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Biomedical subjects

C P Channing

Publications and source records attributed to C P Channing.

At least 19 recordsLinked to original sources
PubMed1990

Inhibitory and stimulatory action of porcine follicular fluid on FSH-induced 125I-hCG specific binding in rat granulosa cells.

In previous studies, follicular fluid (FF) of large antral follicles (LFF) manifested a stimulatory effect on granulosa cell (GC) luteinization in domestic livestock, but was found to have an inhibitory effect on rodent GC. In the present study, the type of LFF effect on the luteinizing of rat GC was reevaluated in two different models. GC obtained from immature hypophysectomized rats treated with diethylstilbestrol were cultured with increasing concentrations of FSH alone or FSH + estradiol-17 beta (E2), either for 3 or 4 successive days of culture (model 1) or for 4 days of culture with medium change after 2 days of culture (model 2). The addition of FSH increased 125I-hCG specific binding in a dose-dependent manner to a maximum of approximately 110-fold (model 1) or 45-fold (model 2) compared with GC culture in medium alone. At the maximal effective dose of FSH, addition of E2 increased the 125I-hCG binding 2-fold (model 1) and 3.5-fold (model 2). 125I-hCG specific binding induced by FSH or FSH + E2 in model 1 was decreased by concurrent treatment (added on the day of cell inoculation) with porcine LFF (approximately 3-fold) or porcine serum (approximately 4.5-fold). In model 2, however, porcine LFF increased FSH-induced 125I-hCG specific binding 3-fold, provided LFF was added only after GC were primed with FSH alone for 2 days. When porcine serum was added instead of porcine LFF, only a permissive action was observed. These data may suggest that an initial GC differentiation is indispensible for obtaining the FF stimulatory effect on FSH-induced 125I-hCG specific binding in rat GC.

Animals↗
PubMed1989

Increasing 125I-human chorionic gonadotrophin specific binding in human granulosa cells by follicle-stimulating hormone and follicular fluid.

It has been reported that success at in-vitro fertilization/embryo transfer (IVF/ET) treatment is increased by follicular fluid (FF) re-injected into the abdomen. In the present study a possible direct effect of FF on human granulosa cell (GC) progesterone (P4) secretion and LH/human chorionic gonadotrophin (HCG) receptor content was studied in the presence and absence of FSH. Human GC cultured for 8 days in medium alone showed a 40-fold decrease in P4 secretion. Addition of human FSH increased P4 secretion and [125I]HCG specific binding by 12- and 8-fold, respectively, compared to human GC cultured in medium alone. The effect of FF was evaluated in a heterologous system by the addition of FF from large antral porcine follicles (LFF) to human GC in culture. The decline in human GC-P4 secretion after 8 days of culture was not altered by either porcine serum alone or porcine LFF alone. However, the concomitant addition of FSH and LFF significantly increased [125I]HCG specific binding, but did not alter the FSH-induced P4 secretion when both parameters were compared to GC cultured in FSH + porcine serum. Furthermore, the addition of HCG alone significantly increased P4 secretion 33- and 70-fold in GC pre-cultured with either FSH alone or FSH + LFF respectively compared with the stimulatory effect of HCG on GC pre-cultured in medium alone. These results may suggest that FSH and LFF increase the functional content of LH/HCG receptor in luteinized human GC.

Animals↗
PubMed1988

Characterization of follicular fluid stimulatory factor upon FSH-induced granulosa cell differentiation.

The modulatory role of follicular fluid (FF) upon gonadotropin-induced granulosa cell (GC) differentiation has been previously demonstrated. In the present study, the stimulatory factor of large-antral FF (LFF) was partially characterized. Addition of ovine FSH to porcine GC culture increased 125I-hCG specific binding about 6-fold and progesterone (P4) secretion 15-fold. The FSH-induced 125I-hCG specific binding was increased 1.9-fold by LFF and decreased 7.7-fold in the presence of small-antral FF (SFF). It is suggested that SFF may contain an inhibitory factor which is overcome by a stimulatory factor accumulated in FF with follicular growth. An initial purification of LFF stimulatory factor was achieved by two-step ethanol elution. The obtained LFF purified fraction increased the FSH-induced 125I-hCG specific binding 1.5-fold and P4 secretion 4.3-fold compared to GC cultured with FSH + LFF. Addition of LH alone increased the P4 secretion 2.3-, 3.4-, and 6 fold in GC precultured with FSH alone, FSH + neat LFF, and FSH + LFF purified fraction, respectively, as compared to GC precultured in plain medium. Therefore, the LFF stimulatory increase of 125I-hCG specific binding may represent an increase of functional LH receptors. Freezing and thawing of LFF 4 times, exposing LFF to urea for 24 h, heating at 60 C for 20 min, and tryptic digestion each abolished the LFF stimulatory effect upon the FSH-dependent increase of 125I-hCG specific binding and P4 secretion. Thus, the LFF stimulatory activity could be attributed to one or more proteins.

Animals↗
PubMed1985

Demonstration of a gradient in inhibin activity, estrogen, progesterone, and delta 4-androstenedione in follicular fluid, ovarian vein blood, and peripheral blood of normal women.

Ovarian vein serum from 3 subjects during the late follicular phase of the menstrual cycle had detectable inhibin activity, whereas ovarian vein serum of 12 other subjects during the early follicular phase and luteal phase had no detectable inhibin activity in a rat anterior pituitary cell culture assay. Subjects having detectable inhibin activity (102 +/- 47 U/100 microliters) had 1257 +/- 582 U/100 microliters inhibin activity in FF, whereas subjects having no detectable inhibin activity had FF levels of 711 +/- 203 U/100 microliters of inhibin activity. Estrogen levels of FF and ovarian vein serum of the group having detectable inhibin activity in ovarian vein serum were 282 +/- 239 ng/ml and 4.8 +/- 1.77 ng/ml, respectively. The estrogen content of FF and ovarian vein blood of the group having nondetectable inhibin activity in ovarian vein blood was 127 +/0 45 ng/ml and 3.03 +/- 0.6 ng/ml, respectively.

Adult↗
PubMed1985

Effect of calcium ion on the maturation of cumulus-enclosed pig follicular oocytes isolated from medium-sized graafian follicles.

Porcine follicular oocytes from medium-sized follicles (3-5 mm in diameter) were cultured in modified Hank's balanced salts solution (MHBS) to which pyruvate, lactate, and glucose were added as an energy source. Bovine serum albumin (0.4%) was added as a protein source and the oocytes were cultured for 42 h at 37 degrees C in 5% CO2 in air. In this medium porcine oocytes underwent 80-90% nuclear maturation after 42 h. Oocytes were cultured in MHBS with various amounts of CaCl2 as well as in the presence of verapamil, a Ca2+ channel blocker, and the divalent cationophore A23187. It was found that the lowest concentration of Ca2+ required for oocyte maturation was around 0.0265-0.053 mM. Such a requirement for Ca2+ in the culture medium extended through metaphase II. If Ca2+ was omitted during the final 4 h of culture, the metaphase II chromosomes appeared extremely condensed or degenerated. Verapamil at a level of 0.2 mM inhibited germinal vesicle breakdown or resulted in degeneration, whereas lower concentrations did not affect oocyte maturation. In the presence of 0.02 mM verapamil, the maturation of cumulus-enclosed oocytes was not affected, whereas at the same dose of verapamil the maturation of denuded oocytes was inhibited. Less than 3.8 X 10(-7) M divalent cationophore did not inhibit oocyte maturation. Maturation was inhibited by 3.8 X 10(-7) and 3.8 X 10(-6) M divalent cationophore. In conclusion, maintenance of oocytes in a nondegenerated state also requires the constant presence of Ca2+ in the culture medium.

Animals↗
PubMed1985

Stimulatory action of follicular fluid components on maturation of granulosa cells from small porcine follicles.

Follicular fluid obtained from large (6-12 mm) porcine follicles (LFF) was investigated to determine its stimulatory activity on progesterone secretion and on follicle stimulating hormone (FSH) induction of 125I-human chorionic gonadotropin (hCG)-luteinizing hormone (LH) binding sites in porcine granulosa cells in a 4-day culture. Incubation of granulosa cells harvested from small porcine follicles (1-2 mm) with 50% LFF led to stimulation of LH/hCG binding sites and progesterone secretion. After partial purification of pooled LFF or proteins precipitated with 90% ethanol on Sephadex G-100 the greatest stimulatory activity was found in the second protein peak eluted from the column. Chromatography of part of the active fraction on DEAE Sephacel using a continuous gradient of NH4HCO3 yielded seven protein fractions. The second fraction, which eluted early, contained the majority of the stimulatory activity which was purified about 32-fold compared to native LFF. In contrast, addition of follicular fluid recovered from small porcine follicles inhibited FSH induction of LH/hCG binding sites and progesterone secretion. It can be concluded, that maturation of granulosa cells from small follicles may be enhanced by protein(s) present in LFF, but not in fluid recovered from less mature follicles.

Animals↗
PubMed1985

Direct evidence for de-novo synthesis of LH receptors in cultured pig granulosa cells in response to FSH.

FSH plus insulin, cortisol, and thyroxine (IFT) stimulated incorporation of dense isotope-containing (2H, 13C, 15N) amino acids into soluble 125I-labelled hCG binding sites. Evidence of new synthesis of binding sites appeared as early as 3 h after the beginning of the pulse-labelling period. By 48 h the majority of detectable soluble 125I-labelled hCG binding sites appeared to be newly synthesized. Studies with FSH + IFT and puromycin indicated that FSH + IFT stimulated synthesis of new LH/hCG binding sites, and that internalization or degradation of LH/hCG binding sites may also require protein synthesis.

Animals↗
PubMed1985

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells.

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.

Animals↗
PubMed1979

Effects of follicular development on the ability of cultured porcine granulosa cells to convert androgens to estrogens.

Porcine granulosa cells from different stages of follicular development were examined for their ability to convert androgens to estrogens and their ability to secrete progesterone. Granulosa cells from all stages of follicular development can convert androgens to estrogens, and the addition of exogenous androgen is required for estrogen secretion. Granulosa cells obtained from medium and large follicles have a greater capacity to convert androgens to estrogens than do granulosa cells obtained from small follicles. The addition of FSH brought about an increase in estrogen secretion in the presence of androgen only in granulosa cells from large follicles. LH attenuated estrogen secretion in granulosa cells from medium follicles treated with testosterone. Granulosa cells from medium follicles were unable to secrete estrogen from days two to four irrespective of treatment. Androgens augment FSH stimulated progesterone secretion in granulosa cells from medium follicles from days two to four, and the addition of follicular fluid from small follicles stimulates progesterone secretion in the presence of FSH and androgens.

Androgens↗
PubMed1979

Action of porcine follicular fluid oocyte maturation inhibitor in vitro: possible role of the cumulus cells.

To study the mode of action of porcine follicular fluid oocyte maturation inhibitor isolated cumulus-enclosed or mechanically denuded pig oocytes were used. Two types of culture media were employed, a complex containing 15% pig serum (TC199A) and a defined, minimal medium (BMOC). The maturation of cumulus-enclosed oocytes was comparable in the two types of culture media, but only in the complex medium did the cumulus cells remain functional in terms of morphology and progesterone secretion. The low molecular weight portion of follicular fluid partially inhibited oocyte meiosis and cumulus progesterone secretion in 199A. No inhibition of oocyte maturation was seen when follicular fluid was added to BMOC. Since denuded oocytes did not respond to follicular fluid in either culture medium, it is suggested that the cumulus cells may mediate the action of the follicular fluid oocyte maturation inhibitor.

Animals↗
PubMed1979

Inhibitory effect of charcoal-treated porcine follicular fluid upon serum FSH levels and follicular development in the rhesus monkey.

Injection of charcoal treated porcine follicular fluid into 4 intact, 4 long term castrate and 2 postmenopausal rhesus monkeys brought about a selective decline in serum FSH levels. Luteinizing hormone levels were not significantly decreased. Intraperitoneal injection of 4 ml porcine follicular fluid every 8 hours for the first 4 days of the menstrual cycle led to a decrease in follicle and granulosa cell growth observed at that midcycle. Treatment with porcine serum did not exert this effect.

Animals↗
PubMed1979

Evidence that the pituitary gland is the site of inhibitory action of porcine follicular fluid upon FSH secretion in the rat.

This study has examined the effects of porcine follicular fluid (PFF) on the adenohypophyseal secretion of LH and FSH following LHRH treatment. The animal model selected for study the the proestrous rat in which preovulatory LH and FSH surges were blocked with phenobarbital. To elicit the release of gonadotropins from this preparation, two different i.v. LHRH infusion protocols were used. The first elicited a proestrous-like LH and FSH surge while the second infusion schedule evoked the predominant release of FSH with only minor changes in LH. PFF suppressed LHRH-induced FSH secretion in both preparations but had only negligible effects on LH secretion. Interestingly, the suppressive effects of PFF were variable and depended upon the mode in which LHRH was presented.

Animals↗
PubMed1979

Effect of removal of carbohydrate residues upon the half life and in vivo biological activity of human chorionic gonadotropin.

The effect of progressive enzymatic removal of various carbohydrates on the half life (T 1/2) and biologic activity in vivo of hCG was studied in rats. The cleavage of carbohydrates such as galactose, glucosamine and mannose, in addition to sialic acid, reduced the T 1/2 from 60--90 for native hCG to less than 3 minutes. The hCG derivatives had no inherent ability to cause ovulation, but in PMSG, Nembutal-treated immature rats, the hCG derivatives with carbohydrate residues internal to sialic acid removed blocked the stimulatory effect of native hCG upon ovulation.

Animals↗