Melatonin: daily cycle in plasma and cerebrospinal fluid of calves.

Melatonin was measured by radioimmunoassay in jugular vein plasma and lateral ventricle cerebrospinal fluid collected from calves at 12 times of the day and night. Melatonin in cerebrospinal fluid increased 17-fold from an average (+/- standard error) of 38 +/- 8 picograms per milliliter during the day to an average of 637 +/- 133 picograms per milliliter during the night (P less than .001). Plasma concentrations of melatonin increased sixfold from an average, per milliliter, of 19 +/- 4 picograms during the day to 121 +/- 24 picograms during the night (P less than .001).

Spontaneous benign prostatic hyperplasia in the beagle. Age-associated changes in serum hormone levels, and the morphology and secretory function of the canine prostate.

This paper is a cross-sectional study of spontaneous benign prostatic hyperplasia (BPH) in a single canine species. The effects of aging and hormonal changes on the growth, histology, and glandular secretory function of the canine prostate were studied in 42 male beagles ranging in age from 8 mo to 9 yr. The beagle prostate enlarges for at least 6 yr, whether normal or hyperplastic. In contrast, prostatic secretory function, determined by ejaculate volume and total ejaculate protein, declines markedly after 4 yr of age. These reciprocal growth and functional changes in the prostate are closely associated with a progressive increase in the incidence of BPH, which is already apparent in some dogs by age two. With age there is a modest decrease in serum androgen levels with no apparent change in serum 17 beta-estradiol levels. This suggests that the growth and functional changes that are associated with the development of BPH and are initiated very early in life reflect an altered sensitivity of the prostate to serum androgens or a response to the relative decrease in the serum androgen to estrogen ratio.

Cloning and characterization of an ovine intracellular seven transmembrane receptor for progesterone that mediates calcium mobilization.

Classically, progesterone has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors (nPR) and subsequent modulation of gene expression. However, there is increasing evidence for rapid, nongenomic effects of progesterone in a variety of tissues in mammals, and it seems likely that a membrane PR (mPR) is causing these events. The objective of this study was to isolate and characterize an ovine mPR distinct from the nPR. A cDNA clone was isolated from ovine genomic DNA by PCR. The ovine mPR is a 350-amino acid protein that, based on computer hydrophobicity analysis, possesses seven transmembrane domains and is distinct from the nPR. Message for the ovine mPR was detected in hypothalamus, pituitary, uterus, ovary, and corpus luteum by RT-PCR. In CHO cells that overexpressed a mPR-green fluorescent protein fusion protein, the ovine mPR was localized to the endoplasmic reticulum and not the plasma membrane. Specific binding of 3H-progesterone to membrane fractions was demonstrated in CHO cells that expressed the ovine mPR but not in nontransfected cells. Furthermore, progesterone and 17 alpha-hydroxy-progesterone stimulated intracellular Ca2+ mobilization in CHO cells that expressed ovine mPR in Ca2+-free medium (P < 0.05) but not in CHO cells transfected with empty vector. This rise in intracellular Ca2+ is believed to be from the endoplasmic reticulum as intracellular Ca2+ mobilization is absent when mPR transfected cells are first treated with thapsigargin to deplete Ca2+ stores from the endoplasmic reticulum. Isolation, identification, tissue distribution, cellular localization, steroid binding, and a functional response for a unique intracellular mPR in the sheep are presented.

Differences in the rates of internalization of 125I-labeled human chorionic gonadotropin, luteinizing hormone, and epidermal growth factor by ovine luteal cells.

The rate of internalization and degradation of radioiodinated hCG, ovine LH (oLH), human LH (hLH), and mouse epidermal growth factor (mEGF) was studied in suspensions of ovine luteal cells. Hormone bound to the plasma membrane was quantified by treating the cells with acidic buffer (pH 3.0), and the radioactivity removed from the receptor was measured. The radioactivity remaining in the cell pellet was considered to have been internalized. The extent of degradation of the radioactive hormones was determined by subjecting the radioactivity that was released into the medium during the incubation periods to precipitation with 20% trichloroacetic acid. The non-precipitable radioactivity was considered to have been degraded. The required time for loss of half of the radioactivity that had been bound to the cell membrane at time zero was 22.8 +/- 2.3 h for hCG, 23.3 +/- 1.1 h for asialo-hCG, 15.1 +/- 1.4 h for hLH, 0.4 +/- 0.2 h for oLH, and 0.3 +/- 0.1 for mEGF. The quantities of radioactivity that had been internalized (intracellular plus degraded) were greater at earlier times (15-120 min) for oLH or mEGF than for hCG, asialo-hCG, or hLH. These data suggest that ligands bound to different receptors on ovine luteal cells are internalized and degraded at different rates by the same cell and that ligands that bind to the same receptor (hCG, hLH, and oLH) can also be internalized and degraded at very different rates.

Internalization and degradation of human chorionic gonadotropin in ovine luteal cells: effects of inhibition of protein synthesis.

Studies were undertaken to investigate the possibility that receptors for LH in monolayer cultures of enzymatically dissociated ovine luteal cells are recycled. Cultured cells (3-10 X 10(5) total steroidogenic cells/dish) were incubated with or without cycloheximide (CHX; 10(-4) M) and 2 X 10(6) cpm [125I]iodo-hCG in the presence or absence of 30 micrograms nonradioactively labeled hCG for 0, 12, 24, 36, or 48 h. At each time point, the amounts of radioactivity bound to the cells (bound [125I]iodo-hCG), located intracellularly (intracellular [125I]iodo-hCG), and degraded and returned to the medium as [125I]monoiodotyrosine (degraded [125I]iodo-hCG) were determined. The number of receptors for LH was determined by Scatchard analysis. Cell viability was also monitored by: 1) trypan blue dye exclusion, 2) the ability of the cells to synthesize protein, and 3) basal and hCG-stimulated secretion of progesterone. More than 90% of the cells remained viable after 48 h of culture, and CHX had no effect on cell viability. Protein synthesis in CHX-treated cells was inhibited by more than 90%. Basal and hCG-stimulated secretion of progesterone were also inhibited by CHX. Treatment with CHX increased the amounts of membrane-bound and internalized [125I]iodo-hCG and decreased the amounts of [125I]iodo-hCG that were degraded. When the quantities of radioactivity in these three fractions (plasma membrane-bound, internalized, and degraded) were added together to obtain a value for the total amount of [125I]iodo-hCG that had been bound to receptor during the 48-h time course (total receptor-associated [125I]iodo-hCG), the value for control cells was not significantly different from the value for CHX-treated cells. Furthermore, the total receptor-associated [125I]iodo-hCG was approximately 2-fold greater than the amount of [125I]iodo-hCG required to saturate receptors at time zero. These data indicate that synthesis of new receptors is not required for the continued binding, internalization, and degradation of [125I]iodo-hCG. Further, the data are compatible with the hypothesis that receptors for LH are recycled or that a portion of the total receptor population is in an unavailable form when the cells are intact but are available for binding after homogenization of the cells.

Radioimmunoassay of serum concentrations of melatonin in sheep exposed to different lighting regimens.

A specific and sensitive double-antibody radioimmunoassay for melatonin (N-acetyl-5-methoxytryptamine) has been developed utilizing rabbit antisera to a bovine serum albumin conjugate of N-succinyl-5-methoxytryptamine and utilizing N-3-(4-hydroxyphenl)-propionyl-5-methoxytryptamine for radioiodination. The least detectable concentration of melatonin standard was 10 pmolar (2.3 pg/tube) with 50% inhibition resultinhibition curves obtained with increasing quantities of melatonin or increasing quantities of chloroform extracts of ovine sera were parallel. The immunoreactivity found in ovine sera c-migrated with [3H]melatonin on silica gel G when developed with chloroform:methanol (9:1). N-Acetylserotonin, 5-methoxytryptamine, serotonin, tryptophan, 6-hydroxymelatonin, 6-methoxytetrahydroharmalan, and several other indole and beta-carboline compounds do not influence the estimation of melatonin in the radioimmunoassay. Concentrations of melatonin could be accurately determined when 31 to 1000 pg were added to 1 ml ovine serum. Serum samples with melatonin concentrations of 1000 pg/ml, 500 pg/ml and 75 pg/ml had intra-assay coefficients of variation of 9.1%, 8.6%, and 17.4%, respectively. The respective inter-assay coefficients of variation were 22.7%, 18.1%, and 37.1%. Ewes exposed to a 12 h light: 12 h dark lighting regimen demonstrated a circadian rhythm in serum concentrations of melatonin. Concentrations ranged from 10-30 pg/ml during periods of light to 100-300 pg/ml during periods of dark. During exposure to continuous light, the circadian rhythm was abolished and concentrations of melatonin were maintained at 10-50 pg/ml. When exposed to conditions of continuous dark the circadian rhythm persisted. A precipitous drop in serum concentrations of melatonin resulted when ewes experiencing peak melatonin concentrations were exposed to light. Concentrations returned to peak levels when the lights were turned off 3.5 h later.

Adenosine 3',5'-monophosphate-binding capacity in small and large ovine luteal cells.

The photoaffinity analog [32P]8-azidoadenosine cAMP ([32P]8-N3cAMP) was used to study available cAMP-binding sites on cAMP-dependent protein kinases in homogenates of ovine small (12-22 microns) and large (greater than 22 microns) luteal cells. Binding of the analogs to specific proteins was detected by autoradiography after sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and was quantitated by liquid scintillation counting. In homogenates of untreated small and large cells, only the type I isoenzyme of the regulatory subunit (R1) of protein kinase had a measurable number of available cAMP-binding sites. Small luteal cells were incubated for 2 h with increasing concentrations of ovine LH (oLH), cholera toxin, or forskolin, and media were collected for quantification of cAMP and progesterone. Cells were harvested and homogenized, and intracellular cAMP content and photoincorporation of [32P]8-N3cAMP into RI were measured. Treatment of small luteal cells with oLH, cholera toxin, and forskolin resulted in dose-dependent increases in cAMP in both the cells and the incubation media and in the progesterone content of the media. These increases were accompanied by a dose-dependent decrease in photoincorporation of [32P]8-N3cAMP into RI of small cells, which was correlated (P less than 0.05) with the increase in progesterone secretion. A time-dependent decrease (P less than 0.05) in photoincorporation in small cells incubated with oLH (100 ng/ml), cholera toxin (1000 ng/ml), or forskolin (5 microM) preceded an increase (P less than 0.05) in progesterone secretion by these cells. Large ovine luteal cells incubated with increasing concentrations of cholera toxin or forskolin demonstrated a dose-dependent decrease in photoincorporation of [32P]8-N3cAMP into RI. The time-dependent decrease (P less than 0.05) in photoincorporation in large cells incubated with cholera toxin (1000 ng/ml) or forskolin (5 microM) was not followed by enhanced progesterone secretion. These observations are consistent with a role for cAMP involvement in progesterone secretion by ovine small luteal cells and suggest a lack of cAMP involvement in progesterone synthesis by large cells.

Internalization of ovine luteinizing hormone/human chorionic gonadotropin recombinants: differential effects of the alpha- and beta-subunits.

The rate of internalization and degradation of radioiodinated hCG and ovine LH (oLH) as well as homologous and heterologous recombinants of their subunits was studied in suspensions of ovine luteal cells. Hormone bound to the plasma membrane was quantified by treating the cells with acidic buffer (pH 3.0) and quantification of the radioactivity that was removed from receptor. The radioactivity remaining in the cell pellet was considered to be intracellular. The extent of degradation of the radioactive hormones was determined by subjecting the radioactivity released into the medium during incubation to precipitation with 20% trichloroacetic acid. The non-precipitable radioactivity was considered to have been degraded. Radioactivity was lost from the cell membrane with a t1/2 of 16.8 +/- 2.5 h for radioiodinated hCG, 22.8 +/- 3.8 h for the alpha-subunit of hCG recombined with radioiodinated beta-subunit of hCG, 8.9 +/- 4.5 h for the alpha-subunit of oLH recombined with the radioiodinated beta-subunit of hCG, 0.5 +/- 0.1 h for the alpha-subunit of hCG recombined with the radioiodinated beta-subunit of oLH, 0.5 +/- 0.1 h for radioiodinated oLH, and 0.7 +/- 0.2 h for the alpha-subunit of oLH recombined with the radioiodinated beta-subunit of oLH. In general, the levels of radioactivity that were intracellular and the rate of degradation of the hormone were inversely related to the quantities of hormone that remained on the plasma membrane. The preparations that contained the beta-subunit of hCG were all internalized at a slower rate (t1/2 = 9-22 h) and consequently were degraded more slowly than any of the preparations that contained the beta-subunit of oLH (t1/2 = 0.5-0.7 h). When the radioiodine in oLH was present in the beta-subunit, intracellular levels of radioactivity were higher and degradation occurred more rapidly than when the radioiodine was in the alpha-subunit. Although the differences were less dramatic, hCG with radioiodine in the beta-subunit also reached higher levels intracellularly, but was degraded to a lesser extent at 16 and 24 h than was hCG with radioiodine in the alpha-subunit. These data demonstrated a dramatic difference (approximately 30-fold) in the rate of loss of oLH vs. that of hCG from the membrane of ovine luteal cells. Further, it appears that the beta-subunit of each of the two hormones is the major factor in determining the rate of internalization of the intact hormone. The differences is how the two hormones are processed at the receptor level may help explain the known differences in their steroidogenic potencies.

Cytochalasins and colchicine increase the lateral mobility of human chorionic gonadotropin-occupied luteinizing hormone receptors on ovine luteal cells.

To determine whether cytoskeletal proteins restrict the mobility of LH-receptor complexes occupied by hCG, we measured the rate of lateral diffusion and the fraction of mobile receptor-hormone complexes by fluorescence photobleaching recovery on luteal cells treated with microtubule and microfilament disruptors and on portions of the plasma membrane separated from the underlying cytoskeleton (blebs). Enzymatically dispersed ovine luteal cells obtained on day 11 from superovulated ewes were pretreated for 1 h at 37 C with cytochalasin B (20 micrograms/ml), cytochalasin D (20 micrograms/ml), or colchicine (40 micrograms/ml). These drugs had no effect on the lateral diffusion of cell membrane glycoproteins labeled with tetramethylrhodamine isothiocyanate-derivatized succinyl Concanavalin-A. However, drug treatment caused a significant increase in the diffusion coefficient and mobile fraction of hCG-occupied LH receptors compared to values obtained on untreated cells. hCG-occupied LH receptors on untreated cells exhibited a mobile fraction of 10.3%, and these mobile complexes had a diffusion coefficient of 3.3 X 10(-11) cm2 sec-1.hCG-occupied LH receptors on cytochalasin B-treated cells had a diffusion coefficient of 9.0 X 10(-11) cm2 sec-1, with 26% fluorescence recovery. Cytochalasin D and colchicine caused significantly greater increases in hCG mobility to 22.0 and 22.5 X 10(-11) cm2 sec-1, respectively, although the mobile fraction remained at about 25-30%. That binding of hCG to the LH receptor restricts lateral mobility through interactions of the hormone-occupied receptor complex with the cytoskeleton is further indicated by results obtained on membrane blebs. The fraction of mobile hCG-occupied LH receptors on blebs was 52.3%, and complexes had a diffusion coefficient of 1.7 X 10(-10) cm2 sec-1. The hormone-induced restriction of LH receptor lateral diffusion had no effect on the lateral mobility of tetramethylrhodamine isothiocyanate-epidermal growth factor-labeled receptors.

Relationship between membrane potential and progesterone release in ovine corpora lutea.

When slices of ovine luteal tissue were perfused with medium containing luteinizing hormone (LH), the output of progesterone was increased significantly (P less than 0.01) in eleven of twelve experiments. However, addition of LH to the medium did not influence the luteal cell membrane potential. The addition of 47 mM potassium to the medium resulted in increased progesterone output (P less than 0.01) and depolarization of the luteal cell membrane within 2 min. Progesterone output decreased to approximate pretreatment levels within 2 min of the return to normal potassium levels in the perfusion medium. High levels of potassium further increased the output of progesterone from tissue stimulated with LH. Perfusion of the slices with sodium-free medium also resulted in increased (P less than 0.01) progesterone output within 2 min, which returned to pretreatment levels within 2 min after normal sodium levels were restored to the medium. Perfusion of the slices with sodium-free medium did not influence the membrane potential. Perfusion of the tissue with LH, 47 mM potassium, or sodium-free medium had no effect on progesterone output if the medium was calcium-free and/or contained 2 mM EGTA. These data suggested that the calcium ion plays an important role in mediating the steroidogenic response of ovine luteal tissue to LH. A second series of experiments was designed to ascertain if luteal cells were coupled electrically. Sixty-six pairs of luteal cells separated by 150-300 mum were penetrated with electrodes and the membrane potential of both cells was studied. One cell of each pair was hyperpolarized by passage of 0.4 nA current into the cell, but in no case was there an effect on the membrane potential of the other penetrated cell. Likewise, when five cells were injected iontophoretically with Procion Yellow there was no evidence of diffusion of the dye to adjacent cells. There was no evidence obtained in this study which suggested that ovine luteal cells were coupled electrically.

Sequential patterns of circulating LH and FSH in female sheep during the early postnatal period: effect of gonadectomy.

To determine the basis for the marked daily variability in circulating luteinizing hormone (LH) in the female lamb shortly after birth and to determine whether circulating follicle-stimulating hormone (FSH) exhibits similar variability during this period of life, serum levels of both gonadotropins were monitored frequently (20-min intervals) over a 3- or 6-hr period each week for the first 9 weeks of life. Similar measurements of circulating LH and FSH were made in lambs ovariectomized at 2 weeks of age to assess whether the ovary influences the secretion of these gonadotropins. Pulsatile release of LH, but not FSH, was observed in both groups of lambs. Although mean concentrations of circulating FSH in intact female lambs were similar to basal levels of the cyclic adult, mean concentrations of serum LH were much higher. Because of the discontinuous release of LH in intact lambs after 4-5 weeks of age, patterns of LH were often indistinguishable from those of the long-term ovariectomized adult. At 9 weeks of age the frequency of episodic LH release in intact lambs ranged from 0.33-1.1 pulses/hr. Castration at 2 weeks of age produced a concomitant, but delayed, rise in mean serum LH and FSH beginning 4-5 weeks later. By 9 weeks of age serum LH, but not FSH, had attained levels comparable to those of the castrated adult, and the mean frequency of LH release (about hourly) was similar for both groups. The results indicate that by 9 weeks of age the ovary of the lamb influences the secretion of LH and FSH. Because in the 9-week-old intact female lamb episodic release of LH occurs producing high (castrate) levels of this gonadotropin while circulating concentrations of FSH remain stable and within basal levels of the adult cycling female, it is suggested that acute concomitant release of LH and FSH does not occur at this age and that separate negative feedback loops for the control of LH and FSH secretion may exist during early postnatal life.

Hormone-independent activation of adenylate cyclase in large steroidogenic ovine luteal cells does not result in increased progesterone secretion.

The role of cAMP in controlling steroidogenesis in small and large ovine luteal cells was examined. Corpora lutea collected from superovulated ewes (9-11 days postovulation) were dissociated, and the two cell types were purified by elutriation. Both cell types were incubated for 0.5, 1, 2, and 4 h at 37 C with ovine LH (100 ng/ml), cholera toxin (100 ng/ml), or forskolin (50 microM). At each time point, progesterone levels were measured in the medium. Adenylate cyclase activity and cAMP concentrations in the cells and incubation medium were also determined. Progesterone secretion by small cells was significantly stimulated by ovine LH (up to 7.3-fold), cholera toxin (up to 4.2-fold), and forskolin (up to 4.5-fold) during the 4-h incubation. Intracellular levels of cAMP were significantly elevated in the small cells by ovine LH (up to 2.5-fold) and forskolin (up to 5.6-fold). Accumulation of cAMP in medium after incubation of small cells was also significantly stimulated by ovine LH (up to 215-fold), cholera toxin (up to 93-fold), and forskolin (up to 1105-fold). Adenylate cyclase activity, however, was only significantly stimulated by cholera toxin (2.6-fold) and forskolin (3.8-fold). None of the treatments stimulated progesterone secretion by large cells at any time (less than 1.6-fold). Intracellular levels of cAMP in the large cells were not elevated after treatment with ovine LH, but were elevated in cells treated with cholera toxin (up to 2.8-fold) and forskolin (up to 2.6-fold). Accumulation of cAMP in the medium was markedly increased with forskolin treatment (106-fold). Adenylate cyclase activity was found to be significantly stimulated by cholera toxin (2.2-fold) and forskolin (up to 5.1-fold), but not by ovine LH (less than 1.1-fold). Steroid secretion in the small cells appears to be enhanced by elevated intracellular cAMP levels. However, treatments that result in dramatic increases in intracellular levels of cAMP failed to influence the secretion of progesterone in large cells.

Hormonal regulation of messenger ribonucleic acid encoding steroidogenic acute regulatory protein in ovine corpora lutea.

Steroidogenic acute regulatory protein (StAR), proposed to be involved in the transport of cholesterol to the inner mitochondrial membrane, has recently been cloned from MA-10 cells. Using reverse transcription-polymerase chain reaction, we generated a complementary DNA encoding 404 base pairs of StAR from ovine luteal tissue to perform studies regarding regulation of the messenger RNA (mRNA) encoding this protein. In Exp 1, ewes were hypophysectomized (HPX) on day 5 of the estrous cycle and administered saline or physiological regimens of LH and/or GH until collection of luteal tissue on day 12 of the estrous cycle (n = 4/group). Luteal concentrations [mean +/- SEM; femtomoles per microgram poly(A)+ RNA] of mRNA encoding StAR were lower (P < 0.05) in the HPX plus saline-treated ewes (26.4 +/- 7.3) than in day 12 pituitary-intact ewes (n = 4; 77.7 +/- 9.3). Replacement of LH (59.1 +/- 13.1), GH (59.1 +/- 12.8), or LH and GH (69.9 +/- 4.5) in HPX ewes increased (P < 0.05) concentrations of mRNA encoding StAR to values not different from those in day 12 controls. In Exp 2, ewes on day 11 or 12 of the estrous cycle were injected with prostaglandin F2 alpha (PGF2 alpha) to induce luteal regression. Corpora lutea were collected 4, 12, or 24 h after injection (n = 4-5/time point) and from untreated control ewes (n = 4) or 24 h after injection of saline (n = 4). Treatment with PGF2 alpha decreased (P < 0.05) concentrations of progesterone in serum 4, 12, and 24 h after injection. Concentrations of StAR mRNA were decreased (P < 0.01) to 47%, 19%, and 8% of control values 4, 12, and 24 h after PGF2 alpha injection, respectively. In Exp 3, ewes received ovarian arterial infusions of saline, PGF2 alpha, or phorbol 12-myristate 13-acetate (PMA), and luteal tissue was collected 0 (no infusion), 4, 12, or 24 h later (n = 3-4/group). Treatment with PGF2 alpha or PMA decreased (P < 0.05) concentrations of progesterone in serum 4, 12, and 24 h postinjection. Steady state concentrations of mRNA encoding StAR (P < 0.05) were 36% and 25% of the control value 12 and 24 h after PGF2 alpha injection. Injection of PMA decreased (P < 0.05) concentrations of StAR mRNA to 75% and 50% of control values at 4 and 12 h, but concentrations of mRNA encoding StAR were not different from control values at 24 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Secretory granules and progesterone secretion by ovine corpora lutea in vitro.

To study the relationship between formation and release of Golgi-derived secretory granules and progesterone secretion, slices of ovine luteal tissue were incubated in the presence of LH and/or calcium ionophore A23187. Increases in progesterone secretion in response to LH and/or ionophore were accompanied by a concomitant release of secretory granules. In contrast, in the presence of colchicine, LH-stimulated progesterone secretion was significantly reduced (P less than 0.01), granule formation appeared to be blocked, and there was little evidence of exocytosis. In addition, unusual pleomorphic membrane-bounded saccules containing an electron-dense material were abundant throughout the centrospheric region of cells treated with colchicine. Because of the close parallelism between formation and release of Golgi-derived secretory granules and progesterone secretion, it appears that progesterone secretion may be coupled to exocytosis of secretory granules. Although the exact content and function of the secretory granules described remains to be elucidated, the data obtained are compatible with the notion that they may contain a progesterone carrier protein.

Sequential patterns of circulating luteinizing hormone and follicle-stimulating hormone in female sheep from early postnatal life through the first estrous cycles.

Patterns of circulating LH and FSH were examined during several 6-h periods in developing female sheep from 3 weeks of age through the first estrous cycles (30--40 weeks of age) and early pregnancy. With the onset of pulsatile LH secretion, beginning 11 weeks after birth, circulating LH increased to levels 2- to 5-fold greater than those observed in the adult. Thereafter, high and variable levels of circulating LH persisted through young adulthood when ovarian cyclicity including ovulation became manifest. Although prior to the initial estrus changes in frequency of LH release (number of LH discharges/6-h period) appeared to occur randomly from week to week, after first estrus, changes in frequency became predictable. During the early (day 1) and late (day 15) luteal phases of the first estrous cycles, when circulating progesterone was low (less than 0.6 ng/ml), the frequency of LH release was increased (5--7 discharges/6 h) while during the mid-luteal phase (day 7--12), when circulating progesterone was high (2--4 ng/ml), the frequency was diminished (0--2 discharges/6 h). Massive and sustained discharges of LH which resembled preovulatory surges were observed only shortly before first estrus. In contrast to the pulsatile release pattern of LH, concentrations of circulating FSH within the 6-h periods were relatively constant and within the range found in the adult. These findings lead to the following conclusions: a) changes in negative feedback control are not directly responsible for the onset of ovarian cyclicity in the sheep as indicated by the lack of differences in mean concentrations of circulating LH and FSH before and after first ovulation; b) the onset of the surge mode of gonadotropin secretion occurs only shortly before first ovulation; and c) progesterone may play an inhibitory role in regulating tonic LH secretion during the first estrous cycles.

Differences in the lateral mobility of receptors for luteinizing hormone (LH) in the luteal cell plasma membrane when occupied by ovine LH versus human chorionic gonadotropin.

Receptors for LH are internalized by ovine luteal cells 50 times slower when occupied by hCG than when occupied by ovine LH (oLH). To determine if differences in the rate of internalization were due to differences in the lateral mobility of the hormone-receptor complexes in the cell membrane, the diffusion coefficients of oLH- and hCG-LH receptor complexes were measured using fluorescence photobleaching recovery methods. Tetramethylrhodamine isothiocyanate (TRITC)-labeled oLH and hCG, which retained full ability to bind to receptor, were bound to LH receptors on enzymatically dispersed ovine luteal cells. Molecules labeled with TRITC within a 3-micron 2 region of the cell surface were bleached by a 500-msec pulse of 3 mW laser light at a wavelength of 514.5 nm. The laser beam intensity was then attenuated 20,000-fold, and fluorescence from the bleached area was measured by single photon counting as unbleached fluorescent hormone-receptor complexes diffused into the region. Data were analyzed on-line by a NOVA 3/12 computer. The oLH-LH receptor complex had a diffusion coefficient of 1.9 +/- 1.0 X 10(-10) cm2/sec-1, a value comparable to that of cell surface proteins nonspecifically labeled with succinylated Concanavalin A. Fluorescence recovery after photobleaching was 35%. In contrast, hCG-LH receptor complexes were immobile on the time scale of the experiment, implying that the diffusion coefficient was substantially less than 1 X 10(-11) cm2/sec-1. Deglycosylated hCG-TRITC bound to LH receptor had a diffusion coefficient (1.1 +/- 0.1 X 10(-10) cm2/sec-1) similar to that of receptors occupied by oLH. Thus, it appears that the carbohydrate portion of the hCG molecule plays a role in decreasing the mobility of the receptor for LH. These data demonstrate that the rate of lateral movement of the LH receptor in the plasma membrane of luteal cells appears to be modulated by the nature of the bound hormone.

Mammary blood flow and endocrine changes during parturition in the ewe.

The temporal relationship between changes in mammary blood flow (MBF) and changes in the concentration of plasma prolactin, progesterone, estradiol-17beta, and cortisol, was examined in chronic sheep preparations undergoing spontaneous labor (Group I) or labor induced by an infusion of dexamethasone (1 mg/24 h) to the fetus (Group II). In Group I, an increase in prolactin (45 to 489 ng/ml), a decrease in progesterone (15 to 4 ng/ml), and an increase in MBF (97 to 365 ml/min) occurred at about the same time, whereas increases in estradiol-17beta (80 to 211 pg/ml) and cortisol (9 to 39 ng/ml) followed the change in MBF. A similar pattern of changes in MBF and hormonal concentrations occurred over a shorter period when premature labor was induced in the animals in Group II. These findings suggest that changes in plasma prolactin and progesterone concentrations play an important role in the regulation of MBF at the time of parturition.

Steroidogenic acute regulatory protein and peripheral-type benzodiazepine receptor associate at the mitochondrial membrane.

Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) have both been implicated in the transport of cholesterol across mitochondrial membranes in steroidogenic cells. Therefore, we hypothesized that StAR and PBR were associated in this process. To test this hypothesis, we measured fluorescence energy transfer (FET) between these proteins by fusing enhanced green fluorescent protein (GFP, donor fluorophore) and yellow fluorescent protein (YFP, acceptor fluorophore) to the C-terminus of ovine StAR (37GFP) and ovine PBR (PBRYFP), respectively. These intrinsically fluorescent proteins were stably transfected into Cos-7 cells and determined to be biologically active. For FET to occur the appropriate fluorescent molecules need to be <100 A from each other. We observed 22.0 +/- 0.9% energy transfer efficiency for 37GFP and PBRYFP, a 4.9 fold increase above non-specific energy transfer between free GFP and PBRYFP (p <.0001). Thus, it appears that StAR and PBR are closely associated in mitochondrial membranes and that these molecules may interact in the transportation of cholesterol.

Validation of a heterologous radioimmunoassay for insulin-like growth factor-I in bovine serum.

A quantitative, repeatable, heterologous radioimmunoassay (RIA) for insulin-like growth factor-I (IGF-I) was developed for bovine serum. Untreated serum could not be assayed due to interference by IGF-I-binding protein. Serum acidified to pH 3.6 in glycine-HCl (0.1 mol/l) for 24 h at 37 degrees C and neutralized with NaOH produced inhibition curves non-parallel to the [Thr59]-IGF-I standard. Neutralization by 40-fold dilution of acidified serum samples with assay buffer produced inhibition curves nearly parallel to the IGF-I standard. Complete parallelism was achieved by utilizing preprecipitated normal rabbit serum-sheep anti-rabbit gamma-globulin to separate antibody-bound 125I-labelled IGF-I from free 125I-labelled IGF-I. Recovery of IGF-I (1.3-52.3 fmol) added to serum was quantitative. The sensitivity of the RIA (n = 6) was 8.25 +/- 0.17 (S.E.M.) fmol. Intra- and interassay coefficients of variation were 3.03 and 4.95% respectively. Serum IGF-I levels measured in beef calves at weaning were positively correlated with weaning weight, total weight gain from birth to weaning and average daily weight gain. In conclusion, a heterologous RIA for IGF-I in beef serum which is sensitive, accurate, precise and repeatable has been developed.

Response of the corpus luteum to luteinizing hormone.

The response of steroidogenic tissues to tropic hormones is regulated in part by specific receptors in the target cells for the stimulatory hormone. As a result of hormone binding to receptor the enzyme adenylate cyclase is activated with a resultant increase in intracellular levels of cAMP. Enhanced protein kinase activity then leads to increased steroidogenesis via several possible mechanisms, including direct activation of components of steroidogenic enzyme systems via phosphorylation. The initial effects of tropic hormones such as LH are dependent upon the number of receptors present on the surface of the target cell. Numerous factors influence the number of LH receptors in the corpus luteum. A model is presented for the mechanisms involved in the loss and renewal of LH receptors in the luteal cell. The life of the LH receptor on luteal cells appears to be a single binding of hormone. The hormone-receptor complex is then internalized by endocytosis and the hormone is degraded in lysosomes. After internalization the receptor is also degraded in lysosomes or recycled via the Golgi apparatus. New or recycled receptors for LH are incorporated into the limiting membrane of protein containing secretory granules. One of the actions of LH is enhancement of the exocytosis of these secretory granules with incorporation of the limiting membrane (and the LH receptors?) of the granule into the plasma membrane of the cell. These proposed mechanisms explain the increase in the number of receptors for LH seen immediately after stimulation of the luteal cell with massive doses of LH and also explain the "down-regulation" of LH receptors 24 hr after administration of LH.