Search PubMed⌕ Search

Biomedical subjects

R L Stouffer

Publications and source records attributed to R L Stouffer.

At least 73 records · Page 4Linked to original sources
PubMed1994

Radioligand binding assay of progesterone receptors in the primate corpus luteum after in vivo treatment with the 3 beta-hydroxysteroid dehydrogenase inhibitor, trilostane.

We and others have detected progesterone receptors (PR) in the primate (macaque and human) corpus luteum by immunocytochemistry. However, we have been unable to measure PR in the corpus luteum by conventional ligand binding assay, presumably because high endogenous concentrations of progesterone (P) in luteal tissue prevented specific binding of radiolabeled hormone to PR during the assays. To test this hypothesis, we treated monkeys with the 3 beta-hydroxysteroid dehydrogenase inhibitor, Trilostane, to reduce levels of endogenous P before conducting binding assays for PR in luteal tissue. To obtain adequate tissue for saturation analysis, rhesus monkeys (n = 6) were superovulated by treating them with hFSH beginning at menses (day 1) for 6 days, then with hFSH and hLH (days 6-9), followed by hCG (day 10). Trilostane (600 mg) was given 5 days after hCG treatment (day 15), and binding assays were conducted 18 h later. Trilostane significantly reduced mean (+/- SE) serum levels of P from 97.8 +/- 16 to 2.7 +/- 1.3 nmol/L within 18 h (P < 0.001). Strong nuclear staining of PR was detected by immunocytochemistry in both Trilostane-treated and control (not Trilostane-treated) tissue, but ligand binding was measurable only in Trilostane-treated monkeys. Scatchard transformations of saturation curves revealed high affinity binding of [3H] R5020 in luteal cytosol and nuclear extracts with an approximate dissociation constant (Kd) of 4.8 and 1.37 nmol/L, respectively. Also, the PR-specific monoclonal antibody, JZB-39, shifted a [3H]R5020-bound luteal macromolecule on sucrose gradients. Mean cytosolic and nuclear binding of [3H]R5020 were 0.31 +/- 0.09 and 0.06 +/- 0.02 fmol/micrograms DNA, respectively. Similar binding of [3H]R5020 was demonstrated in corpora lutea obtained during spontaneous menstrual cycles after Trilostane treatment (n = 3). These results show unequivocally that the PR in macaque luteal tissue can bind P with high affinity and suggest a receptor-mediated action of P in the primate corpus luteum.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Acute administration of a 3 beta-hydroxysteroid dehydrogenase inhibitor to rhesus monkeys at the midluteal phase of the menstrual cycle: evidence for possible autocrine regulation of the primate corpus luteum by progesterone.

Colocalization of progesterone receptors and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), a key enzyme in progesterone biosynthesis, in macaque luteal cells suggest that progesterone has an autocrine role in the regulation of primate luteal function. To test this hypothesis, we administered trilostane, a 3 beta HSD inhibitor, to rhesus macaques at the midluteal phase of spontaneous menstrual cycles to rapidly and reversibly reduce progesterone production. Animals received trilostane (600 mg/dose; treated group; n = 5) or vehicle (control group; n = 4) orally on days 6-7 of the luteal phase. Trilostane significantly (P < 0.05) elevated pregnenolone levels within 1 h of treatment compared to those in vehicle-treated animals; after 1 day of treatment, the mean pregnenolone level (173 nmol/L) was 86-fold greater than the control value. Pregnenolone levels dropped after cessation of drug administration and became indistinguishable from control levels by day 13. Trilostane significantly reduced serum progesterone levels within 3 h of initial administration (P < 0.01), and levels remained near baseline (1.0 nmol/L) throughout the 2 days of treatment. Progesterone levels also remained low after cessation of trilostane treatment in four of five monkeys, and trilostane-treated animals experienced a shorter luteal phase than vehicle-treated animals (7.8 +/- 0.2 vs. 16 +/- 1 days; P < 0.01). Histological analysis (n = 3/group) revealed indexes of premature structural luteolysis by 4 days after the onset of trilostane administration. Exposure to trilostane had no effect on the percentage of luteal cells expressing progesterone receptors, as determined by immunocytochemistry. Serum LH levels were not different between treatment and control groups throughout the experimental period. As trilostane dramatically reduced serum progesterone and induced premature menses without major concurrent alteration in serum cortisol, we conclude that trilostane ios an effective, rapidly acting inhibitor of 3 beta HSD in the macaque corpus luteum during the midluteal phase of the menstrual cycle. Progesterone production did not typically resume after cessation of trilostane treatment despite continuing gonadotropin support luteolysis. Thus, progesterone or a related metabolite may be required to maintain the function and structural integrity of the primate corpus luteum during the normal menstrual cycle.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Steroid reduction during ovarian stimulation impairs oocyte fertilization, but not folliculogenesis, in rhesus monkeys.

OBJECTIVE: To test the hypothesis that steroids locally modulate and may be required for normal follicular function and gametogenesis in primates, the effects of steroid reduction during gonadotropin-stimulated folliculogenesis was studied in rhesus monkeys. DESIGN: Animals received human FSH (hFSH; days 1 to 6) and hFSH+human LH (hLH; day 7) to promote multiple follicular growth, and then received hCG (day 8) for ovulatory maturation. Four animals received trilostane (3 beta-hydroxysteroid dehydrogenase inhibitor) on days 1 to 8 or no inhibitor (controls; n = 4). Follicles were aspirated 34 hours after hCG. MAIN OUTCOME MEASURES: Follicular growth, serum E2, P, and pregnenolone, oocyte nuclear maturity, and IVF. RESULTS: Trilostane markedly reduced E2 to levels as low as 7% of controls throughout the follicular phase. Pregnenolone was 66-fold greater during trilostane treatment relative to controls. In both groups, P was at baseline during follicular stimulation but was reduced for 72 hours after hCG in trilostane-treated animals. Despite E2 suppression, follicular growth and oocyte nuclear maturity were unaltered by trilostane. Trilostane hindered the fertilizability of metaphase II oocytes (15%) in three of four animals compared with controls (65%). Metaphase I oocytes that required > 8 hours to complete meiosis in vitro failed to fertilize in the same three of four receiving trilostane relative to controls (31%). CONCLUSIONS: Follicular growth and oocyte meiosis did not require high or increasing E2 levels. Levels of follicular products other than E2 may be of value in determining the progress of ovarian stimulation protocols. However, the acquisition of oocyte competence for fertilization may require steroids.

3-Hydroxysteroid Dehydrogenases↗
PubMed1993

Gonadotrophic and local control of the developing corpus luteum in rhesus monkeys.

The actions of the mid-cycle gonadotrophin surge to convert the pre-ovulatory follicle into the corpus luteum are not well understood in primate species. In experiments titrating the surge duration required in macaque monkeys during artificial in-vitro fertilization (IVF)-related cycles, short (< or = 14 h) LH surges similar to those in rodents and domestic animals failed to initiate peri-ovulatory events. Attenuated (24 h) surges, one-half the duration of spontaneous surges in primates, reinitiated oocyte meiosis and initial luteinization of granulosa cells. However, only surges of > or = 48 h sustained luteal development and function to produce luteal phases of approximately 13 days. Three approaches-immunocytochemistry of steroid receptors, binding of radiolabelled steroid, and reverse transcription-polymerase chain reaction of mRNA-indicate that progesterone receptor (PR), but not oestrogen receptor, is expressed in the macaque corpus luteum. Studies on cells collected before and after the gonadotrophin surge identified a novel action of LH to induce PR expression in luteinizing granulosa cells. Thus, the LH surge requirements vary between non-primate and primate species, as well as between peri-ovulatory events, and the LH surge may promote cellular recognition of paracrine and autocrine factors (e.g. progesterone) that become predominant in the developing corpus luteum.

Animals↗
PubMed1993

Comparison of functional response of rat, macaque, and human ovarian cells in hormonally defined medium.

A serum-free medium has been developed which supports in vitro function by ovarian cells derived from rat, monkey, and human tissue. This granulosa cell medium (GCM) consists of Dulbecco's Modified Eagle's Medium: Ham's F-12 medium (1:1, v:v) supplemented with insulin, transferrin, aprotinin, selenium, fibronectin, penicillin, and streptomycin. Ovarian cells from three species were compared: rat, macaque, and human. Four types of ovarian cultures were examined: 1) purified granulosa cell cultures and 2) co-cultures containing granulosa-theca-stroma cells, 3) luteal cells, and 4) granulosa-lutein (harvested from in vitro fertilization cultures) cells. Each cell type was characterized by its response to FSH or hCG when cultured in GCM. Morphologic responses to FSH were observed in GCM in rat granulosa and granulosa-theca-stroma cell cultures, macaque and human granulosa-lutein cells, and human granulosa-theca-stroma cell cultures. The FSH-stimulated cells retracted and became rounded, leaving long intercellular connections. Luteal cells did not retract in response to FSH, and the cells remained firmly attached to the fibronectin matrix. Steroidogenic regulation of the GCM-cultured ovarian cells was monitored following stimulation of the cultures with FSH. The ability of the cells to aromatize testosterone was first examined. Rat granulosa cell cultures and granulosa-theca-stroma cell cultures, macaque granulosa-lutein cell cultures, and human granulosa-theca-stroma cell cultures all accumulated estradiol when given FSH and testosterone for 48 h. Moreover, these cell types as well as human luteal cells were able to metabolize 25-hydroxy [1,2-3H]cholesterol to various steroid metabolites. The data indicate that GCM supports normal granulosa cell morphologic response to FSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Isolation of ovine luteal cell subpopulations by flow cytometry.

Differences in the characteristics of small and large luteal cells, as reported by various laboratories, may be due to species diversity and/or methodological differences in cell preparation. To evaluate whether the method of cell separation affects the properties of luteal cell subpopulations, we sorted and characterized sheep luteal cells by flow cytometry via methods previously used to investigate luteal cell subtypes from the macaque corpus luteum. Corpora lutea were obtained from superovulated ewes on Day 10 after hCG injection and enzymatically dissociated. Dispersed cells were shipped overnight on ice from the University of Arizona to the Oregon Regional Primate Research Center. Viability of cells upon arrival was > or = 80%. When dispersed cells were analyzed by flow cytometry based on forward and 90 degrees light scatter, three distinct subpopulations (P1, P2, P3) were identified. In P1, 35.5 +/- 2.1% of cells, most (97.0 +/- 0.6%; n = 3) of which were 15-22 microns in diameter, stained positive (+) for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. The remainder of P1 cells were 3 beta-HSD negative and < or = 22 microns. The size distribution of P2 was similar to that of P1, but P2 contained more (53.3 +/- 4.2%; n = 4) 3 beta-HSD (+) cells. P3 consisted mostly (88.5 +/- 4.6%; n = 3) of 3 beta-HSD (+) cells > 25 microns in diameter. Cell subpopulations were incubated (n = 6) at 37 degrees C for 3 h with or without hCG (0.1-100 ng/ml), prostaglandin E2 (PGE2; 500 ng/ml), or dibutyryl (db)-cAMP (5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Native and modified (acetylated) low density lipoprotein-supported steroidogenesis by macaque granulosa cells collected before and after the ovulatory stimulus: correlation with fluorescent lipoprotein uptake.

We recently reported that uptake of fluorescent-tagged low density lipoprotein (DiI-LDL) by macaque granulosa cells (GC) was greatly enhanced within 27 h of an ovulatory stimulus (hCG injection). The present study was designed to determine whether increased DiI-LDL uptake correlated with an increased capacity for LDL-supported steroidogenesis. We also tested whether modified [acetylated (ac)] LDL or high density lipoprotein (HDL), which are not ligands for the LDL receptor, supported progesterone (P) production. Beginning at menses, adult female rhesus macaques were treated with human (h) FSH and hLH for 9 days to promote development of multiple follicles. On day 10, monkeys were injected with hCG (1000 IU) or received no ovulatory stimulus. Large follicles were aspirated on day 10 (nonluteinized GC) or 27-34 h after hCG administration (luteinizing GC). GC (2 x 10(4)/0.2 ml) were cultured in Dulbecco's Modified Eagle's Medium-Ham's F-12 plus insulin, transferrin, H2SeO3, and aprotinin, with 0-100 micrograms hLDL, ac-hLDL, or hHDL. P concentrations in medium were determined by RIA. LDL (1-25 micrograms/ml) dose-dependently increased (up to 15-fold; P < 0.05) basal and hCG-stimulated P production by luteinized GC on days 1-8 of culture. However, LDL (25 micrograms/ml) did not alter basal P production by nonluteinized GC and increased (2-fold; P < 0.05) hCG-stimulated P secretion only on days 4-8. Basal and hCG-stimulated P production by luteinized GC were initially (days 1-2) increased (up to 2-fold; P < 0.05), but later (days 6-8) suppressed (P < 0.05) in a dose-dependent manner by 1-100 micrograms ac-LDL/ml. Ac-LDL did not alter basal or hCG-stimulated P production by nonluteinized GC. HDL (1-100 micrograms/ml) did not alter P production by either luteinized or nonluteinized GC. The number of viable luteinized GC on day 8 was reduced (30-50%; P < 0.05) after exposure to 10 micrograms ac-LDL/ml or more, whereas only the highest dose (100 micrograms/ml) of LDL or HDL reduced cell survival. Ac-LDL did not alter the survival of nonluteinized GC in culture. Flow cytometric analyses using fluorescent-tagged lipoproteins (DiI-LDL/DiI-ac-LDL) demonstrated the uptake of both native and ac-LDL by luteinized GC. Uptake of DiI-LDL was competitively suppressed in a dose-dependent manner by unlabeled LDL, but not by ac-LDL. In contrast, DiI-ac-LDL uptake was competitively inhibited by both ac-LDL and LDL.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Insulin-like growth factor binding proteins in sera of pregnant nonhuman primates.

Insulin-like growth factors (IGFs) are mitogenic peptides that are important for fetal and maternal tissue growth during pregnancy. They circulate complexed primarily with a serum binding protein, IGFBP-3, which regulates the availability of the IGFs to their target tissues. We previously reported that in pregnant women, serum IGFBP-3 levels, assessed by Western ligand blotting, decline markedly beginning at 6 weeks gestation due to a circulating protease that cleaves IGFBP-3 into a 29-kilodalton (kDa) protein and lower mol wt (M(r)) fragments. In the current study, we compared IGFBP profiles, IGFBP-3 and IGFBP-1 levels, and IGFBP protease activities in sera from pregnant and nonpregnant women, baboons, and rhesus monkeys, using Western ligand blotting, IGFBP-specific immunoassays, IGFBP-3 protease assay, and zymographic gel electrophoresis. Serum IGFBP profiles in nonpregnant human and nonhuman primates were similar and were not cycle-dependent. IGFBP-3 (37-43 kDa), IGFBP-2 (31 kDa), and IGFBP-1 (28 kDa) were identified in all three species using IGFBP-specific human antisera. A 24-kDa IGFBP was also present and is believed to be IGFBP-4. Serum IGFBP-1 levels increased throughout gestation in human and nonhuman primates. Serum IGFBP-2 and putative IGFBP-4 were barely detectable in all three species from midgestation to term, but increased several days postpartum. In contrast, serum IGFBP-3 profiles differed markedly between species during gestation. Rather than the decrease seen in human pregnancy serum, there was an increase in circulating IGFBP-3 levels in nonhuman primates. Furthermore, for both baboon and rhesus monkey, the M(r) of serum IGFBP-3 was about 2 kDa greater in pregnant than in nonpregnant animals, and deglycosylation studies suggested that the higher M(r) forms may be alternatively glycosylated or may have a unique primary structure. As in nonpregnant women, serum IGFBP-3 protease activity was absent in nonpregnant and pregnant baboons. However, rhesus monkey serum contained a calcium-dependent protease that cleaved recombinant human IGFBP-3 into unique fragments, compared to the human pregnancy enzyme. Unlike human pregnancy serum, which proteolyzes IGFBP-3, in human nonpregnancy serum, rhesus serum incubated under similar conditions did not result in proteolysis of rhesus IGFBP-3, suggesting that the IGFBP-3 protease in human pregnancy serum is not present in the circulation of the rhesus monkey. To assess proteolytic activity in these sera, zymographic polyacrylamide gel analysis, using gelatin as a substrate, was performed. A minor band of proteolytic activity (72 kDa) was observed in all three species throughout gestation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Administration of an aromatase inhibitor during the late follicular phase of gonadotropin-treated cycles in rhesus monkeys: effects on follicle development, oocyte maturation, and subsequent luteal function.

Local modulation of follicular and gametogenic functions by ovarian androgens and estrogens in mammalian species has been proposed. This study examined the effects of elevated androgen/estrogen ratios during follicular maturation in vivo by inhibiting aromatase activity in rhesus monkeys. To obviate steroid feedback effects, gonadotropin-treated animals were used. Beginning at menses (day 1), animals received human (h) FSH (60 IU/day, im) on days 1-6, followed by hFSH plus hLH (60 IU/day, im) on days 7-9 to promote the growth of multiple follicles. Ovulatory maturation was induced by hCG (1000 IU, im) on day 10. On days 8-10, four animals received an aromatase inhibitor, 1,4,6-androstatrien-3,17-dione (ATD; 1-1.25 g, orally, twice/day), while five served as controls and received no further treatment. Within 8 h of ATD treatment, a 63% reduction in serum estradiol levels relative to control values was evident, which reached maximal suppression (84%) by day 10. A marked elevation (17-fold) in serum androstenedione and a lesser increase (2.6-fold) in serum testosterone occurred with aromatase inhibition, yielding androstenedione/estradiol (18.0) and testosterone/estradiol (1.9) ratios greater than those in controls (0.6 and 0.3, respectively). ATD treatment did not alter follicular diameters or the total number of follicles per animal (20 +/- 3) relative to control values (16 +/- 3). Of the total cohort classified, the proportion of oocytes collected at prophase I was greater (P < 0.05) after ATD treatment (31%) than in controls (11%). Completion of oocyte meiosis to metaphase II was retarded (P < 0.05) in ATD-treated (4%) compared to control (26%) animals. Furthermore, the in vitro fertilization rate of metaphase II oocytes from ATD-treated animals (9%) was reduced (P < 0.05) relative to that in controls (25%). While basal progesterone production by luteinizing granulosa cells in vitro was similar between groups, the addition of hCG in vitro enhanced progesterone secretion by cells from ATD-treated animals (3.1 +/- 0.3-fold over basal) to a greater extent (P = 0.05) than in controls (1.5 +/- 0.3-fold). Progesterone receptor was detected by immunocytochemistry in nuclei of luteinizing granulosa cells from ATD-treated animals as well as controls. Serum progesterone profiles and the length of the luteal phase were similar between groups. Thus, acute elevation of serum androgen/estrogen ratios in vivo during follicular maturation was detrimental to the gametogenic functions of the primate follicle, but did not alter follicular growth, events of early luteinization, or subsequent luteal function.(ABSTRACT TRUNCATED AT 400 WORDS)

Androstatrienes↗
PubMed1993

Human recombinant activin-A alters pituitary luteinizing hormone and follicle-stimulating hormone secretion, follicular development, and steroidogenesis, during the menstrual cycle in rhesus monkeys.

Activin, a stimulator of pituitary FSH secretion in nonprimate species, may also act in the ovary to modulate follicular development. To examine whether activin has similar actions in primates, female rhesus monkeys (n = 3/treatment) exhibiting regular menstrual cycles received sc injections of either vehicle or 60 micrograms/kg recombinant human activin-A at 0800 and 1600 h for 1 (acute) or 7 (chronic) days beginning in the early follicular phase. The vehicle-treated monkeys displayed menstrual cycles of normal length, with the follicular (11.3 +/- 1.3 days, mean +/- SE) and luteal (16.6 +/- 1.8 days) phases demarcated by midcycle peaks in serum estradiol (E) and bioactive LH. After the first activin injection, levels of human activin A peaked at 90 ng/mL within 1 h and returned to baseline before the second injection 8 h later. Although serum E and FSH levels did not change, LH increased (273%, P < 0.05) within 8 h. Acute activin treatment increased (P < 0.05) serum E within 24 h to levels (1290 +/- 330 pmol/L) typically observed at midcycle. With chronic treatment, serum E peaked on day 2 (2580 +/- 338 pmol/L; P < 0.05), then declined and rose to a second peak (1680 +/- 279 pmol/L) on day 5. During chronic activin treatment, LH levels peaked on day 2 (603 +/- 270 ng/mL; P < 0.05 compared to day 0, 15 +/- 7 ng/mL) whereas FSH increased progressively until day 5 (937 +/- 320 ng/mL; P < 0.05 compared to day 0, 169 +/- 59 ng/mL). After acute or chronic activin, the expected midcycle rises in serum E and gonadotropins were delayed to greater than or equal to day 20 (n = 4) or did not occur before menses (n = 2). Although an enlarged ovary with one greater than or equal to 4-mm follicle was observed by laparoscopy during the late follicular phase in vehicle-treated monkeys, medium-to-large follicles were not visible on ovaries during chronic activin treatment or later at the expected midcycle interval in activin-treated monkeys. Similar hormonal and ovarian events were obtained after activin treatment of amenorrheic monkeys having serum FSH, LH, and E levels that were comparable to those at menses in spontaneous menstrual cycles. Thus, exogenous activin stimulates pituitary LH and FSH secretion and ovarian estrogen secretion during the early follicular phase in intact monkeys. However, acute or chronic activin treatment did not promote complete follicular development and disrupted subsequent events in the menstrual cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Activins↗
PubMed1992

Inhibin production by macaque granulosa cells from pre- and periovulatory follicles: regulation by gonadotropins and prostaglandin E2.

Although inhibin (IN) is secreted by granulosa cells (GC) of preovulatory follicles, the major source of immunoreactive IN circulating during the primate ovarian cycle is the corpus luteum. The aims of this study were (1) to investigate culture conditions for optimal IN production by luteinized GC (LGC) from rhesus monkeys and (2) to compare IN and progesterone (P) production by nonluteinized GC (NGC) and LGC in response to putative agonists. Animals were treated for up to 9 days with human menopausal gonadotropins to promote the development of multiple preovulatory follicles. GC were obtained from large follicles before (NGC) or 27 h after (LGC) an ovulatory injection of hCG. For Aim 1, cells were cultured in Hams F-10 medium +/- hCG (100 ng/ml) with or without the addition of insulin/transferrin/selenium, 10% fetal bovine serum, or 10% Serum-Plus (JRH Biosciences, Lenexa, KS). Medium was changed on Days 1, 2, 4, 6, and 8, and IN and P concentrations were determined by RIA. Basal (unstimulated) IN production by LGC was enhanced and maintained for 6-8 days in the presence of serum, but rapidly declined in the absence of serum. In contrast, basal P secretion declined regardless of exposure to serum. Human CG consistently increased (p less than 0.05) IN production only in the presence of serum but stimulated (p less than 0.05) P production under all conditions. For Aim 2, cells were cultured for 4 days in Ham's F-10 medium + 10% macaque serum +/- hCG (100 ng/ml), hFSH (100 ng/ml), prostaglandin E2(PGE2; 14 microns), or dibutyryl(db)-cAMP (5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The Sulawesi Crested Black Macaque (Macaca nigra) menstrual cycle: changes in perineal tumescence and serum estradiol, progesterone, follicle-stimulating hormone, and luteinizing hormone levels.

Events in the normal menstrual cycle of the endangered Sulawesi Crested Black Macaque (Macaca nigra) were characterized. Daily blood samples were obtained during 10 menstrual cycles from five M. nigra demonstrating regular cycles. The amount of perineal tumescence was scored daily. Serum levels of estradiol and progesterone were determined by RIA, serum LH levels were determined by the mouse Leydig cell bioassay, and serum FSH levels were determined by the rat granulosa cell aromatase bioassay. Cycle length was 39.8 +/- 1.0 days (mean +/- SEM) with an LH surge occurring 25 +/- 1.5 days from the onset of menses. After menses, both LH and estradiol were initially depressed, with estradiol first exceeding 50 pg/ml 8 days before the LH surge. In five cycles, peak estradiol levels (340 +/- 44 pg/ml) occurred on the day of the LH surge (637 +/- 58 ng/ml) and in the other five cycles, on the day before the LH surge. There was a broad increase of FSH in midcycle without a well-defined surge corresponding to the LH surge. Progesterone began increasing on the day of the LH surge and reached peak levels (6.8 +/- 0.96 ng/ml) 8 days later. Maximal perineal tumescence was generally associated with the time of the LH surge, but variation between animals made it impossible to predict accurately the day of the LH surge by perineal tumescence scores alone.

Animals↗
PubMed1992

Gonadotropin surge increases fluorescent-tagged low-density lipoprotein uptake by macaque granulosa cells from preovulatory follicles.

In the primate ovary, luteal steroidogenesis is largely dependent upon cholesterol derived from receptor-mediated uptake of circulating low-density lipoprotein (LDL). However, granulosa cells (GC) of preovulatory follicles possess few LDL binding sites compared to those present in developing and mature corpora lutea. We recently reported (Endocrinology 1991; 129:3247-3253) that uptake of LDL tagged with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) can be monitored in macaque luteal cells by fluorescence-activated flow cytometry. This study was designed to determine whether an ovulatory stimulus induced uptake of DiI-LDL in GC aspirated from preovulatory follicles of macaques undergoing ovarian stimulation. Development of multiple large follicles was stimulated in adult rhesus macaques with human gonadotropin treatment for 9 days. On Day 10, monkeys received either no ovulatory stimulus or 1000 IU hCG to initiate ovulatory events. GC were aspirated on Day 10 in monkeys receiving no ovulatory stimulus (nonluteinized GC) or 27 h or 34 h after hCG injection (luteinizing GC). GC were resuspended in Ham's F-10 medium + 0.1% BSA and incubated with several concentrations (0-25 micrograms/ml) of DiI-LDL (Biomedical Technologies, Stoughton, MA) for various time intervals (2-60 min). DiI-LDL uptake by GC was time- and concentration-dependent. Coincubation of cells with DiI-LDL and unlabeled LDL dose-dependently suppressed the percentage of fluorescent cells. In contrast, coincubation with up to a 250-fold excess of acetylated LDL or high-density lipoprotein did not alter the percentage of fluorescent GC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Administration of human luteinizing hormone (hLH) to macaques after follicular development: further titration of LH surge requirements for ovulatory changes in primate follicles.

After stimulation of multiple follicular development, endogenous LH surges elicited by GnRH or GnRH agonist were of insufficient duration (4-14 h) to evoke oocyte maturation and luteinization in this species. In this study, periovulatory LH surge requirements were further titrated using hLH as the ovulatory stimulus. Beginning at menses, rhesus monkeys were treated with human gonadotropins for 9 days to stimulate follicular growth. To induce ovulatory maturation on day 10, animals received: 1) hCG (1000 IU, im; n = 8); 2) highly-purified, urinary hLH (2542 IU, im; n = 4); or 3) hLH (2542 IU, im) followed by three injections of hLH (200 IU, im) at 8-h intervals (0800, 1600, 2400 h) daily during the luteal phase until menses (n = 3). Oocytes and luteinizing granulosa cells were obtained via follicle aspiration 27 h after the initial hLH or hCG injection. Estradiol and progesterone levels were measured in daily serum samples by RIA. Bioactive LH levels were determined at selected intervals within 36 h of the hLH ovulatory stimulus. Nuclear maturity of oocytes was evaluated as an indicator for reinitiation of meiosis. Luteinizing granulosa cells were processed for indirect immunocytochemistry using a monoclonal antibody to human progesterone receptor. In vitro progesterone production by luteinizing granulosa cells over 24 h was also assessed in the absence and presence of hCG. In all groups, serum estradiol rose to similar peak levels on day 10. After hLH, bioactive LH levels peaked (1262 +/- 79 ng/mL; mean +/- SEM) by 2-6 h, declined thereafter but remained above surge levels (100 ng/mL) for 18-24 h. Within 24 h of hLH injection, serum progesterone increased to 13 +/- 3 nmol/L, but returned to baseline in 1-6 days. In contrast, higher levels of progesterone were observed after hCG (114 +/- 51 nmol/L) and during luteal phase treatment with hLH (137 +/- 25 nmol/L) and the luteal phase was longer (11.5 +/- 0.4 and 14.3 +/- 0.7 days, respectively). Of the total cohort of oocytes aspirated, the proportion of oocytes resuming meiotic maturation (metaphase I plus metaphase II) was similar after hCG (76%) and hLH (74%). However, the proportion of oocytes maturing to metaphase II tended to be less (P = 0.08) after hLH (13%) than hCG (22%). Fertilization rates were similar between the two groups. Progesterone receptor was detected in nuclei of luteinizing granulosa cells from all animals receiving hCG, but only in some given hLH.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Activin-A inhibits progesterone production by macaque luteal cells in culture.

Since inhibin is produced during the luteal phase of the menstrual cycle in women and nonhuman primates, the primate corpus luteum (CL) may be a local site of inhibin/activin action. This study was designed to determine whether inhibin or activin altered steroidogenesis by macaque luteal cells in vitro. Luteal cells were obtained by enzymatic dispersion of CL from rhesus monkeys at midluteal phase of the menstrual cycle. Cells (2 x 10(4)/0.2 mL) were cultured in wells coated with extracellular matrix from bovine corneal endothelial cells in Dulbecco's modified Eagle's medium/F-12 medium (1:1 vol/vol) + insulin (2 ng/mL), transferrin (5 ng/mL), H2SeO3 (0.25 nmol), and aprotinin (10 micrograms/mL). Various concentrations (0-400 ng/mL) of recombinant human-inhibin-A, recombinant human-activin-A or human CG (hCG) (100 ng/mL; CR123) alone or in combination with inhibin or activin were added to the culture media (n = 5 Exp). Media were changed daily for 4 days and progesterone (P) concentrations were determined by RIA. Inhibin exposure did not alter P levels compared to that of control (untreated) cultures. In contrast, activin (10-400 ng/mL) suppressed P production (P less than 0.05) below controls and inhibin-treated cultures by days 3 and 4. Exposure to hCG increased P levels throughout culture (9 x control levels by day 4; P less than 0.05). hCG-stimulated P production was unaltered by inhibin, whereas activin (50-400 ng/mL) reduced (maximal inhibition of 40%; P less than 0.05) hCG-stimulated P production by day 4 of culture. Cell number on day 4 was not altered by any dose of inhibin or activin, but the number of cells staining for 3 beta-hydroxysteroid dehydrogenase was reduced (P less than 0.05) by 32.9 +/- 2.6% in activin-treated cultures. Since P levels declined during culture in all treatment groups, in a second series of experiments (n = 4), luteal cells were cultured for 4 days with or without hCG (100 ng/mL) and low density lipoprotein (LDL; 100 micrograms/mL) +/- 0-400 ng activin/mL. P production in the presence of hCG+LDL was greatly enhanced compared to other treatment groups and was sustained during days 2-4 of culture. Activin at doses of 50-400 ng/mL suppressed (maximal inhibition of approximately 35%; P less than 0.05) hCG+LDL-stimulated P production on days 3 and 4. These results suggest that the primate CL is a target for activin action to suppress luteal cell activities, including gonadotropin-regulated, lipoprotein-mediated steroidogenesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

Progesterone production by monkey luteal cell subpopulations at different stages of the menstrual cycle: changes in agonist responsiveness.

Small (less than or equal to 15 microns diameter) and large (greater than 20 microns diam.) luteal cells of the rhesus monkey have been separated by flow cytometry based on light scatter properties. To determine whether the steroidogenic ability and agonist responsiveness of luteal cell subpopulations vary during the life span of the corpus luteum, small and large cells were obtained at early (Days 3-5), mid (Days 7-8), mid-late (Days 11-12), and late (Days 14-15) luteal phase of the cycle. Cells (n = 4 exp./group) were incubated in Ham's F-10 medium + 0.1% BSA for 3 h at 37 degrees C with or without hCG (100 ng/ml), prostaglandin E2 (PGE2; 14 microM), dibutyryl-cAMP (db-cAMP; 5 mM), or pregnenolone (1 microM). Basal progesterone (P) production by large cells was up to 30-fold that by small cells depending on the stage of the cycle. HCG stimulated (p less than 0.05) P secretion by both small (1.8 +/- 0.2-fold) and large (3.7 +/- 0.7-fold) cells in the early luteal phase. HCG responsiveness declined during the luteal lifespan; P production by small cells was not significantly enhanced by hCG by mid luteal phase, whereas that by large cells was stimulated 1.7 +/- 0.2-fold (p less than 0.05) even at late luteal phase. Cell responses to db-cAMP were similar to those for hCG.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Localization of androgen receptor in the follicle and corpus luteum of the primate ovary during the menstrual cycle.

Ovarian androgens may act locally to modulate follicular and luteal function in various species. This study examined the distribution of androgen receptors within the primate ovary throughout the menstrual cycle. Ovaries were collected from rhesus and cynomolgus monkeys during the early, mid-, and late (n = 3-5 per stage) follicular and luteal phases of the cycle. The tissues were processed for indirect immunocytochemical localization of androgen receptors with a specific monoclonal antibody against human androgen receptor (AN1-15). In addition, ovaries (n = 3) were collected from rhesus monkeys for biochemical detection of androgen receptor using 3H-androgen and AN1-15. Specific immunocytochemical staining, as determined by comparing adjacent tissue sections incubated with either AN1-15 or a nonspecific control antibody, was exclusively nuclear. Androgen receptor was detected in the germinal epithelium and ovarian stroma at all stages of the cycle. The thecal and granulosa cells of growing follicles, and of many but not all atretic follicles, contained androgen receptors. Luteinizing granulosa cells of the periovulatory follicle and luteal cells from the early and midluteal phase stained intensely for androgen receptor. Regressing corpora lutea of the late luteal phase also stained for androgen receptor; however, fully regressed corpora lutea in the early follicular phase of the next cycle did not exhibit receptor staining. Luteal cells that were androgen receptor-positive also stained histochemically for the presence of 3 beta-hydroxysteroid dehydrogenase. Sucrose gradient analysis with radiolabeled androgen demonstrated a shift in the androgen receptor peak in monkey ovarian tissue upon addition of AN1-15, confirming the presence of androgen receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Comparison of the steroidogenic response of luteinized granulosa cells from rhesus monkeys to luteinizing hormone and chorionic gonadotropin.

The dynamics of the steroidogenic response of nonprimate gonadal cells to gonadotropins suggests that the biologic action of pituitary LH differs from that of placental CG. To compare the response to LH and CG in primate species, luteinized granulosa cells (LGCs) obtained from rhesus monkeys following follicle stimulation were cultured in vitro. The pattern and levels of progesterone (P) produced during culture was influenced by the concentration (0-10%) and type (fetal bovine or macaque) of serum in the medium and whether LGCs were plated on plastic or extracellular matrix from bovine corneal endothelial cells. After 2-3 days of culture, LGCs were exposed acutely (15-30 min) or chronically (6 h) to 1 or 100 ng/ml human LH (hLH, NIH 1-2) or hCG (CR123), 50 micrograms/ml ovine LH (oLH, NIH-oLH-25), or incubated in the absence of gonadotropins (controls). After the first 15-30 min, the media were changed at 30-min intervals. Both acute and chronic exposure to hLH, hCG, and oLH increased (p less than 0.05) P concentrations above control levels within 15-30 min. There were no differences in the patterns or levels of P elicited by hLH or hCG over time for each treatment condition. Chronic exposure to 1 and 100 ng/ml hLH or hCG and 50 micrograms/ml oLH sustained P levels above that of controls for the 6-h interval. Acute exposure to 1 ng/ml hLH or hCG failed to maintain elevated P levels throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗