Synergistic action of growth hormone and insulin-like growth factor I (IGF-I) on proliferation and estradiol secretion in porcine granulosa and theca cells cultured alone or in coculture

The effect of growth hormone (GH) on insulin-like growth factor I (IGF-I) secretion and the effects of GH and IGF-I on [(3)H] thymidine incorporation and estradiol (E2) secretion by theca interna (Tc) and granulosa cells (Gc) cultured alone and in coculture were studied in cultured porcine follicular cells, prepared from small (SF), medium (MF) and large (LF) preovulatory follicles. We demonstrated that both Tc and Gc secrete IGF-I and that GH had no effect on IGF-I secretion by Tc but, increased IGF-I secretion by Gc isolated from SF and cultured alone or in coculture. IGF-I stimulated secretion of E2 by all cells, except in Tc derived from SF in which the effect was not statistically significant. The only stimulatory effect of concurrent treatment with GH on E2 secretion was noted in Tc derived from MF. IGF-I increased the [(3)H] thymidine incorporation in all Tc cells but GH did not augment this effect. In Gc, IGF-I stimulated [(3)H] thymidine incorporation in cells derived from SF and MF but not from LF. GH had no stimulatory effect except on Gc derived from LF and grown alone. The highest stimulatory effect was observed in SF. This was smaller in MF and no effect was noted in LF. In conclusion, our work shows that both Gc and Tc are sites of IGF-I production and for the first time shows the stimulatory role of IGF-I in proliferation of Tc cells derived from all types of follicles and augmentation of E2 secretion in Tc derived from MF and LF. The promotion of the mitogenic activity in Tc by IGF-I during all stages of follicular development suggests an important role for theca cells in follicular growth.     

Response of porcine theca and granulosa cells to GH during short-term in vitro culture

In Experiment 1, the influence of exogenous GH on steroid secretion by granulosa and theca interna cells recovered from small (1-3 mm), medium (4-6 mm) and large (8-12 mm) follicles was tested. In the second experiment, theca cells (Tc) and granulosa cells (Gc) obtained from large follicles were cultured separately or in two types, Tc/Gc co-culture, where both types of cells were mixed in one well or Gc and Tc were separated by cell culture membrane inserts. In the third experiment, the influence of GH on the morphology of Gc and Tc cells and activity of Delta(5),3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied. Cells were grown in the control medium (M199+5% of calf serum) or supplemented with 100 ng/ml GH. Testosterone (10(-7) M) was added as the aromatase substrate to granulosa cells cultures. The media were assayed after 48 h of culture for progesterone and oestradiol by RIA. GH added to the culture media had no effect on oestradiol and progesterone secretion by granulosa cells isolated from small and medium follicles while it stimulated both oestradiol and progesterone secretion by Gc isolated from large preovulatory follicles. A stimulatory effect on oestradiol secretion by Tc isolated from all size follicles was observed. GH did not stimulate progesterone secretion by Tc isolated from small follicles but stimulated progesterone secretion by Tc isolated from medium and large preovulatory follicles. Both co-culture systems exhibited synergistic effect on oestradiol secretion. The stimulatory effect on progesterone secretion under the influence of GH was observed in Gc cultured alone and Tc cultured alone. In contrast, the secretion of progesterone was attenuated in both co-culture systems and the addition of GH further augmented this attenuation. A statistically significant increase in oestradiol secretion was observed in all culture conditions. The addition of GH to the culture medium stimulated the activity of 3beta-HSD compared with the control culture from both types of cells. In conclusion, the present studies indicate that there are direct and follicular development stage dependent actions of GH on steroidogenesis of porcine follicular cells.     

Involvement of transforming growth factor alpha in the regulation of rat ovarian X-linked inhibitor of apoptosis protein expression and follicular growth by follicle-stimulating hormone

The expression of X-linked inhibitor of apoptosis protein (XIAP), a member of a family of intracellular antiapoptotic proteins, is induced by FSH during follicular development in vivo. Whether the XIAP up-regulation by FSH (100 ng/ml) is a direct action of the gonadotropin and is important in the control of granulosa cell proliferation during follicular growth is unclear. The overall objective of the present study was to examine whether the FSH-induced XIAP expression and granulosa cell proliferation during follicular development is mediated by the secretion and action of intraovarian transforming growth factor alpha (TGFalpha). In rat follicles cultured for 2 and 4 days, FSH stimulated estradiol production, TGFalpha secretion, XIAP expression, and follicular growth. The theca cells are the primary follicular source of FSH-induced TGFalpha, as indicated by in situ hybridization. Intrafollicular injection of a neutralizing anti-TGFalpha antibody (50-200 ng/ml; immunoglobulin G as control) or addition of estradiol-antagonist ICI 182780 (0.5-100 nM) to the culture media suppressed FSH-induced XIAP expression and follicular growth. The effect of ICI 182780 could be partially reversed by high concentrations of estrogen (250 and 500 nM). Whereas TGFalpha (10-20 ng/ml) significantly increased granulosa cell XIAP content and proliferation in primary granulosa cell cultures, FSH alone was ineffective in eliciting the mitogenic response. Our results support the hypothesis that FSH stimulates granulosa cell proliferation via theca TGFalpha secretion and action in response to increased granulosa cell estradiol synthesis, and that XIAP up-regulation in response to FSH suppresses granulosa cell apoptosis and facilitates FSH-induced follicular growth.     

The role of IGF-I, cAMP/protein kinase A and MAP-kinase in the control of steroid secretion, cyclic nucleotide production, granulosa cell proliferation and preimplantation embryo development in rabbits

The aim of this study was to investigate the actions of insulin-like growth factor I (IGF-I) on the secretory and proliferative functions of rabbit ovarian cells and on early embryogenesis. It was found that addition of IGF-I at a lower concentration (1 ng/ml) stimulated progesterone secretion by cultured rabbit granulosa cells, whilst higher concentrations of IGF-I (10, 100 ng/ml) were inhibitory. IGF-I had no effect on estradiol secretion. Cyclic AMP secretion was slightly increased after addition of IGF-I at 10 ng/ml, but not by higher concentrations. Cyclic GMP secretion was stimulated by IGF-I at 100 ng/ml only. A blocker of protein kinase A, Rp-cAMPS, did not alter progesterone and estradiol secretion but did prevent the action of IGF-I on progesterone secretion. An immunocytochemical study demonstrated that IGF-I significantly increased the proportion of proliferating cell nuclear antigen-positive (PCNA-positive) cells. Rp-cAMP did not change cell proliferation but partially prevented the proliferation-stimulating effect of IGF-I. IGF-I (100 ng/ml) significantly increased the proportion of divided zygotes and the number of embryos reaching the morula/blastocyst stage. Blockers of PKA, Rp-cAMPS and KT5720, reversed the effects of IGF-I on zygote cleavage and embryo development. Addition of IGF-I (100 ng/ml) significantly increased MAPK within the cells (proportion showing immunoreactivity to ERK-1 and ERK-3 antibodies and intensity of a 42 kDa band related to ERK-2). Rp-cAMPS suppressed the basal ERK-2 immunoreactivity but not that of ERK-1 or ERK-3. It completely inhibited the IGF-I-induced activation of ERK-3 but not that of ERK-1 or ERK-2. This in vitro study demonstrates that IGF-I is a potent stimulator of ovarian secretion, proliferation and embryogenesis in rabbit. Its effects are mediated by cAMP/PKA- and, probably by, MAPK-dependent intracellular mechanisms.     

Insulin-like growth factors (IGFs) as autocrine/paracrine regulators of granulosa cell differentiation and growth: studies with a neutralizing monoclonal antibody to IGF-I

Evidence that granulosa cells secrete and respond to insulin-like growth factors (IGFs) suggests, but does not prove, the importance of IGFs as intraovarian regulators. To further assess the role of these peptides in ovarian function, a neutralizing monoclonal antibody to IGF-I was employed to block the actions of IGFs in porcine follicular fluid and in granulosa cell-conditioned medium. In one series of experiments, granulosa cells from immature porcine follicles were cultured in medium containing porcine follicular fluid that had been charcoal-treated to remove steroids. As noted before, fluid from large follicles (LFF) stimulated progesterone production in a dose-dependent manner. The stimulatory effect of LFF (30% v/v) could be inhibited by greater than 50% by the anti-IGF monoclonal antibody. This inhibitory action was specific for the anti-IGF antibody and could be overcome by the addition of excess exogenous IGFs. In another series of experiments, granulosa cells were made dependent on endogenously produced IGFs by culturing them in a serum-free medium without exogenous growth factors. The effects of follicle-stimulating hormone (FSH), estradiol (E2), growth hormone (GH), and combinations thereof on progesterone production were inhibited by approximately 50% by the anti-IGF antibody. The effects of IGFs on indices of cell growth (judged by the criterion of being inhibited by the anti-IGF antibody) were less dramatic. A modest 18% increase in cell number was observed with FSH and E2 treatment in serum-free medium; this effect was virtually abolished by the antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of angiogenin on granulosa and theca cell function in cattle

Angiogenin is a member of the ribonuclease A superfamily of proteins that has been implicated in stimulating angiogenesis but whether angiogenin can directly affect ovarian granulosa or theca cell function is unknown. Therefore, the objective of these studies was to determine the effect of angiogenin on proliferation and steroidogenesis of bovine granulosa and theca cells. In experiments 1 and 2, granulosa cells from small (1 to 5 mm diameter) follicles and theca cells from large (8 to 22 mm diameter) follicles were cultured to evaluate the dose-response effect of recombinant human angiogenin on steroidogenesis. At 30 and 100 ng/ml, angiogenin inhibited (P<0.05) granulosa cell progesterone production and theca cell androstenedione production but did not affect (P>0.10) granulosa cell estradiol production or theca cell progesterone production, and did not affect numbers of granulosa or theca cells. In experiments 3 and 4, granulosa and theca cells from both small and large follicles were cultured with 300 ng/ml of angiogenin to determine if size of follicle influenced responses to angiogenin. At 300 ng/ml, angiogenin increased large follicle granulosa cell proliferation but decreased small follicle granulosa cell progesterone and estradiol production and large follicle theca cell progesterone production. In experiments 5 and 6, angiogenin stimulated (P<0.05) proliferation and DNA synthesis in large follicle granulosa cells. In experiment 7, 300 ng/ml of angiogenin increased (P<0.05) CYP19A1 messenger RNA (mRNA) abundance in granulosa cells but did not affect CYP11A1 mRNA abundance in granulosa or theca cells and did not affect CYP17A1 mRNA abundance in theca cells. We conclude that angiogenin appears to target both granulosa and theca cells in cattle, but additional research is needed to further understand the mechanism of action of angiogenin in granulosa and theca cells, as well as its precise role in folliculogenesis.     

Time dependent and cell-specific action of polychlorinated biphenyls (PCB 153 and PCB 126) on steroid secretion by porcine theca and granulosa cells in mono- and co-culture.

To characterize PCB action on follicular cell steroidogenesis two PCB congeners were selected as model substances. PCB 126 because of its dioxin-like configuration and high toxicity and PCB 153 because it is one of the most commonly detected PCB congeners in breast milk. The direct effect of PCBs was investigated using a culture system of porcine theca and granulosa cells collected from porcine preovulatory follicles. Granulosa and theca cells were cultured in M199 medium supplemented with 1, 10 or 100 pg/ml of PCB 126 or 1, 10 and 100 ng/ml of PCB 153. The media were changed after 48, 96 and 144 h and frozen until further estradiol (E2) analysis. Additionally, progesterone (P4) was measured in the granulosa cells culture medium and testosterone (T) in theca cells culture medium. Decrease of testosterone concentration in the theca cells culture medium was found after 96 and 144 hours in culture by both investigated PCB congeners. A decrease in E2 concentration was found after exposure to PCB 153. These findings suggest different actions of two congeners on the steroid synthesis in theca cells. The lack of an increase in E2 secretion after the exposure to PCB 126 could be due to depletion of androgen precursor. In granulosa cell culture PCB153 decreased E2 secretion and increased P4 secretion suggesting luteinization and disruption of aromatization process. PCB 126 in a doses from 1 to 10 pg had no effect on granulosa cells steroidogenesis. However, the highest dose (100 pg) increased concentration of both E2 and P4. This observation suggest that PCB 126 in a pharmacological doses may affect cell membrane permeability, thereby increasing steroid outflow into the medium. These results suggest time dependent and cell-specific differences in PCB 153 and 126 action on follicular cells steroidogenesis. Further studies are required to elucidate the mechanism of PCBs action on ovarian steroidogenesis.     

Differential regulation of pig theca cell steroidogenesis by LH, insulin-like growth factor I and granulosa cells in serum-free culture

The regulation of pig theca cell steroidogenesis was studied by the development of a physiological serum-free culture system, which was subsequently extended to investigate potential theca-granulosa cell interactions. Theca cells were isolated from antral follicles 6-9 mm in diameter and the effects of plating density (50-150x10(3) viable cells per well), LH (0.01-1.0 ng ml(-1)), Long R3 insulin-like growth factor I (IGF-I) (10, 100 ng ml(-1)) and insulin (1, 10 ng ml(-1)) on the number of cells and steroidogenesis were examined. The purity of the theca cell preparation was verified biochemically and histologically. Co-cultures contained 50x10(3) viable cells per well in granulosa to theca cell ratio of 4:1. Wells containing granulosa cells only were supplemented with 'physiological' doses of androstenedione or 100 ng ml(-1). Oestradiol production by co-cultures was compared with the sum of the oestradiol synthesized by granulosa and theca cells cultured separately. Oestradiol and androstenedione production continued throughout culture. High plating density decreased steroid production (P < 0.01). LH increased androstenedione (P < 0.001) and oestradiol (P < 0.05) synthesis and the sensitivity of the cells increased with time in culture. Oestradiol production was increased by 10 ng IGF-I ml(-1) (P < 0.001) but androstenedione required 100 ng ml(-1) (P < 0.001). Co-cultures produced more oestradiol than the sum of oestradiol synthesized by theca and granulosa cells cultured separately (P < 0. 001), irrespective of the androstenedione dose. This serum-free culture system for pig theca cells maintained in vivo steroidogenesis and gonadotrophin responsiveness. Thecal androstenedione and oestradiol production were differentially regulated and were primarily stimulated by LH and IGF-I, respectively. Theca-granulosa cell interactions stimulated oestradiol synthesis and this interaction was mediated by factors additional to the provision of thecal androgen substrate to granulosa cells.     

In vitro differentiation of bovine theca and granulosa cells into small and large luteal-like cells: morphological and functional characteristics

This study was undertaken to investigate whether bovine granulosa and theca interna cells could be luteinized in vitro into luteal-like cells. Granulosa and theca cells were cultured for 9 days in the presence of forskolin (10 microM), insulin (2 micrograms/ml), insulin-like growth factor I (100 ng/ml), or a combination of these agents. During the first day of culture, granulosa and theca cells secreted estradiol and androstenedione, respectively; progesterone rose only after 3-5 days in culture and reached a maximum on the ninth day of culture. Cells incubated in the presence of forskolin plus insulin exhibited morphological and functional characteristics of luteal cells isolated from the corpus luteum. It was found that cell diameter, basal and stimulated progesterone secretion, and pattern of cell replication for both cell types were comparable to those of luteal cells. Numerous lipid droplets and intensified mitochondrial adrenodoxin staining also indicated active steroidogenesis in luteinized cells. After 9 days in culture, stimulants were withdrawn, and the culture proceeded in basal medium for an additional 5 days; elevated progesterone levels were maintained by luteinized granulosa cells (LGC), whereas in contrast a dramatic drop in progesterone production was observed in luteinized theca cells (LTC). On Day 9, cells were challenged for 3 h with LH (10 ng/ml), forskolin (10 microM), or cholera toxin (100 ng/ml), resulting in a 4-fold increase in progesterone secretion by LTC; the same treatments failed to stimulate progesterone in LGC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autocrine role of transforming growth factor beta1 on rat granulosa cell proliferation

We evaluated the effects of transforming growth factor beta1 (TGFbeta1), alone or in combination with FSH and estradiol, on DNA synthesis in primary cultures of immature rat granulosa cells. 3H-Thymidine incorporation was significantly stimulated by TGFbeta1 (5.6-fold). This effect was enhanced by FSH (20 ng/ml, 27.7-fold) or estradiol (100 ng/ml, 13.4-fold) or by a combination of both hormones (59.2-fold). Measurement of TGFbeta bioactivity showed the presence of significant amounts of TGFbeta in conditioned medium from granulosa cell cultures, and most of the activity was present in the latent form. FSH alone or in combination with estradiol produced a marked suppression of the production of latent and active TGFbeta. Activated conditioned medium from control cultures of granulosa cell elicited a 1.4-fold increase in thymidine incorporation. This effect was markedly amplified by FSH (3-fold) and estradiol (4.3-fold) and by a combination of both (8.7-fold). The peptide containing the cell-binding domain of fibronectin (RGDSPC) partially inhibited thymidine incorporation stimulated by TGFbeta1. Fibronectin did not synergize with FSH, and the interaction between TGFbeta1 and FSH was even observed in the presence of this protein. The conclusions reached were as follows: 1) TGFbeta1 is an autocrine stimulator of rat granulosa cell DNA synthesis, 2) FSH and estradiol produce a suppression of latent and active TGFbeta production but markedly amplify TGFbeta action, presumably at a postreceptor level, and 3) the stimulatory effects of TGFbeta1 may be only partly mediated by the increased fibronectin secretion.     

Lipid synthesis and lipoprotein secretion by chick liver cells in culture: influence of growth hormone and insulin-like growth factor-I

1. Chick liver cells were incubated in unsupplemented medium (control), or medium supplanted with either 1 microgram/ml pituitary derived chicken growth hormone (GH), 50 ng/ml recombinant human insulin like growth factor-I (IGF-I), or 1 microgram growth hormone/ml and 50 ng insulin like growth factor-I/ml (GH + IGF-I). 2. GH supplementation stimulated acetate incorporation into liver cell lipid. Low density lipoprotein (LDL) lipid secretion was increased quantitatively by GH. 3. Cells incubated with IGF-I incorporated more acetate into lipid and secreted more lipid as VLDL and HDL than controls. 4. A metabolic antagonism between GH and IGF-I was evident with respect to lipogenesis. 5. Neither GH nor IGF-I altered, quantitatively, cell protein synthesis or apoprotein secretion.     

Effects of interferon on the steroidogenic functions and proliferation of immature porcine granulosa cells in culture.

Recent studies suggest the relevance of several cytokines to the growth and differentiation of granulosa cells. In the present study, we investigated the effects of interferon (IFN) on the steroidogenic functions and proliferation of immature porcine granulosa cells. Human IFN-alpha inhibited FSH-induced progesterone secretion in a concentration-dependent manner. The effect of IFN-alpha was significant at a concentration as low as 10 pg/ml. Maximal inhibitory concentrations (10-50 ng/ml) of IFN-alpha reduced FSH-induced progesterone secretion by 70%. In contrast, estradiol secretion induced by FSH was significantly enhanced by relatively high concentrations (1-50 ng/ml) of IFN-alpha. IFN-alpha (0.1-10 ng/ml) reduced cAMP generation in response to FSH by as much as 80%, although its effect was not concentration-dependent. The proliferation of cultured granulosa cells was inhibited by IFN-alpha in a concentration-dependent manner. Human IFN-gamma did not affect granulosa cell functions. The stimulation of estradiol secretion and the inhibition of cell proliferation induced by IFN-alpha in cultured porcine granulosa cells in this study are in contrast with the effects of IL-1, which, as we reported previously, inhibited both progesterone and estradiol secretion and stimulated cell growth in these cell cultures. Such differences in the mode of action of cytokines may contribute to the regulation of granulosa cell functions under physiological or pathological conditions.     

Somatomedins (insulin-like growth factors), but not growth hormone, are mitogenic for chicken heart mesenchymal cells and act synergistically with epidermal growth factor and brain fibroblast growth factor

Chicken, ovine or human growth hormones have no mitogenic effect on chicken heart mesenchymal cells, which are proliferatively quiescent at low culture densities in medium containing heparinized, heat-defibrinogenated rooster plasma at 10%. Sm-C/IGF-I (15 ng/ml; 2 nM), MSA/rIGF-II (50 ng/ml; 7 nM), insulin (10,000 ng/ml; 1750 nM) or proinsulin (16,000 ng/ml; 1750 nM), however, cause these cells to increase threefold in number during four days of incubation. While EGF alone at 100 ng/ml causes threefold multiplication at four days and brain FGF causes a sixfold increase, EGF acts synergistically with Sm-C/IGF-I, MSA/rIGF-II, insulin or proinsulin to cause 18-fold multiplication, and brain FGF acts synergistically with IGFs to cause 20-fold multiplication. EGF and brain FGF, however, show no mitogenic synergy. Addition to the plasma-containing culture medium of a monoclonal antibody to Sm-C/IGF-I nearly abolishes the mitogenic effect of added EGF or brain FGF but does not affect the autonomous (mitogenic hormone-independent) proliferation of RSV-infected chicken heart mesenchymal cells. These findings support the somatomedin hypothesis for growth hormone action and suggest that potentiation of the activity of other mitogenic hormones, like EGF and FGF, makes a significant contribution to control of cell proliferation by the GH/IGF axis.     

Mitogen-potentiating action and binding characteristics of insulin and insulin-like growth factors in Chinese hamster fibroblasts

alpha-Thrombin alone is able to stimulate DNA synthesis reinitiation of G0-arrested Chinese hamster lung fibroblasts (CC139) as well as continued growth of these cells in serum-free medium. Although insulin at high concentrations (1-10 micrograms/ml) is not intrinsically mitogenic for these cells, it potently enhances the growth-promoting action of thrombin. The generation time of CC139 cells in the defined medium, transferrin, alpha-thrombin, insulin, is around 15 h. To determine whether this effect of insulin is mediated via putative receptors for the insulin-like growth factors (IGFs) on these cells, we examined the abilities of two IGFs, Multiplication-Stimulating Activity (MSA) and IGF-I, to potentiate the thrombin-induced reinitiation of DNA synthesis. Both IGFs were found to be as effective as insulin for this biological effect; however, much lower concentrations were required to elicit half-maximal response, 100 ng/ml of MSA and 30 ng/ml of IGF-I. Detailed binding studies using 125I-labelled insulin, MSA, and IGF-I revealed that CC139 cells specifically bind all three polypeptides with IC50 values for the corresponding ligands of 1-2 ng/ml, 80-100 ng/ml, and 30-40 ng/ml, respectively. 125I-MSA binding was insulin-insensitive, whereas insulin did compete with 125I-IGF-I for binding to CC139 cells. These results indicate that CC139 cells possess at least two types of IGF receptors, an insulin-insensitive IGF receptor with high affinity for MSA which apparently mediates its biological effect, and an insulin-sensitive IGF-I receptor. Insulin appears to exert its mitogen-potentiating activity in CC139 fibroblasts by interacting with the IGF-I receptor.     

Morphological assessment of the effect of growth hormone on preantral follicles from 11-day-old mice in an in vitro culture system

The present study was carried out to verify that the cells attached to the outside of the basement membrane of mechanically isolated follicles are theca cells and to evaluate the effect of growth hormone (GH) on these cells. Preantral follicles, 100-140 micrometer in diameter, were mechanically isolated from 11-day-old BDF1 hybrid immature mice, divided randomly into two groups, and cultured in vitro. One group was treated with 0.1% collagenase immediately after mechanical isolation in an attempt to remove theca cells attached to the outside of the basement membrane. The other group was untreated. Morphological examination revealed that 86.1% of mechanically isolated follicles before collagenase treatment had at least one theca cell around the basement membrane on the single section. However, after collagenase treatment no theca cells remained on the basement membrane of the follicles. Androstenedione secretion as a result of stimulation by 100 ng/ml hCG was significantly higher in the culture medium of the follicles with theca cells than in those of collagenase-pretreated follicles (p < 0.0001), indicating that the cells attached to the outside of the basement membrane were actually functional theca cells, not interstitial cells. To elucidate the effect of GH on theca cells, preantral follicles cultured in the presence of 1.0 mIU/ml GH were morphologically examined. Preantral follicles mechanically isolated from immature mice showed significant proliferation of not only granulosa cells but also theca cells in the presence of GH. In particular, theca cells, which remained dotted on the basement membrane in a small number just after isolation, proliferated and finally formed complete layers after the culture with GH. This is the first report that GH induced the proliferation of theca cells to form morphologically complete layers around the preantral follicle from 11-day-old mice.     

Presumptive mediators of growth hormone action on insulin-like growth factor I release by porcine ovarian granulosa cells

The role of cAMP/protein kinase A (PKA)- and tyrosine kinase (TK)-dependent intracellular mechanisms in mediating the action of porcine growth hormone (GH) on insulin-like growth factor I (IGF-I) secretion by porcine ovarian granulosa cells was studied. It was observed that GH-induced stimulation of IGF-I secretion was accompanied by an increase in cAMP production. The stimulation of PKA by the addition of either a cAMP agonist or a phosphodiesterase inhibitor to the medium increased IGF-I release by the cells, indicating a direct stimulation of IGF-I release by cyclic nucleotides. Moreover, the stimulatory effect of GH on IGF-I was completely suppressed by the addition of the PKA blocker Rp-cAMPS. Neither TK blocker altered the basal IGF-I level, but both strongly suppressed the GH-induced increase in IGF-I accumulation. Taken together, these findings suggest that cAMP/PKA- and/or TK-dependent pathways may be involved in the mediation of GH action on IGF-I release by porcine granulosa cells.     

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells <Emphasis Type="Italic">in vitro</Emphasis>

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.