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)
β-D-Phosphogalactoside galactohydrolase (β-Pgal) was examined in a number of lactic streptococci by use of the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside-6-phosphate. Specific activity of β-Pgal ranged from 0.563 units/mg of protein in Streptococcus lactis UN, to 0.120 in S. diacetilactics 18-16. Essentially no β-D-galactoside galactohydrolase (β-gal) was found in these organisms when o-nitrophenyl-β-D-galactopyranoside served as the chromogenic substrate. S. lactis 7962 was the one exception found. This organism contained rather high levels of β-gal, and very little β-Pgal could be detected. β-Pgal activity was examined in streptococci that differed widely in both their proteolytic ability and rates of lactic acid production during growth in milk. Differences in proteolytic ability did not influence β-Pgal synthesis; also, the rate of lactic acid production was independent of the level of β-Pgal present in the cell, since the rate of lactic acid production could be increased approximately fourfold without changing the amount of β-Pgal present in the cell. Various carbohydrates were tested as potential inducers of the enzyme. Although galactose, either as the free sugar or combined with glucose in lactose, was the only inducer, noninducing sugars such as mannose or glucose showed some ability to cause fluctuations in the basal level of β-Pgal. Cells growing in mannose or glucose exhibited about 30% of the maximal enzyme levels found in cells growing in lactose or galactose. No gratuitous inducers were found.
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 μg/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 < 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 μg/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. Whereas acute exposure to 100 ng/ml hIll or hCG maintained elevated P levels for 6 h, P concentrations fell immediately after acute exposure to 50 μg/ml oLH. Thus, experiments analogous to those performed on nonprimate cells failed to demonstrate disparate actions of hLH and hCG in a primate species where these are endogenous gonadotropins. However, the steroidogenic response of target cells to gonadotropin preparations from heterologous species (e.g., oLH and hLH) can differ.
A phage-mediated transducing system was used in studying certain physiological characteristics of S. lactis C2 wild type, lactose-negative mutants, and lactose-positive transductants. Lac - mutants, obtained by acriflavine treatment of the wild type, were similar to the wild type in all characteristics tested except they lacked β-D-phosphogalactoside galactohydrolase (β-Pgal) and could not transport [ 14 C]lactose; they also had approximately 10% of the proteolytic ability than wild-type cells. The lactose-fermenting characteristic was transduced from the wild type to Lac - mutants. The Lac + transductants obtained were similar to the wild-type parent with respect to lactose fermentation and level of β-Pgal activity (0.186 U of protein per mg). These transductants, however, had not regained full proteolytic ability and were similar to the Lac - mutant in this respect. Lactic acid production of the transductants in milk was approximately two-thirds that of the wild type. Data suggest that both the lactose-fermenting and proteolytic characters are carried on extrachromasomal particles (plasmids).
The midcycle gonadotropin surge promotes vascular endothelial growth factor-A (VEGF-A) production by granulosa cells in the ovulatory follicle, but it is unclear whether primary regulators of VEGF secretion in other tissues, including hypoxia and growth factors, are also important in the ovary. To address these issues, granulosa cells were collected from rhesus monkeys during controlled ovarian stimulation either before (i.e., nonluteinized granulosa cells, NLGCs) or 27 hours after (i.e., luteinized granulosa cells, LGCs) administration of an ovulatory bolus of hCG, and cultured in fibronectin-coated wells containing a chemically defined media. When NLGCs were transferred to various O2 environments (20%, 5%, or 0% O2) or media containing 100 mM CoCl2, LH (100 ng/ml)-stimulated progesterone (P4) levels were markedly (P < 0.05) suppressed by 0% O2 or CoCl2. VEGF concentrations also declined (P < 0.05) in control, CoCl2, and CoCl2 + LH groups in 0% O2, although CoCl2 modestly increased (75% above control; P < 0.05) VEGF levels in 20% and 5% O2. When NLGCs were cultured in the presence of recombinant human insulin-like growth factor (IGF)-1, IGF-2, or insulin, there was a dose-dependent increase (P < 0.05) in VEGF levels on Day 1 of culture. Whereas optimal doses of IGF-1 or IGF-2 (50 ng/ml), hCG (100 ng/ml), and IGF plus hCG stimulated VEGF levels on Day 1, only the combination of IGF-1 or IGF-2 plus hCG increased VEGF above controls and sustained levels through Day 3 of culture. The synergistic effects of IGF and hCG were also evident in P4 levels, and were not due to changes in DNA content between treatment groups. LGCs produced much higher levels of P4 and VEGF, but the responses to different O2 concentrations and insulin-related factors were qualitatively similar to those of NLGCs. These results suggest that hypoxia is not a primary regulator of VEGF production in primate granulosa cells. However, IGFs may act in concert with the gonadotropin surge to promote VEGF secretion in the ovulatory, luteinizing follicle.
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.Moreover, the aromatase and sidechain cleavage enzymes remain active and under gonadotropin regulation when the cells are maintained in GCM. Lastly, cells isolated from three species (rat, macaque, and human) respond similarly to FSH when maintained in GCM.
