Biomedical subjects

W C Gorospe

Publications and source records attributed to W C Gorospe.

14 recordsLinked to original sources

PubMed1992

Interleukin-6: effects on and production by rat granulosa cells in vitro.

Treatment of FSH-stimulated granulosa cells with increasing amounts of interleukin-6 (IL-6) caused a significant concentration-dependent suppression of progesterone biosynthesis. However, basal progesterone production in non-FSH-stimulated cells remained unresponsive to the cytokine. Quantitation of IL-6 in the conditioned media from untreated granulosa cells by the 7TD1 hybridoma cell bioassay revealed detectable levels of IL-6. Further, FSH treatment caused a significant concentration-dependent increase in IL-6 release. In contrast, both basal and FSH-stimulated IL-6 release could be significantly suppressed by interferon gamma (INF-gamma). The results of the present study suggest: 1) a role for IL-6 in the regulation of progesterone production, 2) that the granulosa cell is a source of IL-6 and 3) that the release of IL-6 by the granulosa cell is a regulated event.

Animals↗

PubMed1991

Production of progestin-stimulatory factor(s) by enriched populations of rat T and B lymphocytes.

We have previously demonstrated that a progestin-stimulatory factor(s) (PSF) is present in the supernate of concanavalin A-activated rat splenocytes. In the absence of FSH, PSF evokes dose-dependent increases in both progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) production by undifferentiated rat granulosa cells while basal estrogen production is unaffected. Because splenocytes are composed predominantly of T and B lymphocytes, we sought to determine if PSF production is restricted to one of these specific cell types. To accomplish this, dispersed splenocytes were prepared from the spleens of adult female rats. Macrophages were removed by standard adherence procedures, and highly enriched populations of T and B lymphocytes were obtained by negative selection panning utilizing monoclonal antibodies to T and B lymphocytes. Purity of the T- and B-cell populations (typically greater than 85-95%) was assessed by single-color flow cytometry. Enriched populations of T cells, B cells, or unseparated lymphocytes were then seeded into tissue culture flasks and stimulated with 2.5 micrograms/ml concanavalin A. Following a 48-h incubation at 37 degrees C, cytokine-rich fractions were prepared from the conditioned medium of these cultures by ammonium sulfate fractionation. The presence of PSF was assessed by the ability of the resulting fractions to specifically stimulate progestin production by undifferentiated rat granulosa cells in vitro. Granulosa cells were treated with increasing doses (ranging from 0-30% v/v) of supernatants from cultures of T cells, B cells, or unseparated splenocytes for 48 h. Media were removed and progesterone, 20 alpha-OH-P, and estrogen levels were quantified by RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

20-alpha-Dihydroprogesterone↗

PubMed1991

Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia.

In the present study, we examined the possibility that granulosa cell death during ovarian follicular atresia occurs by apoptosis (programmed cell death). To investigate this possibility, atresia was induced in immature female rats by injecting 15 IU PMSG. Controls received either vehicle or no treatment. PMSG-treated animals were killed on days 1-5 post-injection while controls were killed on days 1 or 5. The onset of atresia was assessed histologically by light microscopic inspection of 5 microns tissue sections and functionally by quantification of serum progesterone and estrogen levels. Apoptosis is characterized by the cleavage of genomic DNA into oligonucleosomal length fragments by a Ca2+/Mg(2+)-dependent endogenous endonuclease. Such fragments form a distinctive ladder pattern when separated electrophoretically. Accordingly, the occurrence of apoptosis in granulosa cells was assessed by examining the pattern of fragmented DNA in cell lysates after agarose gel electrophoresis. Gels were stained with ethidium bromide and DNA visualized by UV transillumination. The earliest morphological signs of atresia were detected 4 days after PMSG injection as evidenced by degeneration and detachment of granulosa cells from the basal lamina. Serum estrogen increased from basal to levels 7-fold over controls by day 3 after PMSG treatment, falling to control values by day 4 and thereafter. In contrast, progesterone remained basal for the first 3 days, rising to levels 3-fold and 8-fold above controls 4 and 5 days after PMSG treatment, respectively. Such shifts in the ratio of estrogen to progesterone production are known to be characteristic of follicular atresia. Finally, electrophoretic analysis of low mol wt DNA in granulosa cell lysates revealed a definitive ladder pattern of oligonucleosomal length DNA fragments (characteristic of apoptosis) on days 4 and 5 after PMSG injection. This pattern was not detectable on days 1 and 2 after treatment. Lysates obtained 3 days after PMSG treatment showed a faint apoptotic-like pattern of DNA fragments; a result consistent with other systems in which DNA cleavage begins before any morphological signs of death. Interestingly, a ladder pattern of DNA fragments was present in control lysates suggesting that granulosa cell death under normal (vs. induced) conditions of atresia in immature rats occurs by apoptosis. These data demonstrate an intimate association between apoptotic-like events and dying granulosa cells and thus support the possibility that apoptosis is involved in the induction of follicular atresia.

Animals↗

PubMed1990

Effects of cytokines on porcine granulosa cell steroidogenesis in vitro.

Recent evidence indicates that factors produced by immune cells (cytokines) may play a role in ovarian function. To explore this possibility, we examined the effects of conditioned medium obtained from cultures of either unstimulated splenocytes (splenocyte-conditioned medium; SCM) or concanavalin A-stimulated splenocytes (CAS) on estrogen and progesterone production by porcine granulosa cells. Granulosa cells were obtained from small (less than 3 mm) or large (greater than 7 mm) follicles and treated with increasing doses of SCM or CAS in the presence or absence of pFSH (100 ng/ml) for 24 h at 37 degrees C. In granulosa cells obtained from small follicles it was found that both SCM and CAS evoked a dose-dependent increase in estrogen but not progesterone production. Estrogen production was no further enhanced by the presence of FSH. Additionally, SCM was able to augment FSH-stimulated progesterone production by these cells, whereas CAS had no effect. Identical treatment of granulosa cells obtained from large follicles demonstrated that both SCM and CAS caused dose-dependent increases in estrogen as well as progesterone production. In response to CAS, FSH augmented progesterone production but exerted a biphasic on estrogen production (inhibiting at lower doses while stimulating at higher doses). In contrast, SCM had no effect on FSH-stimulated estrogen production. Additional controls indicated that the above results could not be attributed to either concanavalin A or serum. Taken together, these findings suggest that cytokines can exert significant effects over granulosa cell steroidogenesis and further imply that these factors may play an important role in the differentiation and developmental regulation of granulosa cell function.

Animals↗

PubMed1989

Effects of interleukins 1, 2 and 3 on follicle-stimulating hormone-induced differentiation of rat granulosa cells.

A growing body of evidence indicates that substances released by activated immune cells can directly influence the functions of various endocrine cells. In the present study, the direct in vitro effects of interleukins (IL) 1, 2, and 3 on follicle-stimulating hormone (FSH)-stimulated steroidogenesis and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor induction in granulosa cells were examined. In the absence of FSH, none of the interleukins stimulated steroid production or LH/hCG receptor induction during a 2-day culture period. However, in the presence of FSH, both IL-1 alpha and IL-1 beta inhibited, in a dose-dependent manner, progesterone and estrogen production as well as LH/hCG receptor induction in response to FSH. In contrast, both agents augmented 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) production stimulated by FSH. In all cases, less IL-1 beta than IL-1 alpha was required to produce a comparable effect. IL-2 slightly, but significantly, enhanced both FSH-stimulated progesterone and 20 alpha-OH-P production but had no effect on FSH-stimulated estrogen production or LH/hCG receptor induction. IL-3 potentiated the 20 alpha-OH-P response to FSH by up to 65% but had no effect on FSH-stimulated progesterone or estrogen production or LH/hCG receptor induction. These data suggest that the interleukins, which are key mediators of immune responses, may affect mechanisms crucial for the maturation and differentiation of granulosa cells and thus may also play a regulatory role in reproductive function.

Animals↗

PubMed1988

Gamma-interferon inhibits rat granulosa cell differentiation in culture.

A growing body of evidence indicates that factors secreted by cells of the immune system can directly affect a variety of endocrine phenomena. In the present study we examined the direct effects of the cytokine, interferon (IFN), on FSH-stimulated steroidogenesis and LH/hCG receptor induction in rat granulosa cells. We show that gamma-IFN, but not alpha-IFN, inhibits FSH-stimulated progesterone, 20 alpha-hydroxypregn-4-en-3-one and estrogen production as well as gonadotropin-induced LH/hCG receptor formation in a dose-dependent manner. The results suggest that gamma-IFN may play a role in the maturation and differentiation of granulosa cells and thus may serve as a regulatory link between the immune and reproductive endocrine systems.

20-alpha-Dihydroprogesterone↗

PubMed1988

Restoration of the LH secretory response in desensitized gonadotropes.

The time-dependent recovery of gonadotropin-releasing hormone (GnRH) responsiveness in desensitized gonadotropes was examined under conditions of altered membrane fluidity and GnRH exposure. Cultured pituitary cells were treated for 3 h with GnRH (10(-9) M; to provoke homologous desensitization) or vehicle alone (controls). When cells were washed and immediately rechallenged for 3 h with GnRH, gonadotrope responsiveness (assessed by luteinizing hormone (LH) release) was significantly lower in GnRH-pretreated cells than controls. If gonadotropes were allowed to recover in medium alone, membrane fluidity agents 2-(2-methoxyethoxy)-ethyl-8-(cis-2-n-octylcyclopropyl)-octanoate (A2C; 10(-4) M) or cis-vaccenic acid (CVA; 0.5 mM) or a low dose of GnRH (10(-10) M) for up to 48 h prior to rechallenging with GnRH, responsiveness in all cases was significantly lower in GnRH-pretreated cells than controls. However, if cells were treated with either A2C or CVA in the presence of GnRH (10(-10) M) during the recovery period, gonadotrope responsiveness to a subsequent challenge with GnRH was partially restored by 24 h; by 48 h no differences in the LH secretory response to GnRH was detected between GnRH-pretreated cells and controls. The possibility that restoration of the GnRH receptor-linked Ca2+ channel is associated with recovery of the desensitized gonadotrope was also examined. Identical protocols to those described above were used except that the functional integrity of the Ca2+ channel was assessed by measuring LH release in response to increasing doses of maitotoxin (MTX; a specific Ca2+ channel activator). Again, GnRH-pretreated cells were significantly less responsive to MTX than controls when allowed to recover for 48 h in medium alone, A2C (10(-4) M) or GnRH (10(-10) M). However, allowing cells to recover for 48 h under a condition of increased membrane fluidity and basal GnRH levels completely restored the MTX-stimulated LH secretory response in GnRH-pretreated gonadotropes. Taken together, these studies suggest that the physical state of the gonadotrope plasma membrane together with the appropriate hormonal milieu provide an important environment for the gonadotrope to recover from desensitization. Additionally, our results suggest that functional recovery of the GnRH-linked Ca2+ channel may play a requisite role in restoring GnRH responsiveness to the desensitized gonadotrope.

Animals↗

PubMed1988

Lymphokines from concanavalin-A-stimulated lymphocytes regulate rat granulosa cell steroidogenesis in vitro.

Animals with impaired immune function show numerous reproductive disorders. To determine whether immune factors might play a direct role in the regulation of ovarian function, we examined the effects of Concanavalin-A-stimulated lymphocyte supernates (CAS) on steroidogenesis by cultured rat granulosa cells. Granulosa cells from immature estrogen-primed rats were incubated for 48 h with increasing doses of CAS in the presence or absence of FSH. In the absence of FSH, CAS produced a dose-dependent increase in progestin (progesterone and 20 alpha-hydroxyprogesterone) production to a maximum of 190-fold greater than untreated controls, but did not stimulate estrogen production. In the presence of FSH, the stimulatory effect of CAS on progestin production was additive with that of FSH. In contrast, CAS inhibited FSH-stimulated estrogen production in a dose-dependent manner. The maximum inhibition was greater than 90%. Addition of the phosphodiesterase inhibitor isobutylmethylxanthine to CAS-containing cultures significantly enhanced the stimulatory effect of CAS on progesterone production, suggesting that this action may be exerted through a cAMP-mediated pathway. The stimulatory component of CAS was heat labile, acid stable, and required a trypsin-sensitive cell surface recognition site, whereas the inhibitory component was both heat and acid stable. Neither the stimulatory nor the inhibitory actions of CAS were mimicked by treating granulosa cells with supernates from Concanavalin-A-stimulated neonatal cardiac or hepatic cell cultures. Thus, the present studies demonstrate that secretory products of lymphocytes (collectively termed lymphokines) can affect steroidogenesis in cultured rat granulosa cells. These data imply that immune cell factors may play a significant role in the differentiation and maturation of granulosa cells.

1-Methyl-3-isobutylxanthine↗

PubMed1987

Membrane fluidity regulates development of gonadotrope desensitization to GnRH.

Development of GnRH-mediated gonadotrope desensitization was examined under conditions in which membrane fluidity was altered by temperature and/or chemical means. Cultured pituitary cells were preincubated at temperatures ranging from 4 degrees C to 37 degrees C for 3 h with a desensitizing concentration of GnRH (10(-9) M) or with vehicle alone. Cells were then rinsed and responsiveness assessed by a second 3 h incubation with GnRH at 37 degrees C. As preincubation temperatures decreased from 37 degrees C to 23 degrees C, development of desensitization in gonadotropes was progressively reduced. At 23 degrees C and below, gonadotropes failed to become desensitized to GnRH. Decreases in membrane fluidity occurred over the same temperature range as measured directly by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene incorporated into plasma membrane. When membrane fluidity was increased by incubating cells with the membrane mobilizing agent 2-(2-methoxy-ethoxy)-ethyl-8-(cis-2-n-octylcyclopropyl)-octanoate (A2C), low temperature blockade of GnRH-mediated gonadotrope desensitization was reversed. A2C had no measurable effects on either GnRH receptor binding or number and caused no cytotoxic effects. These studies suggest that development of gonadotropine desensitization to GnRH can be regulated by the state of membrane fluidity.

Animals↗

PubMed1987

Agents that decrease gonadotropin-releasing hormone (GnRH) receptor internalization do not inhibit GnRH-mediated gonadotrope desensitization.

Exposure of pituitary cell cultures to GnRH causes gonadotropin release, receptor capping, internalization, and loss as well as altered responsiveness of the target cell. In the present study, the relationship between loss of gonadotrope secretory responsiveness to GnRH (desensitization) and internalization of the GnRH-receptor complex was examined. Pituitary cell cultures were pretreated (30 min) with vinblastine (100 microM, a concentration that prevents measurable receptor internalization) or with medium containing carrier only, incubated with 10(-7) M GnRH (a desensitizing concentration) with or without vinblastine or with medium alone for 60 min, and finally washed and rechallenged for 3 h with increasing concentrations of GnRH to assess the degree of desensitization as determined by LH release. Results indicate that vinblastine had no measurable effect on the ability of GnRH to stimulate LH release or desensitize the cells. In a second series of studies, a GnRH analog (D-Lys6-GnRH) was immobilized to a cross-linked agarose matrix. The covalent link was shown to be stable by biological, immunological, and physical criteria. This product bound to the GnRH receptor and provoked LH release, but was not internalized, as determined by GnRH receptor binding assays. Cultured cells were treated with either 10(-9) M free analog or an equivalent concentration of coupled analog (as measured by LH release) for 3 h. Cells were washed, then rechallenged with GnRH to assess desensitization. Both the free and coupled analogs provoked an equivalent degree of desensitization. While a significant degree of desensitization also occurred in the presence of 3 mM EGTA (conditions that totally inhibited GnRH-stimulated LH release), the loss of responsiveness was not as great as in the absence of EGTA, indicating that partial depletion of available LH may play a role in GnRH-stimulated gonadotrope desensitization. The present findings suggest that GnRH receptor internalization and LH release can be uncoupled and that loss of the GnRH receptor by internalization is not a sufficient explanation for GnRH-mediated desensitization of the gonadotrope.

Animals↗

PubMed1987

Homologous down-regulation of gonadotropin-releasing hormone receptors and desensitization of gonadotropes: lack of dependence on protein kinase C.

The demonstration that GnRH provokes the accumulation of diacylglycerol and the redistribution of protein kinase C to the membrane fraction in gonadotropes suggests a role for this enzyme as a mediator of GnRH action. In the present work we have investigated the possibility that protein kinase C might mediate GnRH-stimulated receptor down-regulation and desensitization. Pretreatment of pituitary cells for 6 h with GnRH (10(-11) - 10(-6) M) caused a biphasic change in GnRH receptor number [the maximum binding (Bmax) for 125I-buserelin binding was increased by 10(-10) M GnRH and reduced by 10(-7) and 10(-6) M GnRH] and caused desensitization (pretreatment with 10(-9) - 10(-6) M GnRH reduced the proportion of cellular LH released in a subsequent challenge with GnRH). Pretreatment for 6 h with 0.2-200 nM phorbol myristate acetate (a protein kinase C-activating phorbol ester) did not cause desensitization, but at 200 nM, did reduce GnRH receptor number. As a further test of the requirement for protein kinase C for GnRH action, cells were depleted of all measurable protein kinase C (and rendered unresponsive to protein kinase C activators) by prior treatment with a high dose of phorbol myristate acetate (500 nM for 6 h followed by 12 h in plating medium). Depletion of protein kinase C did not alter the ability of GnRH to desensitize gonadotropes or down-regulate its own receptors. The demonstration that the effects of GnRH on receptor number and gonadotrope responsiveness are neither blocked by depletion of protein kinase C nor entirely mimicked by activation of protein kinase C suggests that these effects of the releasing hormone are not solely mediated by this enzyme.

Animals↗

PubMed1986

Mechanism of action of gonadotropin releasing hormone.

GnRH interacts with a plasma membrane receptor to provoke gonadotropin release, as well as regulate numbers of its own receptor and target cell responsiveness. Receptor numbers are altered in different physiological states of the animal. Microaggregation of the GnRH receptor mimics all known actions of the releasing hormone, and therefore is viewed as an early step in the molecular mechanism of hormone action. Internalized hormone is neither necessary nor sufficient for stimulation of known releasing hormone actions. Evidence summarized in the present work suggests that Ca2+ serves a role as a second messenger for GnRH-stimulated gonadotropin release, and that it may be involved in receptor up-regulation in response to the releasing hormone. In the former role, diacylglycerols by their action on protein kinase C, in a fashion independent of the Ca2+ -calmodulin system, may act as a signal amplifier.

Animals↗

PubMed1985

Effects of placenta and maternal serum on prolactin secretion in vitro.

The effects of the placenta and maternal sera on the secretion of prolactin (Prl) were examined in vitro. Placentae were obtained on each of Days 8-11 of pregnancy and extracted in 2.0% butanol-saline. To determine if these extracts could inhibit Prl secretion in vitro, dispersed anterior pituitary cells were incubated with placental extracts containing 1.0 placental equivalent obtained on each of Days 8-11 of pregnancy. Prl secretion was not affected by extracts of placentae obtained on Day 8 but was significantly inhibited by placental extracts obtained on Days 9-11 of pregnancy. In fact, progressively more mature placentae induced greater degrees of Prl inhibition. Extracts of placentae that were obtained on each of Days 8-11 of pregnancy, normalized on the basis of protein and tested for a 24-h period in the dispersed pituitary bioassay, caused the same degree of inhibition over Prl release. Additionally, placental protein from any given day (Days 8-11) of pregnancy induced a highly significant dose-dependent inhibition over Prl secretion. Equivalent amounts of a nonspecific protein, bovine serum albumin, had no effect. These findings indicate that the placenta does indeed contain a Prl inhibitory factor whose specific activity remains relatively constant between Days 8 and 11 of pregnancy. To determine if the inhibitory activity is humoral, maternal sera collected on each of Days 8-11 of pregnancy were placed in culture with dispersed pituitary cells at a concentration of 15.0%. Concomitant with gestational maturity, there was a progressively greater inhibition of Prl release. These findings indicate that the placenta may secrete a substance into the blood which suppresses Prl release directly at the level of the pituitary gland.

Animals↗

PubMed1985

Detection of prolactin inhibitory activity in uterine epithelial cell secretions and rat serum.

We have shown that the uterus of the rat contains a substance that diminishes the release of the luteotropic hormone PRL by acting directly at the anterior pituitary gland. This study was designed to determine which cell type(s) within the uterus secretes this PRL inhibitory activity (PIA) and if PIA of uterine origin appears in circulation. Enzymatically dispersed cells from uteri of ovariectomized (OVX) rats were cultured for 24 h in serum-free Dulbecco's Modified Eagle's Medium. Estimation of PIA in the spent media from heterogeneous uterine cell cultures was evaluated after 24 h by the ability to suppress PRL release from confluent monolayers of cultured pituitary cells. Spent media from uterine cell cultures containing 0.125 X 10(6) or 0.25 X 10(6) cells/well failed to significantly suppress PRL secretion. However, media from 0.5 X 10(6) and 1 X 10(6) uterine cells/well induced 36% and 85% inhibition of PRL release, respectively, without affecting basal LH release. To determine which cell type(s) in the uterus is responsible for PIA secretion, whole uteri were partitioned into epithelial, stromal, and myometrial cell fractions by differential enzymatic dissociation. Varying numbers of cells from each fraction were cultured for 24 h. PIA in the spent media from the homogeneous uterine cell cultures was estimated by its ability to suppress PRL release from cultured anterior pituitary cells. Media from epithelial cell cultures suppressed PRL secretion in a dose-dependent fashion. Media obtained from cultures of a comparable number of stromal or myometrial cells had no significant effects on PRL secretion. LH secretion was unaffected by media obtained from any concentration of the various uterine cell types. Since cultured anterior pituitary cells treated directly with crude uterine extract for 24 h recover and secrete PRL at the same rate as untreated controls during a 72-h posttreatment interval, it is unlikely that the uterine PIA is either proteolytic or cytotoxic. To determine if PIA is detectable in peripheral circulation, sera obtained from OVX or OVX hysterectomized (OVX-HYST) donor rats were incubated with cultured anterior pituitary cells for 24 h. In response to the OVX sera, there was significant inhibition of PRL release. However, upon replacement of OVX sera with OVX-HYST sera, the inhibition was significantly reduced. Thus, PIA is higher in sera of rats bearing uteri. Taken together, these data suggest that the epithelial layer of the uterus secretes a PIA which probably reaches the hypothalamo-pituitary axis through the circulation.

Animals↗