Influence of organochlorine pesticides on physiological potency of cultured granulosa cells from bovine preovulatory follicles

In the present study the direct effect of Dichlorodiphenyltrichloroethane (DDT), Methoxychlor (MXC) and Hexachlorocyclohexane (gammaHCH) was investigated on the DNA synthesis (measured by [3H]thymidine incorporation); proliferation (determined by colorimetric MTT[3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide] assay); and steroidogenesis (estradiol 17beta, progesterone determination) of granulosa cells during in vitro culture. The cells were aspirated from preovulatory follicles in PMSG-treated heifers. They were cultivated in phenol red and serum (0.1% FCS) RPMI 1640 medium without or with the 3 pesticides in different concentrations. In summary, the results showed differential inhibition of parameters named above by these pesticides (gammaHCH < MXC < DDT) in comparison with vehicle-treated control. Progesterone synthesis was more inhibited than estradiol 17beta production by 3 chemicals, which resulted from the loss of viability of cells indicated by estimation of cell-shape-factor and viability of cells. Besides the inhibitory effect, there was a stimulatory effect of low concentrations of DDT and MXC on [3H]thymidine incorporation and proliferation.     

Prostaglandin production by dispersed granulosa and theca interna cells from porcine preovulatory follicles

Prepubertal gilts were treated with 750 IU pregnant mares' serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa (GC) and theca interna (TIC) cells were prepared by microdissection and enzymatic digestion from follicles obtained 36, 72 and 108 h after PMSG treatment and incubated for up to 6 h in a chemically defined medium in the presence or absence of arachidonic acid, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and indomethacin. Production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. Both GC and TIC had the capacity to produce prostaglandins, with production by each cell type increasing markedly with follicular maturation. PGE was the major prostaglandin produced by both cellular compartments. Only PGE production by GC was consistently enhanced by addition of arachidonic acid to the incubation medium. Neither cell type was responsive to FSH and LH in vitro. Indomethacin inhibited the production of PGE and PGF by both cell types. These results provide convincing evidence for an intrafollicular source of prostaglandins and indicate that both cellular compartments contribute significantly to the increased production of prostaglandins associated with follicular rupture.     

Gonadotropin surge increases fluorescent-tagged low-density lipoprotein uptake by macaque granulosa cells from preovulatory follicles.

In the primate ovary, luteal steroidogenesis is largely dependent upon cholesterol derived from receptor-mediated uptake of circulating low-density lipoprotein (LDL). However, granulosa cells (GC) of preovulatory follicles possess few LDL binding sites compared to those present in developing and mature corpora lutea. We recently reported (Endocrinology 1991; 129:3247-3253) that uptake of LDL tagged with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) can be monitored in macaque luteal cells by fluorescence-activated flow cytometry. This study was designed to determine whether an ovulatory stimulus induced uptake of DiI-LDL in GC aspirated from preovulatory follicles of macaques undergoing ovarian stimulation. Development of multiple large follicles was stimulated in adult rhesus macaques with human gonadotropin treatment for 9 days. On Day 10, monkeys received either no ovulatory stimulus or 1000 IU hCG to initiate ovulatory events. GC were aspirated on Day 10 in monkeys receiving no ovulatory stimulus (nonluteinized GC) or 27 h or 34 h after hCG injection (luteinizing GC). GC were resuspended in Ham's F-10 medium + 0.1% BSA and incubated with several concentrations (0-25 micrograms/ml) of DiI-LDL (Biomedical Technologies, Stoughton, MA) for various time intervals (2-60 min). DiI-LDL uptake by GC was time- and concentration-dependent. Coincubation of cells with DiI-LDL and unlabeled LDL dose-dependently suppressed the percentage of fluorescent cells. In contrast, coincubation with up to a 250-fold excess of acetylated LDL or high-density lipoprotein did not alter the percentage of fluorescent GC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptors for insulin-like growth factor-I and tumor necrosis factor-alpha are hormonally regulated in bovine granulosa and thecal cells.

Mastitis induces release of tumor necrosis factor-alpha (TNFalpha) and has been linked with reduced reproductive performance. To further elucidate the role and mechanism of action of TNFalpha on ovarian cells, the effect of TNFalpha on insulin-like growth factor-I (IGF-I)-induced steroidogenesis and IGF-I binding sites in granulosa and thecal cells as well as the hormonal regulation of TNFalpha receptors were evaluated. Granulosa and thecal cells were obtained from small (1-5mm) and large (> or =8mm) bovine ovarian follicles, respectively, and cultured for 3-4 days. During the last 2 days of culture, cells were treated with various hormones and steroid production and specific binding of 125I-IGF-I and 125I-TNFalpha was determined. Two-day treatment with 30 ng/ml of TNFalpha decreased (P<0.05) IGF-I-induced estradiol production by granulosa cells and IGF-I-induced androstenedione production by thecal cells. Two-day treatment with 10 and 30ng/ml of TNFalpha decreased (P<0.05) specific binding of 125I-IGF-I to thecal cells, but had no effect on specific binding of 125I-IGF-I to granulosa cells, or on specific binding of 125I-IGF-II to thecal cells. TNFalpha did not compete for 125I-IGF-I binding to granulosa or thecal cells whereas unlabeled IGF-I suppressed 125I-IGF-I binding. Insulin inhibited (P<0.10) whereas FSH had no effect on the number of specific 125I-TNFalpha binding sites in granulosa cells. In contrast, LH increased (P<0.10) whereas insulin had no effect on specific 125I-TNFalpha binding sites in thecal cells. These results suggest that IGF-I and TNFalpha receptors in granulosa and thecal cells are regulated by hormones differentially.     

Morphological and biochemical identification of apoptosis in small, medium, and large bovine follicles and the effects of follicle-stimulating hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured bovine granulosa cells

The first objective of this study was to determine whether the death of bovine granulosa cells (GC) isolated from small ( 8 mm) follicles during follicular atresia occurs by apoptosis. The second objective was to establish an in vitro model system to elucidate the developmental (GC from follicles of different sizes) and hormonal (FSH and insulin-like growth factor-I [IGF-I]) regulation of bovine GC apoptosis during follicular atresia. Bovine ovaries were obtained from a nearby slaughterhouse. Follicles were classified by morphometric criteria as healthy or atretic. Apoptosis in GC from follicles of different sizes was analyzed by both morphological and biochemical methods. Bovine GC were cultured for 48 h at a density of 5 x 10(6) cells/ml in serum-free media at 39 degrees C to determine the effects of FSH and IGF-I on apoptosis. The results showed that apoptosis occurred in GC from all sizes of follicles. Apoptosis in GC was also detected in some healthy follicles. Degenerate GC displayed the morphological characteristics of apoptosis, including nuclei with marginated chromatin, a single condensed nucleus, multiple nuclear fragments, and/or membrane-bound structures containing variable amounts of chromatin and/or cytoplasm (apoptotic bodies). All GC classified as apoptotic on the basis of their morphology contained fragmented DNA measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique. Cells that had undergone apoptosis were observed mainly in GC and in scattered theca cells. Throughout the GC layer, apoptotic cell death was more prevalent among antral GC than among mural GC. Interestingly, morphological results showed that no apoptosis occurred in cumulus cells. A time-dependent, spontaneous onset of apoptosis occurred in GC from small, medium, and large follicles during in vitro serum-free culture. The rate of DNA fragmentation in the culture of GC from small follicles was higher than that from medium and large follicles. FSH attenuated apoptotic cell death in GC from medium follicles more effectively than in those from small follicles. IGF-I also suppressed apoptosis in cultured GC from small follicles. In conclusion, this study showed that 1) GC death during bovine follicular development and atresia occurs by apoptosis; 2) apoptosis occurs in GC and theca cells; however, apoptosis does not occur in cumulus cells even in atretic antral follicles; 3) GC from all small, medium, and large follicles undergo spontaneous onset of apoptosis when cultured under serum-free conditions; and 4) FSH and IGF-I can attenuate apoptosis in cultured bovine GC.     

Cellular distribution and relative amounts of vascular endothelium growth factor mRNA in granulosa cells from human preovulatory follicles.

Cells were obtained from patients undergoing in vitro fertilization. They were cultured and those producing vascular endothelium growth factor (VEGF) were detected by flow cytometry; relative amounts of mRNA were detected by RT-PCR and measured by PCR Elisa after RT-PCR products were biotinylated. Most of the granulosa cells produced VEGF. This production was maintained over 5 days in culture without adding hCG. The two diffusible forms, VEGF 121 and 165, were the most abundant. VEGF 145, which is specific to the reproductive system, was less abundant. VEGF 189, which is not freely secreted, was not produced by granulosa cells; small amounts were only detected in preparations containing leukocytes. TNF-alpha decreased VEGF production; the effect of TNF-alpha was neutralized by 10 nM staurosporine. Thus, the VEGF in human preovulatory follicles is mostly in the granulosa cells. These cells are therefore a major source of VEGF at ovulation and may play a key role in physiological and pathological processes which involve changes in vascular permeability and/or angiogenesis. The data also suggest that TNF-alpha via protein kinase C modulates the production of VEGF.     

Expression of insulin-like growth factor-I and its receptor by SV40-transformed rat granulosa cells

Cellular proliferation is a dominant aspect of ovarian follicular development in the rat, and insulin-like growth factor I (IGF-I) has been proposed as a mediator of cellular growth and differentiation in the ovary. An SV40-transformed rat granulosa cell line (RGA-41S) has been established as a model for studies on dividing cells of granulosa origin. Granulosa cells from the ovaries of immature diethylstilbestrol-treated rats were infected with the tsA255 mutant of SV40, followed by cloning in serum-free medium to select transformed cell lines which were serum independent. At the permissive temperature (33 C), RGA-41S cells exhibited a transformed phenotype and rapidly formed high density multilayers of compact cells that readily overgrew nontransformed cells. At the nonpermissive temperature (40 C) cell replication declined and division ceased after 4 days. Furthermore, at 40 C the cells grew as a monolayer and assumed a tetrahedral shape with a high cytoplasm-to-nucleus ratio, and displayed reduced ability to overgrow nontransformed cells. The transformed ovarian cells did not express detectable gonadotropin receptors and steroidogenic activity but retained their epithelial phenotype as demonstrated by cytokeratin staining of the cytoskeleton, the presence of microvilli, and the formation of tight junctions between cells. In support of the proposed autocrine-paracrine actions of IGF-I in the ovary, assay of conditioned serum-free culture medium revealed secretion of IGF-I-immunoreactive material by RGA-41S cells. HPLC-purified IGF-I immunoreactivity from these cells eluted with the same retention time as recombinant human IGF-I. When hybridized with a 32P-labeled rat IGF-I cDNA probe, poly(A)+ mRNA prepared from RGA-41S cells grown at both temperatures showed the typical three size classes of IGF-I mRNA on Northern blots (7.5, 1.7, and 0.8-1.2 kilobase kb), although the levels were somewhat higher at 33 C. The presence of IGF-I receptors in transformed cells was demonstrated by specific 125I-IGF-I binding to intact cells. Scatchard analysis indicated a single class of high affinity receptors at a density of 10(5) binding sites per cell and a dissociation constant (Kd) = 0.52 x 10(-9) M. Furthermore, hybridization of a 32P-labeled IGF-I receptor probe to Northern blots of poly(A+) RNA prepared from cells grown at 33 C and 40 C revealed an 11-kilobase rat IGF-I receptor mRNA. Physiological concentrations of IGF-I increased [3H]aminoisobutyric acid uptake by RGA-41S cells grown at either temperature, attesting to the retention of responsiveness to IGF-I in these transformed granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Control of proliferation and differentiation of ovine granulosa cells by insulin-like growth factor-I and follicle-stimulating hormone in vitro.

Granulosa cells of antral follicles both proliferate and undergo differentiation. The aim of the present work was to study the mechanisms controlling the balance between proliferation and differentiation in granulosa cells during the development of antral follicles in the ewe. For this purpose, the responses of both activities to insulin-like growth factor-I (IGF-I) and to FSH in vitro were studied comparatively in granulosa cells from small antral follicles (1-3 mm in diameter) and large antral follicles (5-7 mm in diameter). In granulosa cells from large follicles, IGF-I enhanced both basal and FSH-induced progesterone secretion after a 24-h delay period; this effect was lower and further delayed in cells from small follicles. Reciprocally, FSH increased IGF-I-stimulated progesterone secretion in cells from large follicles. IGF-I increased the thymidine labeling index of granulosa cells from small follicles within 24 h and enhanced cell multiplication. In cells from large follicles, this effect was lower and delayed, but IGF-I also enhanced cell survival. Culture at high density of plating inhibited the proliferative response of both types of cells to IGF-I. FSH was without effect on granulosa cell multiplication. These results suggest that the cytodifferentiative and the growth-promoting effects of IGF-I are clearly distinct. We propose that they would be exerted on two distinct granulosa cell subpopulations, nonproliferating and proliferating cells, respectively. Differences in the responsiveness of cells from small and large follicles could be related to differences in the proportion of these two cellular subtypes.     

Effects of growth hormone and insulin-like growth factor-I on development of in vitro derived bovine embryos

The objectives of this study were to determine whether the addition of growth hormone (GH) to maturation medium and GH or insulin-like growth factor-I (IGF-I) to culture medium affects development of cultured bovine embryos. We matured groups of 10 cumulus-oocyte complexes (COCs) in serum-free TCM-199 medium containing FSH and estradiol with or without 100 ng/ml GH. After fertilization, we transferred groups of 10 putative zygotes to 25 microl drops of a modified KSOM medium containing the following treatments: non-specific IgG (a control antibody, 10 microg/ml); GH (100 ng/ml) + IgG (10 microg/ml, GH/IgG); IGF-I (100 ng/ml) + IgG (10 microg/ml, IGF/IgG); antibody to IGF-I (10 microg/ml, anti-IGF); GH (100 ng/ml) + anti-IGF (10 microg/ml GH/anti-IGF); IGF-I (100 ng/ml) + anti-IGF (10 microg/ml, IGF/anti-IGF); no further additions (control). We repeated the experiment six times. Adding GH to the maturation medium increased cleavage rates at Day 3 compared to control (87.3 +/- 1.2% > 83.9 +/- 1.2%; P < 0.05) but had no effects on blastocyst development at Day 8. At Day 8, blastocyst development was greater (P < 0.01) for GH/IgG (24.8 +/- 2.5%) and IGF/IgG (33.7 +/- 2.5%) than for IgG (16.1 +/- 2.1%) and greater for IGF/IgG than for GH/IgG (P < 0.02). Blastocyst development at Day 8 did not differ between anti-IGF (20.4 +/- 1.8%) and GH/anti-IGF (24.1 +/- 1.9%) or IGF/anti-IGF (17.7 +/- 1.9%), but it was greater for GH/anti-IGF than for IGF/anti-IGF (P < 0.05). The Day 8 blastocysts of GH/IgG and IGF-I/IgG groups had a higher (P < 0.01) number of cells than the IgG group. The addition of anti-IGF-I eliminated the effects of IGF-I on cell number but did not alter GH effects. In conclusion, both GH and IGF-I stimulate embryonic development in cattle and GH effects may likely involve IGF-I-independent mechanisms.     

Differential production of steroids by dispersed granulosa and theca interna cells from developing preovulatory follicles of pigs

Dispersed granulosa and theca interna cells were recovered from follicles of prepubertal gilts at 36, 72 and 108 h after treatment with 750 i.u. PMSG, followed 72 h later with 500 i.u. hCG to stimulate follicular growth and ovulation. In the absence of aromatizable substrate, theca interna cells produced substantially more oestrogen than did granulosa cells. Oestrogen production was increased markedly in the presence of androstenedione and testosterone in granulosa cells but only to a limited extent in theca interna cells. The ability of both cellular compartments to produce oestrogen increased up to 72 h with androstenedione being the preferred substrate. Oestrogen production by the two cell types incubated together was greater than the sum produced when incubated alone. Theca interna cells were the principal source of androgen, predominantly androstenedione. Thecal androgen production increased with follicular development and was enhanced by addition of pregnenolone or by LH 36 and 72 h after PMSG treatment. The ability of granulosa and thecal cells to produce progesterone increased with follicular development and addition of pregnenolone. After exposure of developing follicles to hCG in vivo, both cell types lost their ability to produce oestrogen. Thecal cells continued to produce androgen and progesterone but no longer responded to LH in vitro. These studies indicate that several functional changes in the steroidogenic abilities of the granulosa and theca interna compartments occur during follicular maturation.     

Control of in vitro maturation of bovine cumulus-oocyte complex by preovulatory granulosa cells

The present study was designed to investigate (1) the influence of the secretions of follicular cells on the in vitro maturation of bovine cumulus-oocyte complexes (COCs) and (2) the origin of the factors controlling the metabolic function of cumulus cells during the preovulatory period. Preovulatory granulosa cells were collected from synchronized heifers either before or 7–9 hr after the luteinizing hormone (LH) surge, and their secretions were recovered after a 3 hr incubation. Follicular fluids (FFs) originating from the same follicles and sera from the same animals were also collected. The effects of FFs, sera, and secretions of granulosa cells on COC metabolism were compared during 24 hr of culture. FF stimulated cumulus expansion, progesterone secretion, and overall protein synthesis by COCs but decreased the amount of a major protein of 28 kDa. The time at which FF was collected influenced both cumulus expansion and protein synthesis by COCs. The effects of FF on COC metabolism were detected at the lowest protein concentration studied (0.073 mg/ml) and could be mimicked with serum, but only at a protein concentration 100-fold higher. The inhibitory effect of FF and serum on the amount of the 28 kDa protein was reproduced with the secretions of granulosa cells, acting at protein concentrations five- and 500-fold lower, respectively. However, the secretions of granulosa cells enhanced slightly cumulus expansion and had no effect on progesterone secretion and overall protein synthesis by COCs. These results suggest that COC metabolism is influenced both by endocrine and by local factors secreted by granulosa cells in response to gonadotropins. The paracrine control of COC metabolism by preovulatory granulosa cells could be exerted not only via intercellular contacts but also via substances secreted in FF. © 1993 Wiley-Liss, Inc.     

Effect of cell-to-cell contact on in vitro deoxyribonucleic acid synthesis and apoptosis responses of bovine granulosa cells to insulin-like growth factor-I and epidermal growth factor

Follicle development is the result of a balanced ratio between cell proliferation and cell death. Previous studies demonstrated differential mitotic responses to insulin-like growth factor (IGF)-I and epidermal growth factor (EGF) of cumulus cells (CC) and mural granulosa cells (MGC). Because cell-to-cell contact seems to modulate the occurrence of programmed cell death, the present experiments investigated the role of cell association in mediating apoptosis and the mitogenic responses to these growth factors of CC and MGC. Cumulus cells were cultured either as intact cumulus-oocyte complexes (COC) or after dissociation with EGTA + sucrose, in the presence of 50 ng/ml IGF-I, 5 ng/ml EGF, or both. Mural granulosa cells from the same follicles were similarly cultured either as cell aggregates or as dissociated cells. Synthesis of DNA was assessed by measurement of [(3)H]thymidine incorporation during the last 6 h of a 24-h culture in TCM199. Percentages of cells undergoing apoptosis were determined immunohistochemically in intact COC and GC aggregates, before and after dissociation as well as after the culture period. Epidermal growth factor and IGF-I stimulated DNA synthesis in both cell types; however, EGF inhibited the action of IGF-I in intact COC but not in MGC. Compared to nondissociated cells, dissociation resulted in a reduction of the mitogenic response of CC to both growth factors and of MGC to EGF. Unlike the response of intact COC to combined treatment with the two growth factors, dissociated CC displayed additive responses to the two growth factors in combination. Addition of denuded oocytes to cultures of dissociated CC enhanced both basal and growth factor-stimulated DNA synthesis but did not restore the inhibitory effect of EGF on the IGF-I response characteristic of intact COC. A significant proportion of intact MGC aggregates underwent apoptosis after 24 h of culture, while no increase of apoptotic cells was observed in intact COC. A dramatic increase in the percentage of apoptotic cells was observed in both CC and MGC when cell-cell contact was interrupted, and EGF and IGF-I were able to partially prevent its occurrence. Taken together these data showed that CC and MGC exhibit qualitatively and quantitatively different responses to IGF-I when cultured in the presence of EGF both in terms of DNA synthesis and onset of apoptosis. Moreover, the disruption of cell-cell contact was a major factor reducing cell proliferation and inducing apoptosis among both subsets of GC.     

Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles

Plasminogen activators (PAs) have been shown to be synthesized in ovarian follicles of several mammalian species, where they contribute to the ovulation process. The type of PA secreted by granulosa cells is species-specific. In fact, whereas in the rat, gonadotropins stimulate tissue-type PA (tPA) production, the same hormonal stimulation induces urokinase PA (uPA) secretion in mouse cells. To investigate in more detail the hormonal regulation of this system, we used the rat ovary as a model in which we analyzed the production of PAs by theca-interstitial (TI) and granulosa cells obtained from preovulatory follicles after gonadotropin stimulation. In untreated rats, uPA was the predominant enzyme in both TI and granulosa cells. After hormonal stimulation, an increase in uPA and tPA activity was observed in both cell types. Surprisingly, only tPA mRNA increased in a time-dependent manner in both cell types, while uPA mRNA increased only in TI cells and actually decreased in granulosa cells. These divergent results between uPA enzyme activity and mRNA levels in granulosa cells were explained by studying the localization of the enzyme. Analysis of granulosa cell lysates showed that after hormonal stimulation, 60-70% of the uPA behaved as a cell-associated protein, suggesting that uPA, already present in the follicle, accumulates on the granulosa cell surface through binding to specific uPA receptors. The redistribution of uPA in granulosa cells and the differing regulation of the two PAs by gonadotropins in the rat ovary suggest that the two enzymes might have different functions during the ovulation process. Moreover, the ability of antibodies anti-tPA and anti-uPA to significantly inhibit ovulation only when coinjected with hCG confirmed that the PA contribution to ovulation occurs at the initial steps.     

Overexpression of ovine insulin-like growth factor-I stimulates autonomous autocrine or paracrine growth in bovine mammary-derived epithelial cells.

To test the hypothesis that insulin-like growth factor-I (IGF-I) affects the growth of bovine mammary epithelial cells through an autocrine and/or paracrine pathway, a cell line (MD-IGF-I) was originated from MAC-T cells by cotransfection with a construct containing the cDNA for an ovine exon 2-encoded prepro-IGF-I under control of the mouse mammary tumor virus-long terminal repeat promoter. Clone MD-IGF-I contained multiple copies of the plasmid integrated into the genome, expressed the highest level of IGF-I mRNA, and secreted radioimmunoactive IGF-I into the medium. The mitogenic activity of MD-IGF-I cells was stimulated 80% by dexamethasone (DEX). The total DNA in MD-IGF-I cells was 2.5-fold higher than that in parental MAC-T cells in the presence of DEX. Conditioned medium from MD-IGF-I cells, induced with DEX, stimulated [3H]thymidine incorporation into DNA of MAC-T cells and uninduced MD-IGF-I cells. These data provide evidence that IGF-I was secreted into medium by MD-IGF-I cells. It is suggested that IGF-I can stimulate the growth of mammary epithelial cells by an autocrine and/or paracrine mode of action. The MD-IGF-I cell line may be a suitable system to study translational and posttranslational modifications of IGF-I peptides.     

Enhanced secretion of oxytocin from bovine granulosa cells treated with adrenal steroids

Bovine granulosa cells were exposed in vitro to various adrenal steroids (cortisol, cortisone, corticosterone, aldosterone; 1 mumol/l), in the presence and absence of stimulation by ascorbic acid (0.5 mmol/l), to determine the possible effects of these hormones on ovarian oxytocin and progesterone secretion. Only cortisol produced a consistent stimulation of the cells; the response was dose-related over the range 0.01 to 1.0 mumol/l and was greatly enhanced in the presence of ascorbate. The secretion of oxytocin was stimulated to a greater extent and with more consistency than was that of progesterone. Although the secretion of oxytocin could be stimulated by cortisol on the day of treatment, the cells also showed a delayed and persistent response to exposure earlier in the culture. It is concluded that cortisol may directly stimulate the secretion of ovarian oxytocin in the cow and that granulosa cells may respond in such a way as to smooth out the effects of short-term fluctuations in cortisol concentration.     

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.     

Regulation of amino acid transport and protein metabolism in myotubes derived from chicken muscle satellite cells by insulin-like growth factor-I

The effects of insulin and insulin-like growth factor-I (IGF-I) on amino acid transport and protein metabolism were compared in myotubes derived from chicken breast muscle satellite cells. Protein synthesis was assessed by continuous labelling with [³H]-tyrosine. Protein degradation was estimated by the release of trichloroacetic acid (TCA) soluble radioactivity by cells which had been previously labelled with [³H]-tyrosine for 3 days. Amino acid transport was measured in myotubes incubated in Dulbecco's modified Eagle's medium (DMEM) 0.5% bovine serum albumin (BSA) with or without insulin or IGF-I. Subsequent [³H]-aminoisobutyric acid (AIB) uptake was then measured in amino acid-free medium. IGF-I was more efficient than insulin at equimolar concentration (3.2 nmol/l) in stimulating protein synthesis (127 and 113% of basal, respectively) and inhibiting protein degradation (32% and 13% inhibition of protein degradation following 4 h incubation). Half maximal effective concentrations for stimulation of AIB uptake were 0.27 ± 0.03 nmol/l and 34.8 ± 3.1 nmol/l for IGF-I and insulin respectively, with maximal stimulation of about 340% of basal. Cycloheximide (3.6 μmol/l) diminished IGF-I-stimulated AIB uptake by 55%. Chicken growth hormone had no effect on basal AIB uptake in these cells and neither glucagon nor dexamethasone had an effect on basal or IGF-I-stimulated AIB uptake. This study demonstrates an anabolic effect for IGF-I in myotubes derived from primary chicken satellite cells which is mediated by the type I IGF receptor, since the cation-independent mannose 6-phosphate receptor does not bind IGF-II in chicken cells. © 1993 Wiley-Liss, Inc.     

Proliferation and differentiation of rat theca-interstitial cells: comparison of effects induced by platelet-derived growth factor and insulin-like growth factor-I

This study was designed to evaluate mechanisms regulating proliferation of steroidogenically active and steroidogenically inactive theca-interstitial (T-I) cells, and, specifically, to evaluate the effects of platelet-derived growth factor (PDGF) and insulin-like growth factor-I (IGF-I). T-I cells obtained from immature Sprague-Dawley rats were cultured in chemically defined media. Proliferation was assayed by thymidine incorporation and cell counting. Steroidogenically active cells were identified by the presence of 3beta-hydroxysteroid dehydrogenase activity. Flow cytometry facilitated separation of dividing cells (in S and G2/M phases of the cell cycle) from nondividing cells (in G0 and G1 phases of the cell cycle). PDGF alone (0.1-1 nM) produced a dose-dependent increase in DNA synthesis by up to 136%. IGF-I alone (10 nM) increased DNA synthesis by 56%. In the presence of both IGF-I (10 nM) and PDGF (0.1-1 nM), DNA synthesis increased by 108-214%. PDGF (1 nM) increased the total number of T-I cells by 43%; this effect was due to an increase in the number of steroidogenically inactive cells (47%). In contrast, the stimulatory effect of IGF-I (10 nM) was predominantly due to an increase in the number of steroidogenically active cells (163%). Separation of dividing cells from nondividing cells was accomplished with the aid of flow cytometry. In the absence of growth factors, the proportion of steroidogenically active cells was 35% lower among proliferating than resting cells. PDGF (1 nM) decreased the proportion of steroidogenically active cells among both proliferating and resting cells (by 43% and 16%, respectively). In contrast, IGF-I (10 nM) increased the proportion of steroidogenically active cells among proliferating cells by 56%. These findings indicate that differentiated/steroidogenically active cells divide; furthermore, PDGF and IGF-I may selectively stimulate proliferation of individual subpopulations of T-I cells, thereby providing a mechanism for development of structural and steroidogenically active components of the T-I compartment.     

Treatment with recombinant bovine somatotrophin enhances ovarian follicle development and increases the secretion of insulin-like growth factor-I by ovarian follicles in ewes

To investigate the effect of recombinant bovine somatotrophin (rGH) on ovarian folliculogenesis in sheep, 18 mature Scottish Blackface ewes were assigned randomly to two treatment groups. Starting from day 5 of the synchronised oestrous cycle, animals were injected daily with either vehicle (control group) or 12.5 mg rGH (rGH-treated group) for 7 days. Blood samples were collected once daily during the experimental period for the measurement of growth hormone (GH), insulin-like growth factor-I (IGF-I), insulin, follicle-stimulating hormone (FSH), luteinising hormone (LH) and progesterone. At the end of treatment animals were killed and ovaries collected. All follicles at least 1.0 mm in diameter were dissected out and diameters measured to assess follicular populations for individual animals. Five small follicles (1.0–3.4 mm in diameter) and all the large follicles (at least 3.5 mm) from each animal were incubated in 1 ml of Medium 199 for 1 h. Medium was then changed and incubation continued for a further hour. All medium samples were assayed for IGF-I, oestradiol, testosterone and progesterone.Treatment of ewes with rGH had no effect on the total number of follicles at least 1.0 mm in diameter (control, 34.4 ± 2.6; rGH-treated, 31.3 ± 1.4; P > 0.2). However, when follicles were further classified into different size categories (1.0–2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, 5.1–6.0 and over 6.0 mm in diameter), the population of follicles 2.1–3.0 mm in diameter was significantly increased by rGH treatment (control, 9.2 ± 0.7; rGH-treated, 13.8 ± 1.1; P = 0.02). The number of follicles of 3.1–4.0 mm diameter in the rGH-treated group tended to be increased (P = 0.09), whilst the population of follicles 1.0–2.0 mm in diameter was reduced (P = 0.07). Treatment of ewes with rGH significantly increased peripheral concentrations of GH (P < 0.01), IGF-I (P < 0.01), insulin (P < 0.01) and progesterone (P < 0.05). There was no effect of rGH treatment on circulating concentrations of FSH and LH. Both large and small follicles from rGH-treated ewes secreted significantly (P < 0.001) more IGF-I (37.8 ± 2.2 ng ml h⁻¹, n = 50) than follicles from the control group (26.7 ± 1.6 ng ml⁻¹ h⁻¹, n = 73). However, there was no significant effect of rGH treatment on the secretion of oestradiol, testosterone and progesterone by either large or small follicles.It is concluded that treatment of mature ewes with rGH can enhance the development of ovarian follicles to the gonadotrophin-dependent stages. Furthermore, rGH appears to act through increased secretion of ovarian IGF-I, as well as increased peripheral concentrations of IGF-I and insulin.