Biomedical subjects

J V May

Publications and source records attributed to J V May.

At least 19 recordsLinked to original sources

PubMed1996

Mechanisms of hormone and growth factor action in the bovine corpus luteum.

The binding of hormones and growth factors to their cell surface receptors leads to an orderly cascade of events leading to activation of cytoplasmic effector molecules. The mechanism of action of luteinizing hormone involves the stimulation of multiple signal transduction effector systems including adenylyl cyclase and inositol phospholipid-specific phospholipase C (PLC). This results in the formation of second messengers that activate cAMP-dependent, Ca(2+)-dependent and lipid-dependent protein kinases. Prostaglandin F(2alpha) activates PLC which increases intracellular calcium and activates protein kinase C. This results in the activation of a series of protein kinases in the mitogen-activated protein (MAP) kinase cascade, leading to the activation of nuclear transcription factors c-fos and c-jun. Hormone responsive effector systems, therefore, operate by activating families of protein kinases which regulate cell metabolism, secretion, and gene transcription. Growth factors activate specific receptor protein tyrosine kinases which recruit additional signaling molecules (phospholipase Cgamma, phosphatidylinositol 3-kinase, Shc, Grb2, etc.) initiating a cascade of events mediated via MAP kinases. The signaling pathways activated by hormones interact or cross talk with the signaling pathways activated by growth factors. The diversity of cellular signaling mechanisms elicited by hormones and the potential for interactions with signals generated by growth factor receptor tyrosine kinases, may allow fine tuning of cellular responses during the life span of the corpus luteum.

Journal Article↗

PubMed1996

Transforming growth factor beta expression in the porcine ovary: evidence that theca cells are the major secretory source during antral follicle development.

The transforming growth factors beta (TGF beta) have been implicated as important intrafollicular regulators of follicle development in the mammalian ovary. Recent studies in this laboratory have suggested that, when cultured, both porcine theca and granulosa cells secrete TGF beta, primarily TGF beta 1 (May et al., Endocrine 2:1045-1054, 1994). In this report, evidence is presented that during follicle development in vivo, theca but not granulosa cells are the source of follicular TGF beta. Although both theca and granulosa cells secreted TGF beta when attached to culture dishes, only theca cells secreted detectable levels of TGF beta when cells were cultured in serum-free medium without attachment. Granulosa cells secreted little if any TGF beta. This difference in TGF beta secretion was not found to be due to differences in preparation of the two cell types (i.e., mechanical versus enzymatic preparation). These results suggested that theca cells may be the source of TGF beta in the follicle. To ascertain whether TGF beta is actually secreted by follicles, intact hemi-follicle linings consisting of both theca and granulosa cells were cultured in moderate-term, organ explant culture. Hemi-follicle linings secreted TGF beta at a near linear rate for at least 4 days (approximately 300 pg/follicle/day for 6-8-mm-diameter follicles). The level of TGF beta secretion was directly related to the size of the follicle (p < 0.01). Immunoneutralization studies using TGF beta subtype-specific antibodies indicated that the major form of TGF beta secreted by porcine hemi-follicle linings was TGF beta 1. To further investigate the source of TGF beta during follicle development, a combination of molecular biology procedures was employed. Using 243-bp antisense and sense cRNA probes generated from a simian TGF beta 1 cDNA, we performed in situ hybridization on sections of whole porcine ovaries containing antral follicles. Expression of TGF beta 1 mRNA was localized to both theca and granulosa cells with no expression found in the stroma, suggesting that both cell types transcribe TGF beta 1. TGF beta 1 expression was evaluated additionally by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analysis. Oligonucleotide primers were generated from the porcine TGF beta 1 cDNA sequence for RT-PCR, and the PCR product (287-bp sequence) was amplified and used for Northern analysis. RT-PCR of total RNA isolated from theca and granulosa cells indicated that both cell types expressed TGF beta 1 mRNA. This finding was confirmed via Northern blot analysis, which further indicated the presence of two TGF beta 1 mRNAs of 2.5 and 3.5 kb, consistent with previous reports of alternate splicing of the porcine TGF beta 1 gene. These data indicate that both cell types express TGF beta 1 mRNA. To further evaluate TGF beta 1 expression, we attempted to isolate TGF beta 1 protein from freshly collected theca and granulosa cells by partial purification and immunoprecipitation. Interestingly, the growth factor could be extracted only from theca cells, not from granulosa cells, despite the presence of mRNA in both cell types. Taken together, these data suggest that whereas both theca and granulosa cells produce the TGF beta 1 gene product, only theca cells translate and secrete the actual growth factor. Thus, it is likely that the theca is the source of the growth factor during follicle development.

Animals↗

PubMed1995

Effects of epidermal growth factor on the tyrosine phosphorylation of mitogen-activated protein kinases in monolayer cultures of porcine granulosa cells.

We have examined porcine granulosa cells (pGCs) for the presence of immunodetectable mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinases, ERK) and have further studied the effects of epidermal growth factor (EGF) on the activation of these kinases. Cell lysates prepared from untreated monolayer cultures of pGCs were subjected to Western immunoblotting analysis using monoclonal antibodies to ERK1, ERK2 and pan-specific ERK. MAP kinases were detected having mol wts of 87K (ERK87), 54K (ERK54), 44K (ERK1), and 42K (ERK2). Treatment of pGCs with increasing concentrations (1-10 ng/ml) of EGF for 10 min resulted in electrophoretic mobility shifts of ERK1 and ERK2 suggesting hyperphosphorylation. Immunoprecipitation with an antiphosphotyrosine antibody (PY20), followed by Western analysis using pan-ERK, revealed a marked concentration-dependent increase in tyrosine phosphorylation of ERK2 in response to EGF treatment. The mobility shift and tyrosine phosphorylation of ERK2 was observed as early as 1 min after treatment with 10 ng/ml EGF. In-gel myelin basic protein (MBP) kinase assays revealed significant MBP kinase activity associated with ERK1 and ERK2 in total cell lysates and ERK2 in PY20 immunoprecipitates. Although ERK1 displayed a moderate mobility shift in response to EGF, tyrosine phosphorylation of this MAP kinase was not appreciably increased by EGF. Furthermore, PY20 immunoprecipitates demonstrated minimal MBP kinase associated with ERK1 in response to EGF treatment. Electrophoretic migration, tyrosine phosphorylation, and MBP kinase activity of the ERK54 and ERK87 was not effected regardless of EGF concentration or duration of treatment. These data demonstrate for the first time that pGCs contain immunodetectable MAP kinases. EGF, in a concentration- and time-dependent manner, increases tyrosine phosphorylation and MBP kinase activity (i.e. activation) of ERK2, and to a lesser degree ERK1, suggesting that the activation of MAP kinase may mediate the mitogenic action of EGF in pGCs.

Animals↗

PubMed1995

Differential involvement of protein kinase C in the regulation of transforming growth factor-beta (TGF-beta) secretion by porcine theca and granulosa cells in vitro.

Transforming growth factor-beta (TGF-beta) has been implicated in the regulation of ovarian follicle development. Little, however, is known regarding the regulation of TGF-beta expression within the follicle. To investigate this, granulosa and theca cells were isolated from small antral follicles of prepubertal porcine ovaries, maintained in monolayer culture, and treated with gonadotropins or intracellular activators of the protein kinase A and C pathways. TGF-beta secreted into the medium was measured using a proliferation inhibition bioassay with MvLu1 epithelial cells. Over a broad dose range, FSH and LH were ineffective in stimulating TGF-beta secretion relative to controls in granulosa and theca cells, respectively. Additionally, 8-bromo-cAMP, a direct activator of protein kinase A, was ineffective in stimulating TGF-beta secretion in either cell type. In marked contrast, PMA, a stimulator of protein kinase C, dose-dependently stimulated TGF-beta secretion in theca cells. Interestingly, however, PMA had virtually no effect upon granulosa cells. The stimulatory effect of PMA on theca cell TGF-beta secretion was not observed with the inactive derivitive 4 alpha-PMA, and the PMA effect was inhibited by chelerythrine chloride, an inhibitor with high specificity toward protein kinase C. Taken together, these results argue against a direct role of the protein kinase A pathway in the regulation of TGF-beta expression in porcine follicle cells and support direct involvement of the protein kinase C pathway. Moreover, there appears to be marked differences in the regulation of this growth factor between theca and granulosa cells.

Alkaloids↗

PubMed1993

Purified human alpha-fetoprotein inhibits follicle-stimulating hormone-stimulated estradiol production by porcine granulosa cells in culture.

We have investigated the effects of purified alpha-fetoprotein (AFP) on follicle-stimulating hormone (FSH)-stimulated estradiol production by porcine granulosa cells in monolayer culture. Granulosa cells isolated from small follicles of prepubertal pigs were cultured for 2 days in 5% fetal bovine serum for attachment and incubated for 3 days in medium containing androstenedione and various treatments. The media were then collected and assayed for estradiol by radioimmunoassay. Human AFP significantly (P < 0.05) and dose-dependently inhibited FSH-stimulated estradiol production with 313 ng/ml AFP returning FSH-stimulated estradiol to basal levels; human serum albumin was without effect. AFP purified from either term cord blood or midtrimester amniotic fluid dose-dependently inhibited estradiol production stimulated by the combination of FSH and insulin-like growth factor-I. Furthermore, 125 ng/ml AFP inhibited estradiol production stimulated by cholera toxin, forskolin and cAMP. In contrast, extracellular accumulation of cAMP and progesterone production was not inhibited by AFP. These data indicate that physiological concentrations of purified AFP significantly and dose-dependently inhibit FSH-stimulated estradiol production by porcine granulosa cells in culture. Since AFP is known to augment growth factor-mediated cell growth, these data suggest that AFP inhibits differentiated functions (such as aromatase) while enhancing the proliferation of porcine granulosa cells.

Amniotic Fluid↗

PubMed1993

The secretion of transforming growth factor-beta by bovine luteal cells in vitro.

Transforming growth factor-beta (TGF-beta), a multifunctional polypeptide growth factor, is produced by follicular cells in the ovary. However, there is little information indicating that TGF-beta is produced in the post-ovulatory follicle, i.e. the corpus luteum. In the present communication we present evidence that bovine luteal cells secrete large amounts of TGF-beta when maintained in moderate-term monolayer culture. Using TGF-beta 1 and TGF-beta 2 subtype-specific antibodies to neutralize the bioactivity it was found that 80-90% TGF-beta activity in luteal cell conditioned medium (LCCM) is due to TGF-beta 1, whereas < or = 10% TGF-beta activity in LCCM is due to TGF-beta 2. TGF-beta subtype nonspecific antibodies effectively and completely neutralized all TGF-beta activity in LCCM. The ratio of TGF-beta 1:TGF-beta 2 as estimated on the basis of neutralization studies was supported by visual observation of TGF-beta 1 and TGF-beta 2 protein bands on Western blotting. Using a modified and rapid mink lung epithelial cell bioassay and authentic TGF-beta to generate standard curves, the amount of TGF-beta secreted by luteal cells in vitro was quantitated. The concentration of luteal cell secreted proteins in the medium increased with time during 7 days of culture. Likewise, the TGF-beta concentration in LCCM increased during 7 days. To study the effect of duration of culture on the rate of TGF-beta secretion by luteal cells, conditioned medium was collected at 24 h intervals and replaced with fresh nutrient medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Purified human alpha fetoprotein inhibits growth factor-stimulated estradiol production by porcine granulosa cells in monolayer culture.

Purified alpha fetoprotein (AFP) synergizes with transforming growth factor alpha (TGF alpha) and insulin-like growth factor I (IGF-I) to enhance proliferation of porcine granulosa cells (pGC) in primary culture, suggesting a role for AFP in the modulation of growth factor-mediated cell growth. TGF alpha stimulates basal estrogen production by pGC and is in fact more potent than FSH in these cells. In this study, we investigated the effects of AFP on growth factor-stimulated estradiol (E2) production by pGC. Basal production of E2 was not altered by the addition of AFP. AFP dose-dependently inhibited TGF alpha-stimulated E2 production with statistically significant inhibition observed with 2.5 micrograms/ml. We have previously shown that the mitogenic effects of AFP are maximized with TGF alpha+IGF-I. E2 production was even more sensitive to AFP inhibition when the two growth factors were combined. Human serum albumin (HSA; 10 micrograms/ml) was without effect. AFP did not interfere with the E2 RIA, affect the uptake of or display specific in vitro binding of the androgen substrate. Furthermore, human AFP and HSA did not exhibit specific in vitro binding of E2, in contrast to purified rat AFP (positive control). These data indicate that physiological concentrations of purified AFP significantly and dose-dependently inhibit growth factor-stimulated E2 production by pGC in culture. Since AFP is known to increase TGF alpha+IGF-I mediated cell growth, these data suggest that AFP may be inhibiting the differentiated function (steroidogenesis) of pGC while enhancing the proliferation of these cells.

Amniotic Fluid↗

PubMed1992

The regulation of porcine theca cell proliferation in vitro: synergistic actions of epidermal growth factor and platelet-derived growth factor.

An important but poorly understood aspect of mammalian follicle development involves the regulation of theca cell proliferation. To investigate the premise that growth factors regulate theca cell proliferation, porcine theca cells were prepared by collagenase/DN'ase digestion of follicle linings after the removal of the granulosa cells and allowed to attach for 24 h. This method provided a monolayer of theca cells that had little if any granulosa cell contamination and which secreted high levels of androstenedione relative to granulosa cells during moderate-term culture (33-fold difference, P less than 0.01). In medium containing fetal calf serum (10%), theca cells were significantly more responsive to platelet-derived growth factor (PDGF) than epidermal growth factor (EGF) in terms of proliferation (13.4 +/- 0.2-vs. 7.0 +/- 0.1-fold increases relative to the initial cell count, P less than 0.05). This is in contrast to granulosa cells which were significantly more responsive to EGF than PDGF (7.1 +/- 0.1 vs. 4.0 +/- 0.2 fold-increases, P less than 0.05). Since serum has been shown to contain both EGF and PDGF, proliferation studies were performed using plasma-derived serum (PDS) which is growth factor restricted to examine more closely the direct effects of growth factors. In medium containing 0.25% PDS and within experiments, PDGF (1-25 ng/ml) stimulated theca cell proliferation in a dose-dependent manner (2.3-fold increase relative to controls, P less than 0.05) whereas EGF did not. EGF, however, markedly enhanced the proliferative action of PDGF (6.4-fold increase relative to controls, P less than 0.05). Insulin-like growth factor I and low density lipoprotein, factors which enhance markedly the proliferative effects of EGF and PDGF in terms of granulosa cell proliferation, exhibited only a modest synergistic effect with respect to EGF and PDGF upon theca cells (9.5-fold increase vs. a 6.4-fold increase above controls, P less than 0.05). Temporal studies in vitro indicate that theca cell proliferation is low during the first 3-day exposure to growth factors irrespective of treatment (a 2-fold increase over the seeding density). During the second 3-day exposure, however, theca cell proliferation increases 4- to 5-fold. The temporal pattern of theca cell proliferation stimulated by fetal calf serum supplemented with EGF or PDGF and PDS-containing medium supplemented with PDGF, EGF, insulin-like growth factor I, and low density lipoprotein is similar. These results suggest that PDGF is a major mitogen toward porcine theca cells and that EGF greatly enhances its activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Human alpha-fetoprotein purified from amniotic fluid enhances growth factor-mediated cell proliferation in vitro.

Using a primary monolayer culture of porcine granulosa cells (pGC) as an in vitro cell proliferation assay, we have examined the growth-promoting activity of alpha-fetoprotein (AFP) purified from term cord blood and midtrimester amniotic fluid. Increasing concentrations (2.5-20%) of crude human cord blood (CB) increased pGC proliferation, while identical concentrations of crude amniotic fluid (AF) were ineffective. When the cell system was maximally stimulated, AF dose dependently decreased cell proliferation. AFP purified from AF and CB (1.25-5.0 micrograms/ml) was not mitogenic alone, but, in the presence of epidermal growth factor (EGF) + insulin-like growth factor (IGF-I) (10 ng/ml each), AFP dose dependently increased cell proliferation to nearly double that of EGF + IGF-I alone. The response of pGC to the proliferative effects of AF-AFP and CB-AFP were identical at each dose of AFP tested. These results indicate that although crude, pooled midtrimester AF does not display the mitogenic activity seen in cord blood, AFP purified from pooled AF significantly synergizes with growth factors to increase cell proliferation markedly.

Amniotic Fluid↗

PubMed1991

Human mammary tumor cell proliferation: primary role of platelet-derived growth factor and possible synergism with human alpha-fetoprotein.

Human mammary medullary carcinoma cells (passages 16 to 21) were cultured for 2 days to allow for attachment, followed by 6 days of culture in either fetal calf serum, human cord blood, human amniotic fluid, or growth factors in the presence or absence of purified human alpha-fetoprotein (AFP). When growth factors were tested alone, only platelet-derived growth factor produced a significant increase in cell proliferation. Although up to 40% amniotic fluid had no effect on cell proliferation, human cord blood was two-fold more potent than fetal calf serum at similar concentrations. The addition of 10 ng/ml of platelet-derived growth factor increased the proliferative activity of human cord blood 1.5- to 2.5-fold. Ablation of endogenous AFP by affinity chromatography reduced the proliferative activity of cord blood by 75%. Similarly, the mitogenic activity of cord blood plus platelet-derived growth factor was reduced by 56% when AFP was removed. Purified AFP dose-dependently enhanced the proliferative activity of platelet-derived growth factor. This synergistic effect was specific for platelet-derived growth factor. We conclude that platelet-derived growth factor is a major growth factor controlling the proliferation of these tumor cells and that AFP may enhance growth factor proliferative activity and human mammary tumor growth.

Amniotic Fluid↗

PubMed1991

Synergistic action of purified alpha-fetoprotein and growth factors on the proliferation of porcine granulosa cells in monolayer culture.

alpha-Fetoprotein (AFP) is present in high levels in fetal fluids, certain neoplasias, and regenerating liver. Although AFP's physiological role remains an enigma, we have recently demonstrated mitogenic activity for AFP. Using a primary monolayer culture system, we have further investigated the proliferative activity of purified AFP. Porcine granulosa cells from small ovarian follicles were attached for 2 days in Ham's F-12-Dulbecco's Modified Eagle's Medium (1:1) and 5% fetal calf serum, followed by 6 days of culture in medium containing 0.25% plasma-derived serum plus 25 micrograms/ml low density lipoprotein with or without growth factors and/or purified human AFP. In this system AFP alone does not stimulate proliferation. However, when combined with epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I; 10 ng/ml each), AFP (5 micrograms/ml) significantly (P less than 0.01) enhanced growth factor-mediated proliferation 4.5-fold over that of medium controls. Equivalent doses of purified human serum albumin or transferrin demonstrated no effect. The effects of AFP were dose dependent, with significant (P less than 0.05) enhancement of proliferation (2.7-fold over controls) observed with as little as 0.313 micrograms/ml AFP. Increased proliferation was noticed as early as 24 h after the addition of AFP and by 48 h AFP, EGF, and IGF-I had significantly (P less than 0.05) increased proliferation over that seen in medium controls, cells treated with EGF plus IGF-I, or cells treated with 10% fetal calf serum plus EGF, and this trend continued linearly over 5 days of culture. AFP (5 micrograms/ml) significantly increased the proliferative response observed with increasing doses of EGF, IGF-I, or EGF plus IGF-I, but did not appear to alter the dose-response curves. AFP dose-dependently (1.25-5 micrograms/ml) and significantly (P less than 0.05) increased proliferation of porcine granulosa cells in response to platelet-derived growth factor (PDGF) and EGF (25 and 10 ng/ml, respectively), but not to PDGF alone. In contrast, AFP produced no further proliferation of porcine thecal cells in response to PDGF plus EGF. Binding of EGF, IGF-I, or PDGF to purified AFP could not be demonstrated. These results demonstrate that physiological levels of AFP, although not mitogenic alone, can significantly enhance the mitogenic activity of EGF plus IGF-I/PDGF and may function to modulate growth factor-mediated cell proliferation during development and neoplasia.

Animals↗

PubMed1991

A novel mechanism for the induction of aromatase in ovarian cells in vitro: role of transforming growth factor alpha-induced protein tyrosine kinase.

In the developing follicle, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are the primary stimulators of steroidogenesis in granulosa and theca cells. The steroidogenic actions of both these gonadotropins are largely if not exclusively mediated through cAMP. Previous studies have shown that EGF and TGF alpha do not affect basal estrogen production but attenuate FSH-stimulated estrogen production in vitro in rat granulosa cells. Here we present evidence that TGF alpha stimulates basal estrogen production in prepubertal porcine ovarian granulosa and theca cells in culture under defined conditions. In granulosa cells, TGF alpha is more potent than FSH in stimulating estrogen production. LH does not stimulate estrogen production in prepubertal porcine theca cells but rather attenuates that stimulated by TGF alpha. Treatment of follicular cells with genistein (inhibitor of protein tyrosine kinase) attenuates TGF alpha-induced estrogen biosynthesis suggesting that the action of TGF alpha is mediated through protein tyrosine kinase. These studies provide evidence for an alternative cAMP-independent and TGF alpha-induced tyrosine kinase-dependent mechanism for the induction of ovarian aromatase.

Animals↗

PubMed1990

A microcomputer data base system for an in vitro fertilization clinic programmed in BASIC.

A computer program was developed for the IBM personal computer to be used for in vitro fertilization and gamete intrafallopian transfer clinics. This program, written in BASIC, allows input, editing, updating, sorting, and printing of patient data. Statistical functions permit summation of patient data based on various combinations of user-defined treatment cycles, diagnoses, and protocols, thus making possible comparison of pregnancy and other patient data between and among various treatment groups and diagnoses. The statistical information can be continually updated and revised when new data become available on patients (such as confirmation of pregnancy by ultrasound or live births) and at the end of each cycle. The formats used are useful in assimilating individual clinic data for various surveys and other reporting requirements. The program can be easily modified by anyone with minimal training in the BASIC programming language.

Ambulatory Care Facilities↗

PubMed1990

Human alpha fetoprotein enhances epidermal growth factor proliferative activity upon porcine granulosa cells in monolayer culture.

Alpha fetoprotein (AFP) is present at high concentrations in fetal fluids, certain neoplasias, and regenerating liver. Its physiological function remains largely unknown. Using a primary monolayer culture system, we investigated the proliferative activity of human (h) cord blood (CB) and highly purified AFP. hAFP, purified from hCB by Cibacron blue and immunoaffinity chromatography was homogeneous on SDS-PAGE and silver stain. Porcine granulosa cells from ovarian small follicles were cultured (25,000/cm2) for 2 days in medium (Ham's F-12:DMEM, 1:1) + 5% fetal calf serum (FCS) to facilitate attachment, followed by 6 days in medium containing: FCS, hCB or h amniotic fluid (1-20%)+/- EGF (10 ng/ml); or 0.25% plasma-derived serum (PDS) containing human low density lipoprotein (LDL, 25 ug/ml), +/- AFP (0.05-5 ug), and +/- EGF and IGF-I (10 ng/ml). In this system, single growth factors do not stimulate proliferation, a characteristic also exhibited by AFP. When combined with EGF, however, AFP dose-dependently increased proliferation to levels equal to that obtained with 10% FCS (2.3-fold increase vs PDS/LDL controls). When combined with EGF+IGF-I, AFP again dose-dependently increased proliferation to levels equal to that obtained with 10% FCS+EGF (6.7-fold increase vs controls). Purified human albumin used in place of AFP was not effective. TGF-a but not PDGF could replace the proliferative activity of EGF. These results suggest that AFP at physiological levels, although not itself mitogenic, can enhance the mitogenic activity of EGF and TGF-a and may function to modulate growth factor-mediated proliferation during development and neoplasia.

Animals↗

PubMed1990

Regulation of granulosa cell proliferation: facilitative roles of platelet-derived growth factor and low density lipoprotein.

Recent studies suggest that epidermal growth factor (EGF) and/or transforming growth factor-alpha (TGF-alpha) and insulin-like growth factor-I (IGF-I) act synergistically to promote granulosa cell proliferation in vitro suggesting a similar role in vivo. Using a serum-restricted, monolayer culture system containing very low levels of platelet-poor plasma-derived serum (PPPDS), the facilitative roles of platelet-derived growth factor (PDGF) and low density lipoprotein (LDL) with respect to growth factor-stimulated granulosa cell proliferation were investigated. In nutrient medium containing only 0.1% PPPDS, PDGF (1-25 ng/ml) had no effect upon granulosa cell proliferation. When combined with EGF, which alone does not stimulate granulosa cell proliferation, PDGF dose-dependently increased cell proliferation to levels obtained with 10% fetal calf serum (2.4-fold increase relative to controls, P less than 0.05). When combined with EGF and IGF-I, a combination which does stimulate mitosis in granulosa cells, PDGF again dose-dependently enhanced proliferation (P less than 0.05). The extent of proliferation obtained with EGF + IGF-I + PDGF was consistently greater than that obtained with 10% fetal calf serum (P less than 0.05) but significantly less than that obtained with EGF + fetal calf serum, a treatment which stimulates rapid granulosa cell proliferation. LDL has been shown to greatly enhance granulosa cell steroidogenesis by providing exogenous cholesterol. However, cholesterol is also required for plasma membrane biosynthesis and cell growth. LDL alone, had no effect upon porcine granulosa cell proliferation relative to media controls (0.1% PPPDS) nor did it synergize with any single growth factor to induce mitosis. When combined with EGF + IGF-I, and EGF + PDGF, but not PDGF + IGF-I, LDL dose-dependently (1-25 micrograms/ml) enhanced proliferation (P less than 0.05) to levels equivalent to that obtained with 10% fetal calf serum. When combined with EGF, IGF-I, and PDGF, LDL at 10 micrograms/ml enhanced proliferation to an extent equivalent with EGF + fetal calf serum (a 5.4-fold increase relative to media controls). High density lipoprotein did not itself stimulate proliferation nor did it facilitate proliferation mediated by growth factors. When maintained in medium alone (0.1% PPPDS), the cell population doubling time was 8.0 +/- 0.5 days. In the presence of EGF, IGF-I, PDGF, and LDL (10, 10, and 5 ng/ml, and 10 micrograms/ml, respectively) the doubling time was reduced to 2.0 +/- 0.1 days.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

The production of transforming growth factor-beta in the porcine ovary and its secretion in vitro.

Porcine granulosa cells isolated from small (1-3 mm in diameter) follicles proliferate rapidly in culture in response to 10% fetal bovine serum (FBS) and epidermal growth factor (EGF) (10 ng/ml). Transforming growth factor-beta (TGF beta) inhibits FBS/EGF-stimulated proliferation in a dose-dependent manner. We have used this proliferation inhibitory property of TGF beta to assay qualitatively, the presence of this growth factor in conditioned medium from cultured follicle cells as well as in partially purified preparations from porcine ovarian compartments. In addition, the concentration of TGF beta in the theca cell conditioned medium was quantitatively estimated by generating a TGF beta-dose-response curve (inhibition of FBS/EGF-stimulated proliferation of granulosa cells in monolayer culture) using authentic human TGF beta-1. Ovarian thecal cells isolated from small and large size follicles in the pig ovary secrete TGF beta-like activity in vitro. Medium conditioned by thecal cells in primary monolayer culture contains a latent form of TGF beta which can be activated by heat or acid treatment. In contrast, and unlike rat granulosa cells, porcine granulosa cells in primary monolayer culture do not secrete detectable levels of TGF beta-like activity in the medium. Incubation of heat-activated thecal cell conditioned medium with a TGF beta-neutralizing antibody (which recognizes TGF beta-1 and 2) but not nonimmune serum attenuated the TGF beta-like activity in thecal cell conditioned medium suggesting that this activity is due to authentic TGF beta. Since many cell types secrete latent TGF beta in the medium when cultured in vitro, we next investigated whether thecal cell secretion of latent TGF beta was a function of cell culture or whether the ovarian thecal compartment actually contained detectable levels of TGF beta-like activity. To this end, we used an acid-ethanol extraction procedure to isolate thecal proteins from fresh-frozen tissue. The acid-ethanol extracted protein fraction was mixed with trace amounts of 125I-TGF beta for detection and chromatographed on Bio-Gel P-60 column under acidic conditions. Elution of TGF beta bioactivity from the Bio-Gel P-60 column as measured by inhibition of granulosa cell proliferation correlated with the elution of radioiodinated authentic TGF beta. Preincubation of TGF beta-like activity-containing fractions with TGF beta-neutralizing antibody attenuated the proliferation-inhibitory activity in these fractions. TGF beta activity was also observed in fractions extracted from porcine corpora lutea.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Ovarian transforming growth factor-beta (TGF beta): cellular site(s), and mechanism(s) of action.

It is the objective of the experiments reported herein to examine the possible relevance of transforming growth factor-beta (TGF beta) to theca-interstitial cell function, and to further characterize the established interaction of TGF beta with the granulosa cell. In examining the interaction of TGF beta (10 ng/ml) with murine theca-interstitial cells, no significant effect was observed on either basal or human chorionic gonadotropin (hCG)-stimulated androsterone accumulation. In contrast, given murine granulosa cells, TGF beta (10 ng/ml) produced dose- and time-dependent augmentation of follicle-stimulating hormone (FSH)-supported aromatase activity with a minimal and median effective doses of 20 +/- 3 and 123 +/- 24 pg/ml, respectively and a minimal time requirement of less than or equal to 48 h. The ability of TGF beta to augment FSH hormonal action could not be accounted for by alteration(s) of specific FSH binding (13965 +/- 298 and 12614 +/- 694 cpm/4 X 10(5) cells for FSH and FSH + TGF beta). However, TGF beta proved capable of exerting a direct upregulatory effect on stimulatable adenylate cyclase activity, further enhancement occurring at site(s) distal to cAMP generation (dibutyryl cyclic AMP (Bt2cAMP) = 1.4 +/- 0.2 ng/culture; Bt2cAMP + TGF beta = 4.1 +/- 0.6 ng/culture). Taken together, our findings are in keeping with the notion that TGF beta, possibly of intraovarian origin, comprises the central signal of autocrine or paracrine loop(s) capable of amplifying gonadotropin action at the level of the granulosa, but not theca-interstitial cell.

Adenylyl Cyclases↗

PubMed1988

Basic fibroblast growth factor as a regulator of ovarian granulosa cell differentiation: a novel non-mitogenic role.

The role of basic fibroblast growth factor (bFGF) in ovarian granulosa cell differentiation was investigated in vitro. To this end, use was made of a primary culture of rat granulosa cells the differentiation of which was monitored by the acquisition of aromatase activity. Concurrent treatment with highly purified bFGF (10 ng/ml) produced a significant (P less than 0.05), albeit reversible inhibition (88 +/- 6%) of FSH (but not basal)-supported aromatization. Although independent of the FSH dose employed and of cell density, bFGF-attenuated aromatase activity proved dose-dependent, with a projected minimal effective dose of 0.11 +/- 0.03 ng/ml (7.5 +/- 2 pM), and an apparent median inhibitory dose of 0.63 +/- 0.09 ng/ml (43 +/- 6 pM). Unaccounted for by alterations in granulosa cell number, plating efficiency, or viability, the ability of bFGF to attenuate FSH hormonal action proved partly attributable to site(s) of action distal, rather than proximal to cAMP generation. Taken together, these observations indicate that the cytodifferentiative and replicative actions of bFGF in the granulosa cell can be dissociated, and lend additional support to the prospect that bFGF, possibly of intraovarian origin, may play a role in granulosa cell differentiation in the course of their ontogeny.

Animals↗