Effects of applying gamma-aminobutyric acid(B) drugs into the medial basal hypothalamus on basal luteinizing hormone concentrations and on luteinizing hormone surges in the female sheep

Prior investigations have shown that localized infusion by microdialysis of gamma-aminobutyric acid(B) (GABA(B)) agonists into the medial basal hypothalamus of male sheep rapidly increases GnRH and LH pulse amplitude. The objectives of these studies were to determine if infusion of GABA(B) agonists SKF 97541 or baclofen into the medial basal hypothalamus of female sheep would affect basal LH secretion and if infusion of a potent antagonist would alter expression of LH surges induced by injection of estrogen. Infusion of either SKF 97541 (10 or 40 microM) or baclofen (1 mM) into estrogen-treated ovariectomized ewes did not alter basal LH secretory patterns, whereas both drugs significantly elevated mean LH and LH pulse amplitude in ovariectomized ewes during the nonbreeding season. Infusion of the antagonist CGP 52432 (250 or 500 microM) did not affect expression of estrogen-induced LH surges in ovariectomized ewes. These observations support the concept that GABA(B) receptors in the medial basal hypothalamus regulate basal LH secretion but do not regulate the surge mode of LH secretion in the female sheep.     

Estradiol stabilizes estrogen receptor messenger ribonucleic acid in sheep endometrium via discrete sequence elements in its 3'-untranslated region

The preovulatory surge of estrogen up-regulates estrogen receptor-alpha (ER) gene expression in the uterus during the estrous/menstrual cycles of female mammals. Previously, we demonstrated that the 5-fold increase in ER mRNA levels in endometrium of ovariectomized ewes treated with a physiological dose of estradiol (E2) is entirely due to an increase in ER mRNA stability. Our current work confirms that the E2 effect is specific to ER mRNA. The sequence of ER mRNA, cloned from sheep endometrium, shows a high degree of conservation with those of other species, even in the 5'- and the very long 3'-untranslated regions. In a cell-free assay, ER mRNA demonstrates greater stability with endometrial extracts from E2-treated ewes compared with those from untreated ovariectomized ewes. The E2-enhanced stability of ER mRNA was ablated by prior treatment of the extracts with proteinase K, 70 C heat, and oxidizing and alkylating reagents, indicating that a protein is responsible for stabilization of the message. The 3'-untranslated region of ER mRNA contains discrete sequences required for E2-enhanced stability, four of which were identified by extensive deletion mutant analyses. Transfer of two of the four minimal E2-modulated stability sequences conferred E2-enhanced stability to a heterologous RNA. These minimal E2-modulated stability sequences contain a common 10-base, uridine-rich sequence that is predicted to reside in a loop structure. Throughout our studies, estrogen stabilization of ER mRNA in sheep endometrium resembled that of vitellogenin mRNA in frog liver, indicating conservation of this ancient mechanism for enhancing gene expression in response to estrogen.     

Developmental changes of glucocorticoid receptors in the rat pancreas

Glucocorticoids are known to play a role in the maturation of the exocrine pancreas. The exact mechanism of glucocorticoid action in pancreatic ontogeny is, however, not clear. The present study characterized and quantitated the binding of [3H]dexamethasone to cytosol fractions from pancreata of rats at various ages. Trunk blood samples from these rats were also checked for levels of free and bound corticosterone. Specific and saturable bindings for dexamethasone were found in pancreatic cytosol fractions from newborn suckling and adult rats. Competition studies showed a preference for steroids with glucocorticoid activity. Specific binding was relatively low in pancreatic cytosol from newly born and 1-day old pups. A significant rise was seen after day 15. Cytosolic binding capacities were greatest from pancreata obtained from pups at weaning (3rd to 5th weeks). Values then declined toward the adult level. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 7.3 (+/- 1.1) X 10(-8) M and number of binding sites equalled to 1.29 (+/- 0.18) X 10(-13) mole/mg of cytosolic protein in adult rat pancreas. Pancreata from 25- and 15-day old rats had Kds of 3.4 (+/- 0.8) X 10(-8) M and 2.7 (+/- 0.7) X 10(-8) M with the number of binding sites equal to 1.77 (+/- 0.21) X 10(-13) mole/mg protein and 1.31 (+/- 0.16) X 10(-13) mole/mg protein respectively. Total plasma corticosterone concentration was low before day 10. It rose significantly by day 15, peaked at day 25, and then declined after weaning. About 5-15% of corticosterone during weaning and about 20-30% before and after weaning were in the free form. The peak level of dexamethasone binding corresponded to an increase in the plasma corticosterone level during weaning. This suggests a close relationship between plasma corticosterone levels and pancreatic glucocorticoid receptors. Both may, therefore, play a role in pancreatic development in the rat.     

Further characterization of the electromyographic activity of the myometrium and mesometrium in nonpregnant sheep under estrogen supplementation.

The effects of estrogen administration on uterine contractility varies with animal species. In nonpregnant ovariectomized sheep, estrogen administration has been reported either to inhibit, inhibit then stimulate, or only stimulate uterine contractility. The aim of the present study was to determine the effects of prolonged estrogen administration in the electromyographic (EMG) activity recorded from the myometrium and mesometrium in nonpregnant ovariectomized sheep after estrous synchronization by inserting vaginal progesterone sponges 14 days before surgery. Surgery was performed on four ewes under halothane anesthesia. Bilateral oophorectomy was performed, and stainless steel EMG electrodes were sewn to the mesometrium and myometrium in both left and right horns of the uterus. Blood samples were taken at 1000 h from the uterine vein for 13,14-dihydro-15-keto-prostaglandin F2 alpha determination, and from the femoral artery for estradiol determination. Starting on Day 7 after surgery, estradiol 17 beta (50 micrograms/24 h) was infused continuously into the jugular vein. Estrogen administration had a different effect on the EMG activity recorded from myometrium and mesometrium. The myometrial response to estrogen was an increase in the frequency of short EMG events from 19.0 +/- 8.7 to 57.0 +/- 5.0 (p less than 0.05) for events less than 60 sec, and from 2.70 +/- 0.83 to 10.30 +/- 1.36 (p less than 0.05) for events lasting greater than 60 sec but less than less than 180 sec. In contrast, there was no stimulatory effect of estrogen on mesometrial EMG for both types of short events less than 60 sec, and greater than 60 but less than less than 180 sec.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progesterone does not affect the amount of mRNA for gonadotropins in the anterior pituitary gland of ovariectomized ewes

To evaluate the effect of progesterone on the synthesis and secretion of gonadotropins, ovariectomized ewes either were treated with progesterone (n = 5) for 3 wk or served as controls (n = 5) during the anestrous season. After treatment for 3 wk, blood samples were collected from progesterone-treated and ovariectomized ewes. After collection of blood samples, hypothalamic and hypophyseal tissues were collected from all ewes. Half of each pituitary was used to determine the content of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the number of receptors for gonadotropin-releasing hormone (GnRH). The amounts of mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were measured in the other half of each pituitary. Treatment with progesterone reduced mean serum concentrations of LH (p less than 0.001) but ot FSH (p greater than 0.05). Further, progesterone decreased (p less than 0.05) the total number of pulses of LH. We were unable to detect pulsatile release of FSH. Hypothalamic content of GnRH, number of receptors for GnRH, pituitary content of gonadotropins and mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were not affected (p greater than 0.05) by treatment with progesterone. Thus, after treatment with progesterone, serum concentrations of LH (but not FSH) are decreased. This effect, however, is not due to a decrease in the steady-state amount of mRNA for LH beta or alpha subunits.     

Pituitary receptors for gonadotropin-releasing hormone in relation to changes in pituitary and plasma luteinizing hormone in ovariectomized-hypothalamo pituitary disconnected ewes. I. Effect of changing frequency of gonadotropin-releasing hormone pulses

Studies were undertaken to determine if changes in the amplitude of luteinizing hormone (LH) pulses that occur in response to changes in the frequency of gonadotropin-releasing hormone (GnRH) pulses are due to an alteration in the number of GnRH receptors. Ewes were ovariectomized (OVX) and the hypothalamus was disconnected from the pituitary (HPD). Ewes were then given pulses of GnRH at a frequency of 1/h or 1/3 h. Two control groups were included: OVX ewes not subjected to HPD, and HPD ewes that were not OVX. At the end of one week of treatment, blood samples were collected to determine the amplitude of LH pulses. The treated ewes were killed just before the next scheduled pulse of GnRH, and the content of LH and number of GnRH receptors were measured in each pituitary. The amplitude of LH pulses was highly correlated with the amount of LH in the pituitary gland (r = 0.71, p less than 0.01), and both LH content and pulse amplitude (mean + SEM) were higher in ewes receiving GnRH once per 3 h (189.7 +/- 39.3 microgram/pituitary, 10.3 +/- 1.1 ng/ml, respectively) than in ewes receiving GnRH once per h (77.8 +/- 11.4 microgram/pituitary, 5.2 +/- 1.3 ng/ml). The pituitary content of LH was highest in the OVX ewes (260.2 +/- 57.4 micrograms/pituitary) and lowest in the nonpulsed HPD ewes (61.7 +/- 51.2 micrograms/pituitary). The number of GnRH receptors was similar in all groups, and was not correlated with any other variable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelial vasodilator production by uterine and systemic arteries. IV. Cyclooxygenase isoform expression during the ovarian cycle and pregnancy in sheep

Uterine artery endothelial production of the potent vasodilator, prostacyclin, is greater in pregnant versus nonpregnant sheep and in whole uterine artery from intact versus ovariectomized ewes. We hypothesized that uterine artery cyclooxygenase (COX)-1 and/or COX-2 expression would be elevated during pregnancy (high estrogen and progesterone) and the follicular phase of the ovarian cycle (high estrogen/low progesterone) as compared to that in luteal phase (low estrogen/high progesterone) or in ovariectomized (low estrogen and progesterone) ewes. Uterine and systemic (omental) arteries were obtained from nonpregnant luteal-phase (LUT; n = 10), follicular-phase (FOL; n = 11), and ovariectomized (OVEX; n = 10) sheep, as well as from pregnant sheep (110-130 days gestation; term = 145 +/- 3 days; n = 12). Endothelial and vascular smooth muscle (VSM) COX-1 protein levels and uterine artery endothelial cell COX-1 mRNA levels were compared. Using immunohistochemistry and Western analysis, the primary location of COX-1 protein was the endothelium; that is, we observed 2.2-fold higher COX-1 protein levels in intact versus endothelium-denuded uterine artery and a 6.1-fold higher expression in the endothelium versus VSM (P < 0.05). COX-2 protein expression was not detectable in either uterine artery endothelium or VSM. COX-1 protein levels were observed to be higher (1.5-fold those of LUT) in uterine artery endothelium from FOL versus either OVEX or LUT nonpregnant ewes (P < 0.05), with substantially higher COX-1 levels seen in pregnancy (4.8-fold those of LUT). Increases in uterine artery endothelial COX-1 protein were highly correlated to increases in the level of COX-1 mRNA (r(2) = 0.66; P < 0.01) for all treatment groups (n = 6-8 per group), suggesting that increased COX-1 protein levels are regulated at the level of increased COX-1 mRNA. No change in COX-1 expression was observed between groups in a systemic (omental) artery. In conclusion, COX-1 expression is specifically up-regulated in the uterine artery endothelium during high uterine blood flow states such as the follicular phase and, in particular, pregnancy.     

Cytosolic Type II Estrogen Binding Site in Rat Uterus: Specific Photolabeling with Estrone

Abstract

A low affinity (Kd = 30 nM), large capacity (Bmax = 2.6 pmol/g tissue) estrogen binding site was photolabeled from estradiol-stimulated rat uterus cytosol. To maximize levels of this binding site and reduce those of the type I binding site, ovariectomized rats were injected with high doses of estradiol (10μg per day) for four days with the last injection two hours before sacrifice. This treatment depleted type I estrogen receptors from the cytosol (by 90%) and raised levels of type II sites in the nucleus without affecting cytosolic type II levels. The type II estradiol binding sites were distinguished from the type I sites on the basis of their dissociation kinetics, pH-sensitivity and their behavior towards potassium chloride, somatostatin, sodium thiocyanate, sulfhydryl reagents and ammonium sulfate precipitation. These type II binding sites could be covalently photolabeled with tritiated estrone. A molecular weight of 43 kDa was found on SDS PAGE.     

雌激素对兔子宫内膜细胞EGF受体和细胞周期的调控

 由受体放射配基结合分析证明家兔子宫内膜细胞的EGF受体Kd值为0.53nmol/L,每个细胞的最大结合容量为1.11×10~4结合位点。10~(-10)mol/L雌二醇处理24h,细胞的最大结合容量增至2.75×10~4结合位点数/细胞,而Kd值无明显变化,可是,当10~(-5)mol/L雌二醇处理24h,细胞的EGF受体结合率,DNA合成速度率均下降。G_0/G_1期细胞比值明显下降,而G_2+M期和S期细胞明显上升。     

Cell-specific expression of estrogen-responsive genes in the uteri of cyclic, early pregnant and ovariectomized ewes

A single physiological dose of estradiol up-regulates estrogen receptor-alpha(ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), c-fos, cyclophilin, and actin mRNAs in the endometrium of ovariectomized ewes. Therefore, we hypothesized that these genes would be up-regulated by the preovulatory surge of estrogen which occurs on the evening of Day 15 in the estrous cycle of sheep. ER and PR mRNA concentrations increased between Day 15 and Day 1 in cyclic ewes in most endometrial epithelial cells, while GAPDH mRNA increased in epithelial and stromal cells in the deep endometrium. Day 15 pregnant ewes had lower expression of ER, PR, GAPDH, cyclophilin and actin genes. For ER and GAPDH mRNAs, the greatest reduction occurred in the superficial endometrium. Ovariectomized ewes demonstrated concentrations of ER, PR, and GAPDH mRNAs that were similar to those in the cyclic ewes. While concentrations of c-fos mRNA did not differ between groups, those of cyclophilin and actin mRNAs were lower in the pregnant and ovariectomized ewes. In conclusion, ER, PR and GAPDH gene expression rose during estrus in endometrial cells with the highest ER gene expression and were repressed in pregnant ewes in superficial endometrial cells with the greatest PR gene expression.     

125I-labelled hCG binding characteristics in theca interna and other tissues from Romney ewes and from Booroola x Romney ewes with and without a major gene influencing their ovulation rate

Specific receptors for 125I-labelled hCG in ovarian follicle wall were located in the theca interna. No specific binding of 125I-labelled hCG was found in theca externa and/or stromal tissue. The kinetics of 125I-labelled hCG binding to theca interna followed second order kinetics with calculated association rate constants (ka +/- s.d.) of 1.57 +/- 0.16 X 10(6) and 0.57 +/- 0.02 X 10(6) litres mol-1 sec-1 at 37 degrees C and 22 degrees C respectively. Dissociation of specifically bound 125I-labelled hCG from theca interna was minimal at 37 degrees C and 22 degrees C. The binding of 125I-labelled hCG to theca interna could be displaced with PMSG, FSH-P and sheep LH but other sheep pituitary hormones and LH-releasing hormone showed little or no cross-reaction. The calculated binding capacities (Bmax) and equilibrium dissociation constants (Kd) for 125I-labelled hCG binding to theca interna did not differ between Romney ewes and Booroola x Romney ewes with and without the fecundity (F) gene on Day 10 of the oestrous cycle, during anoestrus or at 36 h after an injection of cloprostenol on Day 10 of the oestrous cycle. When the data for Day 10 and anoestrus were pooled, the median (range) Bmax and Kd values in non-atretic follicles (greater than or equal to 3 mm diameter) were 12.0 (5.1-23.5) fmol/mg protein and 0.10 (0.05-0.16) nM respectively. At 36 h after cloprostenol injection the respective median (range) Bmax and Kd values in non-atretic follicles (greater than or equal to 3 mm diam.) increased to 46.9 (28.4-70.3) fmol/mg protein and 0.23 (0.13-0.65) nM respectively. In corpora lutea the hCG binding characteristics were similar in all the above breeds/genotypes. On Day 10 of the cycle, the mean Bmax but not the mean Kd value was significantly higher (P less than 0.01) than the corresponding value at 36 h after cloprostenol injection. In granulosa cells, from follicles of greater than or equal to 5 mm diameter of Romney and Booroola x Romney (++) ewes and from follicles of greater than or equal to 3 mm diameter of Booroola x Romney (F+) ewes, the hCG binding characteristics were similar. In granulosa cells from smaller sized follicles from the above breeds/genotypes, no specific hCG binding was noted.     

Endocrine aging in C57 BL mice--II. Dynamics of estrogen receptors in the hypothalamic-pituitary axis

Specific cytosolic and nuclear binding sites for estrogens were measured in the hypothalamic-pituitary axis (HPA) of young (4-8 months) and old (16-18 months) C57 BL mice in order to determine any age-related alteration in hormone-receptor interaction. Our results indicated no age differences in the affinity (KD = 0.89 +/- 0.03 (SEM) vs 1.09 +/- 0.2 X 10(-9) M), the specificity, the sedimentation profile (6 s) or in the number (98.9 +/- 4.9 vs 84.4 +/- 2.3 fmol/mg protein) of unoccupied estrogen binding sites in the cytosols. Estradiol administration to young mice induced a complete translocation of cytosolic estrogen receptors to the nucleus, and two types of nuclear binding sites were observed: Type I were specific for estrogens with high affinity (KD = 0.51 +/- 0.06 X 10(-9) M) and low binding capacity (115.1 +/- 22.7 fmol/mg DNA) and sedimented in the 4.0 s area, while Type II binding sites showed a much higher capacity and lower affinity for R2858. HPA nuclear suspensions of aged untreated mice showed undetectable (less than 50 fmol/mg DNA) levels of nuclear estrogen receptors and E2 pre-treatment resulted in a significant increase in both types of binding sites. While no significant changes in the physicochemical characteristics of these nuclear receptors were observed, when compared to young animals, aging was manifested by a translocation defect in the HPA of C57 BL mice. These results suggest aging changes in the endocrine regulating centers of the brain with defective activation of estrogen receptors.     

Effect of number of receptors for gonadotropin-releasing hormone on the release of luteinizing hormone

The relationship between number of receptors for gonadotropin-releasing hormone (GnRH) and the ability of the anterior pituitary gland to release luteinizing hormone (LH) was examined in ovariectomized ewes. A GnRH antagonist was used to regulate the number of available receptors. The dose of GnRH antagonist required to saturate approximately 50 and 90% of GnRH receptors in ovariectomized ewes was determined. Thirty min after intracarotid infusion of GnRH antagonist, ewes were killed and the number of unsaturated (i.e., those available for binding) pituitary GnRH receptors was quantified. Infusion of 10 and 150 micrograms GnRH antagonist over a 5-min period reduced binding of the labeled ligand to approximately 50 and 12% of controls, respectively. The effect of reducing the number of GnRH receptors on release of LH after varying doses of the GnRH agonist, D-Ala6-GnRH-Pro9-ethylamide (D-Ala6-GnRH) was then evaluated. One of four doses of D-Ala6-GnRH (0.125, 2.5, 50 and 400 micrograms) was given i.v. to 48 ovariectomized ewes whose GnRH receptors had not been changed or were reduced to approximately 50 or 12% of control ewes. In ewes with a 50% reduction in GnRH receptors, total release of LH (area under response curve) was lower than that obtained for controls (P less than 0.01) at the 0.125-micrograms dose of D-Ala (6.1 +/- 0.7 cm2 vs. 13.5 +/- 0.7 cm2) but was not different at the 2.5-, 50- or 400-micrograms doses of D-Ala6-GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antiestrogen binding within different pituitary cell populations. Comparison with androgen and estrogen receptors

Gonadotrope-enriched populations were prepared from 42-day old male rats by centrifugal elutriation. They contained 4.8 +/- 0.7% of the cells, 51 +/- 10% of the LH and less than 3% of the PRL (n = 4). Gonadotrope-depleted fractions were also obtained that contained most of PRL cells. Specific antiestrogen binding sites (AEBS) were quantitated in these populations after destruction of estrogen receptor. Results showed the presence of a distinct, specific high affinity binding site for antiestrogen in dispersed pituitary cells and in enriched fractions. However, AEBS are not specific of a pituitary cell type. Thus, AEBS appear different from estrogen receptors in pituitary gland: by the thermal stability of AEBS, by the localization of AEBS in particulate material, by the uniform distribution of AEBS in different populations which differ markedly for E2 binding sites. Whereas the ratio of binding AE/E2 averaged 11.4 in the initial cell suspension it reached only 2.9 in the gonadotropes. The dissociation constants for AEBS were in the same range (1.16 - 2.27 X 10(-9) M) for the different populations.     

Characterization of specific high-affinity receptor for human lymphotoxin

The specific cell surface receptors for lymphotoxin (LT) which are expressed on murine fibroblast L.P3 cells, a subline of L929 cells, were found to consist of a single class of specific high-affinity receptors with a dissociation constant (Kd) of 3.8 X 10(-10) M and a density of 5.8 X 10(3) sites/cell. Similarly, murine fibroblast L929 cells, human melanoma A375 cells and human cervical carcinoma HeLa-S3 cells had about 7.2 X 10(3), 3.5 X 10(3), and 6.6 X 10(3) sites/cell with Kd values of 1.4 X 10(-10), 0.5 X 10(-10), and 1.1 X 10(-10) M, respectively. Among the LT receptor-positive cell lines, there was no direct correlation between the level of specific LT binding and the sensitivity to the cytotoxic or cytostatic effect of LT. Cross-linking of 125I-LT to the cell surface receptors with disuccinimidyl suberate, followed by two-dimensional gel electrophoresis of the cell lysate, revealed two kinds of LT-LT receptor complexes with molecular weights of 70 and 97 kDa, and having the same pI value of 6.8. Cell-bound 125I-LT was internalized within 1 h and degraded intracellularly, and finally secreted into the medium within a few hours. Appropriate concentrations of LT and interferon gamma (IFN gamma) showed synergistic cytotoxicity toward murine fibroblast L.P3 cells and human monocytoma U937 cells, but these cytokines were only slightly cytotoxic individually. Preincubation of these cells with IFN gamma increased the total number of LT receptors without any significant change in the dissociation constant or in the molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of chronic infusion of dibutyryl c-AMP or adenosine on luteal function in sheep

Adenosine or vehicle; dibutyryl c-AMP, a c-AMP analogue, or vehicle in two separate experiments were infused through an indwelling cannula every four hours around the ovarian vascular pedicle of ewes unilaterally ovariectomized on day 8 postestrus. Adenosine or vehicle was infused from day 8 through 22 postestrus and dibutyryl-cAMP was infused from day 8 through 20 postestrus or until the ewes returned to estrus. Interestrous intervals were greater (p less than or equal to 0.05) in ewes receiving adenosine (27.3 +/- 2.4 days) than in control ewes (17.2 +/- 1.3 days). The length of the estrous cycle of ewes receiving dibutyryl c-AMP was greater (22.4 +/- 1.1; p less than or equal to 0.05) than in control ewes which averaged 16.7 +/- 0.6 days. Profiles of progesterone were different (p less than or equal to 0.05) for ewes receiving adenosine or dibutyryl c-AMP when compared to their respective controls. In addition, the overall mean concentrations of progesterone were greater (p less than or equal to 0.05) in dibutyryl c-AMP or adenosine-treated ewes than in controls. In a third experiment, infusions of adenosine or dibutyryl c-AMP intrauterine every 4 hours through a cannula from day 8 through 22 postestrus had no effect (p less than or equal to 0.05) on the interestrous interval or profiles of progesterone. It is concluded that dibutyryl c-AMP or adenosine in vivo can delay luteolysis and adenosine and c-AMP may play roles in luteal secretion of progesterone in sheep but are probably not the uterine embryonic antiluteolysin of early pregnancy in sheep.     

Distribution of insulin receptors in human erythrocyte membranes. Insulin binding to sealed right-side-out and inside-out human erythrocyte vesicles

Analyses of insulin binding to human erythrocytes and to resealed right-side-out and inside-out erythrocyte membrane vesicles have revealed that high affinity insulin binding receptors are present on both sides of the erythrocyte membranes. Insulin binding to human erythrocytes was examined with the use of a binding assay designed to minimize the potential errors arising from the low binding capacity of this cell type and from non-specific binding in the assay. Scatchard analysis of equilibrium binding to the cells revealed a class of high affinity sites with a dissociation constant (Kd) of (1.5 +/- 0.5) X 10(-8) M and a maximum binding capacity of 50 +/- 5 sites per cell. Interestingly, both resealed right-side-out and inside-out membrane vesicles exhibited nearly identical specific sites for insulin binding. At the high affinity binding sites, for both right-side-out and inside-out vesicles, the dissociation constant (Kd) was (1.5 +/- 0.5) X 10(-8) M, and the maximum binding capacity was 17 +/- 3 sites per cell equivalent. These findings suggest that insulin receptors are present on both sides of the plasma membrane and are consistent with the participation of the erythrocyte insulin receptors in an endocytic/recycling pathway which mediates receptor-ligand internalization/externalization.     

Investigations on spermicidal activity in the sheep vagina.

This study was conducted to elucidate some of the effects of a synthetic progestagen and natural ovarian hormones on spermicidal activity in the sheep vagina. In the first experiment, parous ewes were treated for 17 days either intravaginally with medroxyprogesterone acetate (MAP) or subcutaneously with progesterone. They were inseminated artificially either on the last day of progestagen treatment or during estrus after progestagen withdrawal. Their vulvovaginal junctions were ligated to prevent the loss of sperm cells by drainage to the exterior. Untreated control ewes were inseminated during either estrus or the luteal phase of the estrous cycle. The ewes were killed 22 hr. after insemination, their vaginas flushed, and intact sperm cells and tailless sperm heads counted. In the second and third experiments, some of the ewes were bilaterally ovariectomized and inseminated several weeks later. Other ewes were ovariectomized and given subcutaneous injections of estradiol, progesterone, or both hormones.In the first experiment, most sperm cells were recovered intact from estrous or luteal phase control ewes. The intravaginal administration of MAP increased both the breakage of sperm cells into heads and tails and the disappearance of sperm cells. The spermicidal effects of MAP were just as great in ewes inseminated on the last day of treatment. as in those inseminated during the ensuing estrus; these results indicated that the peak estrogen secretion that occurs near the beginning of estrus was not necessary for the intensification of spermicidal activity.In the second experiment, ovariectomized ewes were compared to estrous and luteal phase ewes in regard to vaginal spermicidal activity. Sperm breakage and disappearance occurred least in estrous ewes, to a somewhat greater degree in luteal phase ewes, and to the greatest extent in ovariectomized ewes. The results suggested that endogenous ovarian hormones, particularly those in estrous ewes, suppress spermicidal mechanisms in the vagina.In the third experiment, the administration of estradiol and progesterone to ovariectomized ewes prevented the increase in sperm cell disappearance. Neither hormone alone prevented the increase.     

Binding characteristics of 125I-labelled human FSH to granulosa cells from Booroola ewes which were homozygous, heterozygous or non-carriers of a major gene(s) influencing their ovulation rate

At 37 degrees C 125I-labelled human (h) FSH (NIAMDD-hFSH-I-3) bound rapidly to granulosa cells from Booroola and Romney ewes with 50% maximum binding achieved after 3 min and equilibrium being reached within 45 min, irrespective of whether the cells were obtained from the FF, F+ or ++ Booroola genotypes or from Romney ewes. Binding of 125I-labelled FSH followed second order kinetics and there was no effect of follicle diameter (1-2.5 mm vs greater than or equal to 3 mm). Irrespective of breed, genotype or follicle size, the mean (+/- s.e.m.) calculated association rate constant, (ka) was 7.3 (+/- 0.8) x 10(5) litres mol-1 sec-1 (n = 12). Dissociation of receptor bound 125I-labelled hFSH was less than 5% after 30 min and low but variable (i.e. between 0 and 30%) after 2-6 h irrespective of breed, genotype or follicle size. No gene-specific differences were noted in binding specificity between F+ and ++ genotypes: studies were not performed with cells from FF ewes because of insufficient cells. The binding of 125I-labelled hFSH could be displaced with sheep FSH (NIH-FSH-S16; 10% cross-reaction) and FSH-P (2.5% cross-reaction) but other sheep pituitary hormones and hCG showed little or no cross-reaction (less than or equal to 0.1%). The calculated binding capacities (Bmax) and equilibrium dissociation constants (Kd) for 125I-labelled hFSH binding to granulosa cells did not differ between the Booroola genotypes or between Booroola or Romney follicles of different diameter (i.e. 1-2.5 mm; or greater than or equal to 3 mm). The overall mean +/- s.e.m. (n = 24) Bmax and Kd values were 16.7 +/- 0.8 fm/mg protein (i.e. approximately 800 available receptor binding sites/cell) and 1.1 +/- 0.1 nM respectively. Collectively, these findings suggest that the earlier maturation of follicles in FF or F+ ewes compared to ++ ewes is unlikely to be due to gene-specific differences in the FSH binding characteristics of the granulosa cells.