(2--iodo-histamine) as a ligand. Although several purification steps were used, the internal recovery values for tritiated estrogens were found to be 25-50% for E(1) and E(2) and 15-30% for E(1)S. The detection limit of this method was 4.3 fmol/g tissue for E(2), 19.8 fmol/g tissue for E(1) and 11.9 fmol/g E(1)S, respectively. Using tissue from locally advanced breast cancers (n = 14), we found median levels of E(1), E(2) and E(1)S to be 283.8 fmol/g tissue (range 19.8-547.5), 554.1 fmol/g (9.5-3024.2) and 209.4 fmol/g (11.9-753.4), respectively. The method described here is a promising tool to study intra-tumour estrogen fractions in breast tissue biopsies.     

The effect of exercises on the content and reception of the steroid hormones in rat skeletal muscles

Two peaks of hormone concentration in blood and skeletal muscles (SM) were found, immediately after intensive physical exercises (PE) and the late period of rest. Immediately after PE testosterone (T) and estradiol (E2) concentration in SM increased on 36 and 430%, returning to the initial level in 2 h. E2 and androstenedione content in SM increased from 33 +/- 7 to 89 +/- 3 and from 563 +/- 58 to 767 +/- 38 pg/g tissue accordingly in 48 h, returning to the initial level in 72 h. T content increased in blood 3.7-fold in SM 2.2-fold in 72 h after PE. Androgen receptor (AR) contents in SM cytosol increased (on 28%) in 2 h, then returned to the control level and increased again in 72 h (from 0.6 +/- 0.4 to 1.2 +/- 0.2 fmol/mg protein) after PE. Kd did not change significantly and were 0.33 +/- 0.03 nM. The experiments on female rats showed the same tendency, but T increasing was more marked compared to male rats. The obtained results showed that the hormonal regulation of metabolic processes in SM has a cyclic character and is connected with the changing of hormonal content and hormonal SM receptors.     

Relationship between the preovulatory luteinizing hormone (LH) surge and androstenedione synthesis of preantral follicles in the cyclic hamster: detection by in vitro responses to LH

Preantral follicles of cyclic hamsters were isolated on proestrus, estrus and diestrus I, incubated for 3 h in 1 ml TC-199 containing 1 microgram ovine luteinizing hormone (LH) (NIH-S22), and the concentrations of progesterone (P), androstenedione (A) and estradiol (E2) determined by radioimmunoassay. At 0900-1000 h on proestrus (pre-LH surge) preantral follicles produced 2.4 +/- 0.3 ng A/follicle per 3 h, less than 100 pg E2/follicle and less than 250 pg P/follicle. At the peak of the LH surge (1500-1600 h) preantral follicles produced 1.8 +/- 0.2 ng P and 1.9 +/- 0.1 A and less than 100 pg E2/follicle. After the LH surge (1900-2000 h proestrus and 0900-1000 h estrus) preantral follicles were unable to produce A and E2 but produced 4.0 +/- 1.0 and 5.0 +/- 1.1 ng P/follicle, respectively. By 1500-1600 h estrus, the follicles produced 8.1 +/- 3.1 ng P/follicle but synthesized A (1.6 +/- 0.2 ng/follicle) and E2 (362 +/- 98 pg/follicle). On diestrus 1 (0900-1000 h), the large preantral-early antral follicles produced 1.9 +/- 0.3 ng A, 2.4 +/- 0.4 ng E2 and 0.7 +/- 0.2 ng P/follicle. Thus, there was a shift in steroidogenesis by preantral follicles from A to P coincident with the LH surge; then, a shift from P to A to E2 after the LH surge. The LH/follicle-stimulating hormone (FSH) surges were blocked by administration of 6.5 mg phenobarbital (PB)/100 g BW at 1300 h proestrus. On Day 1 of delay (0900-1000 h) these follicles produced large quantities of A (2.2 +/- 0.2 ng/follicle) and small amounts of E2 (273 +/- 27 pg/follicle) but not P (less than 250 pg/follicle).(ABSTRACT TRUNCATED AT 250 WORDS)     

Androgen and estrogen dynamics in the female baboon (Papio anubis)

Androgen and estrogen dynamics were studied in 5 female baboons (Papio anubis) using constant infusions of [3H]androstenedione/[14C]estrone and [3H]testosterone/[14C]estradiol. Blood samples were obtained prior to the infusions and both blood and plasma was used for measurements of androstenedione (A), testosterone (T), dihydrotestosterone (DHT), estrone (E1), estradiol (E2). Plasma was used for measurements of sex-hormone binding globulin (SHBG), and the percents of T and E2 free, bound to SHBG, and to albumin. Blood samples obtained during the infusions were analyzed for radioactivity as purified androgens and estrogens. Metabolic clearance rates (MCR), and transfer factors ([rho]BB; fraction of steroid infused which is converted to and measured in blood as product) and blood production rates were calculated from whole blood data. All urine was collected for 96 h and an aliquot analyzed for radioactivity as the glucuronides of estrone and estradiol and the % peripheral aromatization calculated. The MCR's, calculated in whole blood, of A, E1, E2 and T were 53 +/- 6 1/day/kg, 39.3 +/- 3 1/day/kg, 29.9 +/- 5.2 1/day/kg and 10.1 +/- 2.3 1/day/kg, respectively. Each MCR was different (P less than 0.05) from the others. The PB of E1 was 15 +/- 2 micrograms/day and was not different from that of E2 (12 +/- 3 micrograms/day). The PB of A, 231 +/- 55 micrograms/day, was greater than that of T, 13 +/- 5 micrograms/day. The interconversions of both the androgens (18.9 +/- 3.4% vs 3.9 +/- 1.0%) and the estrogens (48.8 +/- 10.7% vs 4.0 +/- 0.8%) favored the oxidative pathway, i.e. conversion of 17-OH to 17-oxo steroids. The conversion ratio of A to DHT was greater than that of T to DHT (16.4 +/- 2.1% vs 5.3 +/- 0.7%), and A is a more important source of DHT than is T. The percent of T bound to SHBG (80.7 +/- 0.9%) was greater than percent of E2 (36.9 +/- 9.8%) and inversely the percents of T bound to albumin and free (17.5 +/- 0.8% and 1.65 +/- 0.16%) were less than the respective percents for estradiol (60.5 +/- 9.5% and 2.40 +/- 0.27%). The mean SHBG concentration was 54 +/- 6 nM. The peripheral aromatization of androstenedione, 1.36 +/- 0.05%, was greater than of testosterone, 0.18 +/- 0.02%. This difference is, in part, due to the lack of SHBG-binding of androstenedione. The general pattern of androgen and estrogen dynamics is similar to that in women. This similarity is due, in part, to the presence of SHBG in both baboons and women.     

Profiling of urinary steroids by gas chromatography-mass spectrometry detection and confirmation of androstenedione administration using isotope ratio mass spectrometry

Androstenedione (4-androstene-3,17-dione) is banned by the World Anti-Doping Agency (WADA) as an endogenous steroid. The official method to confirm androstenedione abuse is isotope ratio mass spectrometry (IRMS). According to the guidance published by WADA, atypical steroid profiles are required to trigger IRMS analysis. However, in some situations, steroid profile parameters are not effective enough to suspect the misuse of endogenous steroids. The aim of this study was to investigate the atypical steroid profile induced by androstenedione administration and the detection of androstenedione doping using IRMS. Ingestion of androstenedione resulted in changes in urinary steroid profile, including increased concentrations of androsterone (An), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5α-diol), and 5β-androstane-3α,17β-diol (5β-diol) in all of the subjects. Nevertheless, the testosterone/epitestosterone (T/E) ratio was elevated only in some of the subjects. The rapid increases in the concentrations of An and Etio, as well as in T/E ratio for some subjects could provide indicators for initiating IRMS analysis only for a short time period, 2-22 h post-administration. However, IRMS could provide positive determinations for up to 55 h post-administration. This study demonstrated that, 5β-diol concentration or Etio/An ratio could be utilized as useful indicators for initiating IRMS analysis during 2-36 h post-administration. Lastly, Etio, with slower clearance, could be more effectively used than An for the confirmation of androstenedione doping using IRMS.     

Presence of endothelin-1 and endothelin-3 in peripheral tissues and central nervous system of the pig.

The distribution of endothelin (ET) peptides in the pig was studied in a variety of tissues using selective radioimmunoassays combined with reverse-phase high performance liquid chromatography (HPLC). The levels of ET-like immunoreactivity (LI) were overall relatively low. The highest levels of ET-LI were found in blood vessels, cerebral and coronary arteries containing 3190 +/- 910 and 1330 +/- 450 fmol/g, respectively. Veins generally contained higher levels of ET-LI per tissue weight than corresponding arteries. Peripheral sympathetic and sensory ganglia contained a higher concentration of ET-LI than the studied central nervous system (CNS) areas. In the CNS the highest concentration of ET-LI was found in a non-neuronal structure, the choroid plexus. The levels of ET-LI were also relatively high in the respiratory tract (100-400 fmol/g). In the heart, the endocardium contained the highest levels (190 +/- 44 fmol/g). In the kidney, the concentration of ET-LI was 3-fold higher in the medulla than in the cortex. In the gastrointestinal tract all levels were below 100 fmol/g, except for the colon which contained 120 +/- 50 fmol/g. The characterization of ET-LI in extracts of some of these tissues revealed that ET-1 dominated in the lung, spleen and hypothalamus while ET-3 and ET-1 were present in approximately equal amounts in renal medulla and thoracic spinal cord. The HPLC analysis provided no clear-cut evidence for significant presence of vasoactive intestinal contractor, ET-2 or big ET-1(1-39) in the lung, spleen, kidney, spinal cord or hypothalamus. It is concluded that mature ET-1 and ET-3 are the predominant ET peptides in peripheral tissues and CNS.     

Estradiol binding components in intestinal mucosa of female rats.

The presence of estrogen binding components (EBC) in intestinal mucosa of female rats was investigated by competitive-binding assay using radiolabelled and nonlabelled estradiol 17 beta (E2). EBC were found exclusively in the nuclear fraction and were absent from the cytosolic and from the microsomal fractions. Two types of nuclear EBC with different binding characteristics and capacities were found: Kd1 = 4.8 +/- 0.8 nM, n1 = 18.4 +/- 4.2 fmol/mg protein (n1 = 83.4 +/- 12.5 fmol/mg DNA) and Kd2 = 31.1 +/- 6.8 nM, n2 = 91.1 +/- 18.5 fmol/mg protein (412.7 +/- 80.0 fmol/mg DNA). Type 1 component showed slightly greater affinity for estrogens as compared to progesterone and dexamethasone whereas type 2 component bound other competitors with even greater affinity than E2.     

Steroid dynamics in the rabbit

Male rabbits were infused at a constant rate with 3H-androstenedione/14C-estrone (n = 5) or 3H-testosterone/14C-estradiol-17 beta (n = 3) for 3 1/2 hr and blood samples were obtained over the last hour and analyzed for radioactivity as androstenedione (A), testosterone (T), estrone (E1), estradiol-17 beta (E2 beta) and estradiol-17 alpha (E2 alpha). The mean value for the metabolic clearance rate of androstenedione (MCRA) was 85 +/- 10 l/day/kg, which was significantly greater than the mean MCRE1 59 +/- 10 l/day/kg. MCRT, 42 +/- 8 l/day/kg, and MCRE2 beta, 45 +/- 9 l/day/kg were not different. The conversion ratio of androstenedione to testosterone (CRA,T) was greater than CRT,A but for the estrogens, CRE2 beta, E1 was greater than CRE1,E2 beta. CRE2 beta, E2 alpha was greater than CRE1,E2 alpha. The overall aromatization of androstenedione to estrone, the fraction of 3H-androstenedione infused into the blood and measured as 3H-estrone in blood [( rho]A,E1BB) was 0.0005 +/- 0.0001 and for [rho]T,E2 beta BB was 0.0012 +/- 0.0006. In the rabbit both sex hormone binding globulin (SHBG) and albumin binding may effect the MCRs, and peripheral aromatization of androgens occurs to a far lesser degree than in humans and primates.     

Effects of androstenedione administration on epitestosterone metabolism in men

Androstenedione is a steroid hormone sold over-the-counter to individuals who expect that it will enhance strength and athletic performance. Endogenous androstenedione is the immediate precursor of testosterone. To evaluate the metabolism of oral androstenedione, we randomly assigned 37 healthy men to receive 0 (group 1), 100 mg (group 2), or 300 mg (group 3) of androstenedione in a single daily dose for 7 days. Eight-hour urines were collected 1 day before the start of androstenedione, and on days 1 and 7. Using gas chromatography-mass spectrometry, we measured excretion rates of glucuronide-conjugated epitestosterone, its putative precursor (E-precursor), and metabolites (EM-1 and EM-2), and we evaluated possible markers of androstenedione administration. Day 1 and 7 rates were not different: the means were averaged. The means (microg/h) for groups 1, 2, and 3, respectively were, for epitestosterone 2.27, 7.74, and 18.0; for E-precursor, 2.9, 2.0, and 1.5; for EM-1/E-precursor 0.31, 1.25, and 2.88; for EM-2/E-precursor 0.14, 0.15, and 1.15; for testosterone/epitestosterone (T/E) 1.1, 3.5, and 3.2. Epitestosterone, EM-1, and EM-2 excretion was greater in groups 2 and 3 versus group 1 (0.0001 < P < 0.03), as were EM-1/E-precursor, EM-2/E-precursor, and T/E. E-precursor excretion was lower in groups 2 (P = 0.08) and 3 (P = 0.047) versus group 1. Androstenedione increases excretion of epitestosterone and its two metabolites, while decreasing that of its precursor. Elevated ratios of EM-1- and EM-2/E-precursor, and the presence of 6alpha-hydroxyandrostenedione are androstenedione administration markers.     

Esterification-deesterification of estradiol by human mammary cancer cells in culture

Formation of lipoidal derivatives of estradiol-17 beta (E2) esterified to long-chained fatty acids has been reported to occur in estrogen target tissues. Employing human breast cancer cells in culture, we have detected the rapid synthesis of such compounds upon exposure of the cells to concentrations of [3H]E2 as low as 1 nM. When exposed to 10 nM [3H]E2 in the culture medium, synthesis of E2-lipoidal derivative (E2-L) reached 270 fmol/mg DNA in 2 h in the estrogen receptor positive MCF-7 human mammary cancer cell line. Higher rates (approximately 900 fmol/mg DNA in 2 h) were reached in 2 estrogen receptor negative human mammary cancer cell lines; MDA-MB-231 and MDA-MB-330. E2-L was the major form of estrogen in the latter cells at this time interval (E2-L/E2 approximately 3.0). Far higher concentrations of E2 were found in MCF-7 cells compared to 231 and 330 cells, and, in contrast to the latter, this was mostly specifically bound. Upon subsequent withdrawal of E2 from the medium, intracellular concentrations of E2-L decreased very rapidly in the first 5 h period, then declined more slowly to approximately 50 fmol/mg DNA at 24 h. Intracellular concentrations of E2 were maintained over this time period. E2-L was not present in the medium. Thus, accumulation of E2-L in cells upon continuous exposure to E2 represents the net result of esterification and deesterification reactions. These hydrophobic E2-derivatives may then be involved in the "capture" of E2 for transport through membranes and subsequent regeneration of E2 to maintain occupancy of the nuclear receptor.     

Role of porcine endometrial estrogen sulfotransferase in progesterone mediated downregulation of estrogen receptor

Estrogen sulfotransferase (EST) is a progesterone (Pg) induced secretory endometrial enzyme which may effect estrogen receptor levels by esterifying estradiol-17 beta (E2) to an inactive, sulfate form. The effects of this enzyme were studied using specific inhibitors of EST that do not bind to estrogen receptor (ER): 4-nitroestrone 3-methyl ether and 4-fluoroestrone 3-methyl ether. A 1 h pulse with 4 nM E2 caused ERn (i.e. E2-bound, chromatin-bound receptor) to increase 40% in incubations of proliferative gilt endometrium (no EST activity), while the same E2 treatment of secretory endometrium (high EST activity) caused no increase in ERn. ERn accumulation was completely restored in these experiments by preincubating secretory endometrium with 4 microM 4-fluoroestrone 3-methyl ether. Gilt endometrial explants cultured 7 days with 1 nM E2 plus 1 microM Pg (which induced EST activity) possessed half the ERn as explants devoid of EST activity which were cultured in E2 alone. The addition of 10 microM 4-nitroestrone 3-methyl ester to the cultures of secretory endometrium restored ERn to the levels seen in minces cultured with E2 alone. Furthermore, ovariectomized gilts injected daily with 250 micrograms E2 plus 25 mg Pg had much lower ERn (0.06 fmol/micrograms DNA) than gilts injected with E2 only (0.21 fmol/microgram DNA). ERn was restored completely by supplementing the E2 plus Pg injections with 0.5 g 4-nitroestrone 3-methyl ether administered by vaginal suppositories.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of steroids and release of prostaglandins by spherical pig blastocysts in vitro

Levels of pregnenolone and progesterone in spherical pig blastocysts (near 4 and 15 microM respectively) exceeded respective levels in histotroph by about 400-fold. When blastocysts were cultured for 5 days in a synthetic medium containing pregnenolone sulfate (1 microM), daily rates of release of pregnenolone, progesterone, androstenedione, testosterone, oestrone and oestradiol were determined to be near 320, 45, 26, 27, 0.8 and 9.2 fmol per blastocyst respectively. Daily outputs of progesterone and testosterone (fmol per blastocyst) diminished (P less than 0.05) to 1.3 and undetectable levels (less than 2) respectively in the presence of Trilostane (94 microM). Increasing the content of pregnenolone sulfate in the culture medium (to 4.5 microM) resulted in higher daily rates of release of pregnenolone and progesterone (to near 1740 and 380 fmol per blastocyst respectively), verifying activity of 3 beta-hydroxy-delta 5-steroid dehydrogenase, and of arylsulfatase, in tissues of intact spherical pig blastocysts. Prostaglandin E2 was the predominant prostaglandin (PG) released by cultured blastocysts (about 1 fmol per blastocyst per hour), hourly rates of release of PGH2 (derived) and PGF2 alpha being near 0.1 and less than 0.06 fmol per blastocyst respectively. The data establish a capacity for spherical pig blastocysts to release a range of steroids and PGs of possible significance to embryonic growth and development in vivo.     

Effects of clomiphene citrate on the pituitary gland in chronically estrogenized rat

Effects of clomiphene citrate (clomiphene) on the pituitary gland of chronically estrogenized ovariectomized rats were investigated. Estradiol-17 beta (E2) pellet implanted subcutaneously in castrated rats for 7 days caused significant increases in pituitary weight and serum prolactin (PRL) level but suppressed serum luteinizing hormone (LH) level. In the estrogenized rats about 40% of estrogen receptor (ER) found in whole pituitary cells (65 +/- 7 fmol/10 mg tissue) was observed in the nucleus, while 60% of ER was present in the cytosol fraction. A single injection of 5 micrograms E2 translocated cytosol ER immediately to nuclear compartment; amounts of ER found in cytosol and nuclear fractions were 16 +/- 1 and 37 +/- 4 fmol/10 mg tissue, respectively, at 1 h. However, the distribution of ER returned to the pre-injection level within 4 h. In the non-estrogenized castrated rats, the nuclear retention of ER was significantly longer than that in the estrogenized rats. A single administration of 200 micrograms clomiphene in the estrogenized rats, on the other hand, increased nuclear ER gradually. Nuclear ER reached the peak level at 4 h (62 +/- 5 fmol/10 mg tissue) and the level remained almost unchanged for 24 h. Cytosol ER decreased and reached a nadir at 4 h (4.3 +/- 0.3 fmol), and the replenishment of cytosol ER could not be detected for 24 h. Similar patterns of cytosol and nuclear ER following the clomiphene injection were also found in the castrated rats. The clomiphene administration in the estrogenized rats resulted in a significant reduction of the pituitary weight 48 h after the administration. The present results seem to show the antiestrogenic action of clomiphene in the pituitary gland.     

Identification of angiotensin II receptor and determination of its subtypes in rabbit and guinea pig fetal tissue]

Membrane angiotensin II receptors were measured in trophoblastic tissues using a 2-step procedure. The first step consisted of the relative measurement performed at a fixed 125I[Sar1 Ile8]AII concentration of 0.15 nM in order to determine which tissues had a sufficient number of binding sites for studying the competition curves. The second consisted of determining the maximal binding (Bmax) and the dissociation constant (Kd) for [Sar1 Ile8] AII and the receptor subtypes in these tissues. The relative binding measurement revealed a significant number of occupied sites in rabbit fetal placenta and chorion (159 +/- 17 and 51 +/- 10 fmol/mg proteins) and in guinea pig chorion (132 +/- 12). The mean values of the other trophoblastic tissues were 3-10-fold lower in the 2 species. The competition curves obtained from tissues with high angiotensin II binding receptors showed the predominance of the AT2 subtype in rabbit fetal placenta (AT1/AT2 = 25/75) and of the AT1 receptor in guinea pig chorion (97/3) and in rabbit chorion (90/10). The [SAR1 Ile8] AII affinity (Kd) obtained from Scatchard plot analysis was 1.2 +/- 0.2 nM (n = 5) in fetal placenta and 1.2 (n = 1) in rabbit chorion and 0.5 +/- 0.1 (n = 3) in guinea pig chorion. In these tissues, the respective Bmax values were 1,281 +/- 115 (n = 5), 263 (n = 1) and 1,188 +/- 134 fmol/mg proteins (n = 3). These findings indicate that rabbit fetal placenta and chorion and guinea pig chorion are the most important sites of action for the renin-angiotensin system present in trophoblastic tissues.     

Effects of the intensity of the suckling stimulus and ovarian steroids on pituitary gonadotropin-releasing hormone receptors during lactation

These studies examined whether the decrease in pituitary responsiveness to gonadotropin-releasing hormone (GnRH) observed during lactation in the rat results from a change in pituitary GnRH receptors. GnRH binding capacity was determined by saturation analysis using D-Ala6 as both ligand and tracer. During the estrous cycle, the number of GnRH binding sites increased from 199 +/- 38 fmol/mg protein on estrus to 527 +/- 31 fmol/mg protein on the morning of proestrus, whereas there was no change in receptor affinity (Ka, 6-10 X 10(9) M-1), During lactation, females nursing 8 pups on Days 5 or 10 postpartum had 50% fewer GnRH receptors (109-120 fmol/mg protein) than observed during estrus or diestrus 1 (199-242 fmol/mg protein) although receptor affinity was similar among all the groups. No deficits in pituitary GnRH receptors were observed in females nursing 2 pups on Day 10 postpartum. Removal of the 8-pup suckling stimulus for 24 or 48 h resulted in a dramatic increase in GnRH receptor capacity by 24 h from 120 +/- 16 to 355 +/- 39 fmol/mg protein. The rise in GnRH receptors after pup removal was accompanied by an increase in serum luteinizing hormone (LH) and estradiol concentrations. To assess the role of ovarian steroids in determining GnRH receptor capacity during lactation, females were ovariectomized (OVX) on Day 2 postpartum. Suckling of a large litter (8 pups) completely blocked the postcastration rise in serum LH and in pituitary GnRH receptors on Day 10 postpartum (OVX+ 8, 77 +/- 12 fmol/mg protein; OVX+ 0, 442 +/- 38 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteome analysis of rat serum proteins adsorbed onto synthetic octacalcium phosphate crystals

The present study was designed to determine which proteins are selectively adsorbed onto two bone substitute materials, octacalcium phosphate (OCP) and hydroxyapatite (HA) crystals, from rat serum by proteome analysis. Ground crystals of synthetic OCP and commercially available sintered HA, with the same surface area, were incubated in rat serum proteins at 37 °C for 24 h. The proteins from the crystals extracted with guanidine–HCl–EDTA were listed on the basis of the results of liquid chromatography tandem mass spectrometry (LC/MS/MS). A total of 138 proteins were detected from OCP; 103 proteins were detected from HA. Forty-eight proteins were from both crystals. A quantitative analysis of the proteins detected was performed for the extracted two bone formation-related proteins apolipoprotein E (Apo E), a protein known to promote osteoblast differentiation, and complement 3 (C3). HA adsorbed C3 (3.98 ± 0.03 fmol/μg protein) more than OCP (1.81 ± 0.07 fmol/μg protein) did, while OCP adsorbed Apo E (2.42 ± 0.03 fmol/μg protein) more than HA (1.21 ± 0.01 fmol/μg protein) did even after deleting the high-abundance proteins, such as albumin. The results demonstrated that OCP exhibits a similar property but distinct capacity with HA in adsorbing bone formation-related proteins from the serum constituents.     

Development of preovulatory follicles expected to form short-lived corpora lutea in beef cows

Oestrus, expected to be followed by a short luteal phase, was induced in post-partum cows by weaning their calves at 35 days after parturition. Ovaries containing the first preovulatory follicles (Type F) formed after parturition were collected 3 h after the onset of oestrus. For comparison, preovulatory follicles (Type C) were collected 3 h after the onset of oestrus in normally cycling cows. The number of granulosa cells was determined and the concentrations of receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in granulosa cells and for LH in theca cells were measured. Concentrations of oestradiol-17 beta, testosterone, androstenedione and progesterone in follicular fluid were also measured. Type F follicles contained about twice the number of granulosa cells (based on DNA) as did Type C follicles (45.8 +/- 11.3 and 24.5 +/- 3.9 micrograms DNA/follicle, respectively; P less than 0.05) but these cells had fewer receptors for LH (0.13 +/- 0.02 vs 0.29 +/- 0.03 fmol/micrograms DNA; P less than 0.01) and FSH (0.61 +/- 0.08 vs 1.3 +/- 0.29 fmol/micrograms DNA; P less than 0.08) than did those from Type C follicles. Additionally, there were fewer receptors for LH in theca tissue from Type F than from Type C follicles (28.3 +/- 5.2 vs 51.3 +/- 6.1 fmol/follicle; P less than 0.01). Concentrations of oestradiol-17 beta (475.8 +/- 85.6 vs 112.9 +/- 40.0 ng/ml; P less than 0.01) and androstenedione (214.1 +/- 48.7 vs 24.7 +/- 7.7 ng/ml; P less than 0.01) in follicular fluid were higher in Type C than in Type F follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased aromatase activity in pubic skin fibroblasts from patients with isolated gynecomastia [Poster 47]

Aromatase activity (AR) was studied in pubic skin fibroblasts from 8 patients with isolated gynecomastia (PSFG) and 5 normal subjects (PSFC) by incubation in the presence of [³H]androstenedione (A) and isolation of aromatized metabolites. After 4 h of incubation, no E₂ formation could be measured in PSFC in contrast to low but significant amounts in PSFG [0.03 ± 0.02 (SEM) fmol/μg DNA, p < 0.01]. In addition, 16-OHE₁ represented 62.5% of total aromatized metabolites in PSFG versus 39% in PSFG. After 24 h, there was no significant difference in E₂ formation between the two groups whereas total aromatization remained significantly higher (p < 0.05) in PFSG; E₃+ epiE₃ represented 50% of the metabolites in both groups.     

Mechanism of induction of cellular DNA synthesis by the adenovirus E1A 12S cDNA product

The mechanism of induction of DNA synthesis in quiescent rat 3Y1 cells by the adenovirus E1A gene was investigated using the 3Y1 derivative cell lines g12-21, gn12RB1, and gn12RB2. The g12-21 cells express the E1A 12S cDNA and the latter two cells express both the E1A 12S cDNA and the human retinoblastoma susceptibility (Rb) gene at different levels in response to dexamethasone (dex). The cDNA sequences of E1A-inducible cell cycle-dependent genes, clone 3 and clone 16, were isolated by differential screening of a cDNA library constructed from dex-treated g12-21 cells. The quiescent 3Y1 cells induced c-fos and c-myc expression within 2 h after serum stimulation and expressed clone 16 and clone 3 transiently at around 8 h before the onset of DNA synthesis (10 h). In contrast, the quiescent g12-21 cells treated with dex expressed a high level of E1A at 6 to 8 h after treatment and expressed clone 16 and clone 3 at around 8 h without stimulation of c-fos and c-myc expression, suggesting that E1A bypasses the cell cycle early in G1. The half-maximal rate of DNA synthesis was reached in a much shorter time in dex-treated g12-21 cells (12 h) than in serum-treated 3Y1 cells (18 h), suggesting that E1A also bypasses the cell cycle at the G1/S boundary. The gn12RB1 and gn12RB2 cells were unable to induce DNA synthesis in response to dex presumably due to lower levels of E1A expression, although gn12RB2 but not gn12RB1 cells could express clone 16 and clone 3. These results suggest that the level of E1A required for bypass at the G1/S boundary is higher than that required early in G1.