Modulating peripheral gonadotrophin levels affects follicular expression of mRNAs encoding insulin-like growth factors and receptors in sheep

Evidence suggests that the insulin-like growth factor (IGF) system is involved in follicular growth and development in sheep. However, little information exists as to the role that key peripheral factors play in regulating the expression of IGF components within the follicle. The present study investigated the regulatory effects of FSH and LH on gene expression for IGF ligands and receptors in ovine follicles, using bovine follicular fluid (bFF) and gonadotrophin-releasing hormone antagonist (GnRHa) model systems to perturb endogenous gonadotrophin secretion. Gene expression studies were carried out using in-situ hybridisation with sheep-specific ribonucleotide probes. Treatment of ewes with bFF had no effect on IGF-I mRNA levels. However, IGF-II mRNA levels, particularly in small follicles, and follicular type II IGF-R gene expression significantly increased following bFF administration (P<0.001). Conversely, there was a significant (P<0.001) decrease in type I IGF-R mRNA levels after only 12h of bFF treatment, especially in healthy follicles, although this was transient and was followed by a significant (P<0.01) increase in gene expression levels by 60 h of bFF treatment. Treatment of ewes with GnRHa resulted in a significant increase in mRNA levels encoding IGF-I (P<0.001), IGF-II in early atretic and large follicles (P<0.05), and type II IGF-R in healthy and early atretic follicles (P<0.001). In contrast, GnRHa administration decreased type I IGF-R gene expression levels after 60 h of treatment (P<0.001). These data highlight the roles that endogenous FSH and LH play in regulating IGF ligand and receptor gene expression in the sheep follicle.     

Co-expression of messenger ribonucleic acids encoding IGF-I, IGF-II, type I and II IGF receptors and IGF-binding proteins (IGFBP-1 to -6) during follicular development in the ovary of seasonally anoestrous ewes

In recent years, it has become apparent that components of the insulin-like growth factor (IGF) system are involved in the regulation of ovarian follicular development in sheep. The majority of previous studies have concentrated on investigating only a select few components and not the whole system. The aim of the present study was to use five seasonally anoestrous ewes to investigate the expression of mRNA encoding all 10 components of the sheep IGF system among various-sized follicles within the ovary, using sheep-specific ribonucleotide probes and in situ hybridisation. IGF-I mRNA expression was low and did not vary with follicle size. IGF-II mRNA expression was significantly higher (P < 0.05) in small follicles compared to large follicles. Both IGF receptors had significantly higher (P < 0.05) levels of mRNA expression in small follicles, with the type I receptor being expressed to a slightly greater extent than the type II receptor. IGFBP-2, -3, -4 and -5 gene expression followed a similar pattern to IGF-II and the IGF receptors, whereby expression decreased with increasing follicle size. Similar to IGF-I, IGFBP-6 mRNA expression showed little variation with follicle size. IGFBP-1 mRNA expression was observed at low and constant levels, albeit in small and medium-sized follicles only. These data demonstrate that all of the components of the IGF system are produced in the ovine follicle, and for some of the components, their gene expression varied with stage of follicle development. This study further emphasises the importance of IGF-II as the major IGF in the autocrine and paracrine regulation of follicle development in sheep.     

A role for LH in the regulation of expression of mRNAs encoding components of the insulin-like growth factor (IGF) system in the ovine corpus luteum

Evidence suggests the insulin-like growth factor (IGF) system may be involved in luteal maintenance and regression. However, previous studies have only investigated a few components of the system, primarily in bovine and non-ruminant species. The present study investigated gene expression for the components of the IGF system in ovine corpora lutea (CL) at various key stages of the oestrous cycle (Experiment 1), and the possible regulatory effects of LH on IGF gene expression in ovine CL using a GnRH antagonist model system (Experiment 2). Experiment 1 revealed that IGF-I (P<0.001), type I (P=0.008) and II (P=0.005) IGF-Rs and IGFBP-5 (P<0.05) mRNA levels were significantly elevated in early regressing CL. In contrast, IGF-II levels were high in CL but did not vary throughout the oestrous cycle, while IGFBP-2, -3, -4 and -6 mRNA levels were highest throughout the luteal phase but lower in regressing CL (P<0.05). IGFBP-1 mRNA could not be detected in any CL. Abrogation of LH action following GnRH antagonist administration (Experiment 2) resulted in a significant increase in expression for IGF-I (P<0.001), type II IGF-R (P=0.004) and IGFBP-5 (P<0.05) after only 12h, but these increases were transient. IGF-II, type I IGF-R and IGFBP-2, -3, -4 and -6 mRNA levels remained unaffected by GnRH antagonist treatment. These data highlight the role that LH plays in regulating IGF-I gene expression and lends further support that IGF-I may be a key luteotrophic factor in sheep.     

Follicular and luteal expression of insulin-like growth factors I and II and the type 1 IGF receptor in the bovine ovary.

The localization of mRNAs for insulin-like growth factors I (IGF-I) and II (IGF-II) and the type 1 IGF receptor (IGF-1R) in bovine follicles and corpora lutea was determined using in situ hybridization on sectioned ovaries collected from nonpregnant, cyclic Holstein cows in either the follicular (n = 3) or luteal (n = 5) phases of the cycle. Concentrations were measured as absorbance units of individual regions or follicles from autoradiographs. There was intense follicular expression of mRNAs encoding IGF-II and IGF-1R. For mRNA encoding IGF-II, expression was significantly higher in smaller follicles (< 5 mm diameter, P < 0.01) and, in this size range, expression was significantly greater in healthy compared with atretic follicles (P < 0.01). For mRNA encoding IGF-1R, there was no effect of size but concentrations were again significantly greater in healthy compared with atretic follicles of < 5 mm. In medium (5-10 mm) and large (> 10 mm) follicles, there was no effect of health for expression of either IGF-II or IGF-1R. mRNA encoding IGF-II was found exclusively in the theca, whereas mRNA encoding IGF-1R was confined to the granulosa layer. IGF-I expression was not detectable in 83% of the 53 follicles examined. In the remaining 17% of follicles, expression was very low and was unrelated to size or state of atresia. mRNAs encoding IGF-I, -II and IGF-1R were all present in the corpus luteum, whereas only those for IGF-II and IGF-1R were found in ovarian stroma. These data indicate that the insulin-like growth factors play a significant role in follicular and luteal development in the bovine ovary. Locally produced IGF-II is probably an important regulator of follicular growth, whereas most of the IGF-I present in follicular fluid is likely to be derived from the circulation.     

Expression of mRNA encoding insulin-like growth factors I and II and the type 1 IGF receptor in the bovine corpus luteum at defined stages of the oestrous cycle

Previous studies have implicated insulin-like growth factors I and II (IGF-I and -II), in the regulation of ovarian function. The present study investigated the localization of mRNA encoding IGF-I and -II and the type 1 IGF receptor using in situ hybridization to determine further the roles of the IGFs within the bovine corpus luteum at precise stages of the oestrous cycle. Luteal expression of mRNA encoding IGF-I and -II and the type 1 IGF receptor was detected throughout the oestrous cycle. The expression of IGF-I mRNAvaried significantly during the oestrous cycle. IGF-I mRNA concentrations were significantly higher on day 15 than on day 10, and IGF-I mRNA in the regressing corpus luteum at 48 h after administration of exogenous prostaglandin was significantly greater than in the early or mid-luteal phase (days 5 and 10). In contrast, there was no significant effect of day of the oestrous cycle on expression of mRNA for IGF-II and the type 1 IGF receptor in the corpus luteum. Expression of IGF-II mRNA was localized to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. mRNA encoding the type 1 IGF receptor was widely expressed in a pattern indicative of expression in large and small luteal cells. These data demonstrate that the bovine corpus luteum is a site of IGF production and reception throughout the luteal phase. Furthermore, this study highlights the potential of IGF-II in addition to IGF-I in the autocrine and paracrine regulation of luteal function.     

Acute dietary restriction in heifers alters expression of genes regulating exposure and response to gonadotrophins and IGF in dominant follicles

Dietary restriction in growing cattle and severe negative energy balance in lactating cows have been associated with altered gonadotropin secretion, reduced follicle diameter, reduced circulating oestradiol concentrations and anovulation. Therefore, we hypothesised that acute dietary restriction would influence the fate and function of the dominant follicle by altering the expression for genes regulating gonadotrophin and IGF response in ovarian follicles. Newly selected dominant follicles were collected 7-8 days after prostaglandin F(2α) (PGF) administration from heifers (n=25) that were individually fed a diet supplying 1.2 maintenance (M; control, n=8) or 0.4 M (restricted, n=17) for a total duration of 18-19 days. Heifers within 0.4 M were ovulatory (n=11) or anovulatory (n=6) depending on whether the dominant follicle present at PGF ovulated or became atretic following luteolysis. Control animals were all ovulatory. Acute dietary restriction decreased IGF-I (P<0.001) and insulin (P<0.05) in circulation; oestradiol (P<0.01) and IGF-I (P<0.01) in follicular fluid; and mRNA for FSHR (P<0.01) in granulosa cells but increased mRNA for IGFBP2 (P<0.05) in theca cells of the newly selected dominant follicle. However, this only led to anovulation when dietary restriction also decreased mRNA for CYP19A1 (P<0.05), IGF2 (P<0.01) and IGF1R (P<0.05) in granulosa cells and LHCGR (P<0.05) in theca cells of follicles collected from heifers fed 0.4 M. These results suggest that the catabolic environment induced by dietary restriction may ultimately cause anovulation by reducing oestradiol synthesis, FSH-responsiveness and IGF signaling in granulosa, and LH-responsiveness in theca cells of dominant follicles.     

Growth and the initiation of steroidogenesis in porcine follicles are associated with unique patterns of gene expression for individual componentsof the ovarian insulin-like growth factor system

Ovarian follicular growth and steroidogenesis are controlled by the interaction of insulin-like growth factors (IGFs) and gonadotropins. The objective was to determine the temporal and spatial relationships for gonadotropin receptor, steroidogenic enzyme, and IGF system gene expression during the development of preovulatory porcine follicles. Sows (n = 18) were weaned and follicles were monitored by transrectal ultrasonography. Ovaries were collected from sows when the mean diameter of the preovulatory follicular cohort was approximately 2, 4, 6, or 8 mm. mRNA were measured by in situ hybridization for individual follicles within the preovulatory cohort (3 to 5 follicles per sow). Patterns of gene expression detected by in situ hybridization were confirmed by RNase protection analyses of pooled RNA samples. The amount of LH receptor mRNA and steroidogenic enzyme mRNA (17alpha-hydroxylase and aromatase) increased as the mean diameter of the follicular cohort increased from 2 to 6 mm, but then decreased abruptly for 8-mm follicles. Estradiol concentrations in follicular fluid closely followed the expression patterns of steroidogenic enzymes and LH receptor mRNA. FSH receptor mRNA was present in cohorts of 2-mm follicles but declined in 4-mm follicles and was undetectable in 6- and 8-mm follicles. The expression of IGF-I and type I IGF receptor mRNA were similar for follicles of 2, 4, 6, and 8 mm. In contrast, IGF-II mRNA progressively increased in follicles collected from 2-, 4-, and 6-mm cohorts, and then decreased slightly at 8 mm. Type II IGF receptor mRNA was greatest in 8-mm follicles. IGF binding protein-2 (BP-2) mRNA decreased as follicles achieved progressively larger sizes during the preovulatory period (2 to 8 mm), whereas the IGFBP-4 mRNA remained relatively low for follicles in 2- to 6-mm cohorts but then increased markedly in 8-mm follicles. In summary, temporal and spatial patterns of gene expression for gonadotropin receptor, steroidogenic enzyme, and IGF system genes were characterized in preovulatory porcine follicles by using in situ hybridization and RNase protection analyses. The unique patterns of gene expression suggest interdependence among specific genes that may be essential for preovulatory follicular development.     

Regulation of the genes for insulin-like growth factor (IGF) I and II and their receptors by steroids and gonadotropins in the ovary

Insulin-like growth factors (IGFs) I and II are two single-chain polypeptide hormones that are structurally related to each other and to proinsulin. Among the large number of growth factors involved in ovarian physiology, IGF-I and IGF-II are considered to be important progression factors for ovarian follicular development. To explore the ovarian expression of IGF-I, IGF-II and their receptor genes, a solution hybridization/RNase protection assay, was used. IGF-I mRNA was seen in the granulosa cells, and IGF-II mRNA in the theca-interstitial compartment. To study the hormonal regulation of the IGF-I and IGF-II gene, immature (21-day-old) hypohysectomized rats were treated with FSH (10 μg/day),GH (150 μg/day) and diethylstilbestrol (DES subcutaneous implant/5 days). Estrogen differentially regulated ovarian IGF-I and IGF-II gene expression. In concert with GH, estrogen up-regulated ovarian IGF-I mRNA, but significantly decreased hepatic IGF-I gene expression. Both IGF receptors (type I and type II) as well as the insulin receptor gene, were expressed in both ovarian cells. The expression of the type IIGF receptor gene (but not the type II IGF gene) was up-regulated by FSH and estrogen in vivo. In conclusion, these studies may serve to better understand the auto paracrine role of IGF, and their receptors in the pathophysiology of follicle recruitment, oocyte maturation and potentially embryo development.     

Effect of follicle size on in vitro production of steroids and insulin-like growth factor (IGF)-I,IGF-II,and the IGF-binding proteins by equine ovarian granulosa cells

Little is known regarding the hormonal regulation of granulosa cell steroidogenesis and the ovarian insulin-like growth factor (IGF) system in the mare. The objectives of this study were to determine, first, if estradiol, insulin, and/or FSH affect steroid production by equine granulosa cells (experiment 1) and, second, if the components of the IGF system are produced by equine granulosa cells in culture as well as whether estradiol, insulin, and/or FSH affects IGF and/or IGF-binding protein (IGFBP) production by equine granulosa cells (experiment 2). Granulosa cells from small (6-15 mm), medium (16-25 mm), and large (25-48 mm) follicles were collected from cyclic mares (n = 14), cultured for 2 days in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 days in serum-free medium with or without added hormones. In experiment 1, large-follicle granulosa cells produced less progesterone and more estradiol than did medium- and/or small-follicle granulosa cells (P < 0.05). Progesterone production was inhibited (P < 0.05) by FSH and insulin in small- and medium- but not in large-follicle granulosa cells; estradiol was without effect. Insulin increased (P < 0.05) estradiol production in small- and medium-follicle granulosa cells but had no effect in large-follicle granulosa cells. In experiment 2, IGF-I production was inhibited (P < 0.05) by insulin across all follicle sizes but was not affected by estradiol or FSH. Granulosa cells of medium and large follicles produced more IGF-II than did granulosa cells of small follicles (P < 0.05). Insulin and FSH inhibited (P < 0.05) IGF-II production by granulosa cells of large and medium but not of small follicles; estradiol was without effect. Only IGFBP-2 and -5 were produced by equine granulosa cells. Production of IGFBP-2 was less (P < 0.10) in granulosa cells of large versus those of small and medium follicles, whereas medium-follicle granulosa cells produced more (P < 0.05) IGFBP-5 than did small- or large-follicle granulosa cells. Averaged across follicle sizes, estradiol increased (P < 0.05) IGFBP-2 production, FSH increased (P < 0.10) IGFBP-2 and -5 production, and insulin was without effect. These results indicate that IGF-I, IGF-II, IGFBP-2, and IGFBP-5 are produced by equine granulosa cells and that insulin, FSH, and estradiol play a role in the regulation of steroidogenesis and the IGF system of equine granulosa cells.     

IGF-Ⅰ及其受体、IGF结合蛋白-2和LH受体mRNA在卵泡中的表达

利用原位杂交和原位DNA－3’末端标记的方法研究了胰岛素样生长因子河（IG－I）、IGF－I受体、IGF结合蛋白－2、和促性腺激素受体的信使核糖核酸（mRNA）在不同生长与闭锁阶段的大鼠卵巢卵泡中表达的变化。结果表明：IGF－I主要在正常生长的初级卵泡、窦前卵泡和小窦状卵泡中表达。在各生长与成熟阶段的卵泡中都检测到IGF－I受体mRNA,闭锁卵泡的IGF－I受体表达降低。窦前与窦状的生长和闭锁卵泡均表达IGFBP－2。促卵泡激素（FSH）受体在窦前和小窦状卵泡的表达水平比其在大卵泡中的高。窦前与小窦状卵泡仅在膜细胞中表达黄体生成素（LH）受体mRNA,大卵泡的膜细胞与颗粒细胞均表达LH受体,在闭锁卵泡中仅在膜细胞中观察到LH受体的信号。综上结果,提示IGF－I,IGF－I受体和FSH受体在窦前和小窦状卵泡中的协同表达对卵泡的早期发育有重要作用。LH受体mRNA特异地在大卵泡的颗粒细胞中表达可能与优势卵泡选择相关。     

Involvement of growth hormone (GH) and insulin-like growth factor (IGF) system in ovarian folliculogenesis

During the last decade, involvement of growth hormone (GH), insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in ovarian folliculogenesis has been extensively studied. This review provides an update on the GH, IGF system and their role in ovarian follicular development. In vitro studies and knockout experiments demonstrated an important role of GH in preantral follicle growth and differentiation through their binding with GH receptors, which are located both in the oocyte and follicular somatic tissues. Furthermore, GH stimulates the development of small antral follicles to gonadotrophin-dependent stages, as well as maturation of oocytes. With regard to the IGF system, IGF-I has no effects on primordial follicle development, but both IGF-I and IGF-II stimulate growth of secondary follicles. Depending on the species studies and method used, these proteins have been detected in oocytes and/or somatic cells. In antral follicles, these IGFs stimulate granulosa cell proliferation and steroidogenesis in most mammals. The bioavailability of IGFs is regulated by a family of intrafollicular expressed IGF binding proteins (IGFBPs). Facilitation of IGF can be increased through the activity of specific IGFBP proteases, which degrade the IGF/IGFBP complex, resulting in the production of IGFBP fragments and release of attached IGF.     

Effect of a high maternal dietary intake during mid-gestation on components of the utero-placental insulin-like growth factor (IGF) system in adolescent sheep with retarded placental development

The aim of the present study was to investigate the effects of administering a high plane diet during early to mid-gestation on the uterine and placental insulin-like growth factor (IGF) system and on systemic IGF-I concentrations in pregnant adolescent ewes with restricted placental growth. Embryos recovered from superovulated ewes inseminated by a single sire were transferred in singleton to the uterus of adolescent recipients. After transfer ewes were offered a high (H) or moderate (M) amount of a complete diet calculated to promote rapid or normal maternal growth rates, respectively. Five ewes from each group were switched from either M to H or H to M diets at day 52 of gestation. Maternal and fetal blood samples and placental tissues were collected from all animals at day 104. Ewes on the high plane diet from mid-gestation (HH, MH groups) had restricted placental mass (P < 0.01) and tended to have smaller fetuses. This was associated with increased maternal plasma IGF-I concentrations (P < 0.001). The pattern of expression of components of the IGF system in the uterus and placenta was studied by in situ hybridization. IGF-I mRNA concentrations were below the limit of detection. IGF-II mRNA expression was high in the fetal mesoderm and present in maternal stroma, but was not influenced by nutritional treatment. In contrast, IGF binding protein 1 (IGFBP-1) mRNA expression was higher (P < 0.05) and IGFBP-3 mRNA expression was lower (P < 0.05) in the endometrial glands of ewes in HH and MH groups. In the fetal trophoblast, IGFBP-3 mRNA expression was higher in the MH group. Type 1 IGF receptor expression was increased (P < 0. 01) in the luminal epithelium of the HM group and IGFBP-2 mRNA expression was highest in the placentome capsule of ewes in the HH group. Together, these results indicate that reprogramming of the uterine and placental IGF axis by maternal nutrition could contribute to placental growth retardation in growing adolescent sheep.     

Tissue-specific expression of messenger ribonucleic acids for insulin-like growth factors and insulin-like growth factor-binding proteins during perinatal development of the rat uterus

Insulin-like growth factor (IGF)-I and IGF-II play a number of important roles in growth and differentiation, and IGF-binding proteins (IGFBPs) modulate IGF biological activity. IGF-I has been shown previously to be essential for normal uterine development. Therefore, we used in situ hybridization assays to characterize the unique tissue- and developmental stage-specific pattern of expression for each IGF and IGFBP gene in the rat uterus during perinatal development (gestational day [GD]-20 to postnatal day [PND]-24). IGF-I and IGFBP-1 mRNAs were expressed in all uterine tissues throughout this period. IGFBP-3 mRNA was not detectable at GD-20 but became detectable beginning at PND-5, and the signal intensity appeared to increase during stromal and muscle development. IGFBP-4 mRNA was abundant throughout perinatal development in the myometrium and in the stroma, particularly near the luminal epithelium. IGFBP-5 mRNA was abundantly expressed in myometrium throughout perinatal development. IGFBP-6 mRNA was detected throughout perinatal development in both the stroma and myometrium in a diffuse expression pattern. IGF-II and IGFBP-2 mRNAs were not detected in perinatal uteri. Our results suggest that coordinated temporal and spatial expression of IGF-I and its binding proteins (IGFBP-1,-3,-4,-5, and -6) could play important roles in perinatal rodent uterine development.     

Insulin-like growth factor (IGF)-binding protein-4 proteolytic degradation in bovine, equine, and porcine preovulatory follicles: regulation by IGFs and heparin-binding domain-containing peptides

We recently showed that insulin-like growth factor-binding protein-4 (IGFBP-4) proteolytic degradation in ovine preovulatory ovarian follicles is IGF-dependent and regulated by the heparin-binding domain (HBD) from IGFBP-3 and from connective tissue growth factor (CTGF), heparan/heparin-interacting protein (HIP), and vitronectin. The present study investigated regulation of IGFBP-4 proteolytic degradation in porcine, bovine, and equine ovarian preovulatory follicles. Follicular fluid from such preovulatory follicles contains proteolytic activity, degrading exogenous IGFBP-4. An excess of IGF-I enhanced IGFBP-4 degradation. In contrast, IGFBP-2 or -3 or monoclonal antibodies against IGF-I or -II dose-dependently inhibited IGFBP-4 degradation, and IGF-I or -II reversed this inhibition in a dose-dependent manner. Heparin-binding peptides derived from the C-terminal domain of IGFBP-3 or -5 inhibited IGFBP-4 degradation. Other heparin-binding peptides derived from CTGF, HIP, and vitronectin also inhibited IGFBP-4 degradation, except in porcine follicles. Finally, IGFBP-3 that was mutated in its HBD was less effective at inhibiting IGFBP-4 degradation. Thus, in bovine, porcine, and equine preovulatory follicles, IGFBP-4 proteolytic degradation both depends on IGFs and is inhibited by peptides containing HBD. Overall, these results suggest that during terminal development of follicles to the preovulatory stage in domestic animal species, the increase in IGF bioavailability might enhance IGFBP-4 degradation. In contrast, in atretic follicles, the decrease in IGF bioavailability, resulting partly from the increase in IGFBP-2 (sow, heifer, mare) and IGFBP-5 (heifer) expression would participate in the decrease of IGFBP-4 degradation. In bovine atretic follicles, IGFBP-5 would also strengthen the inhibition of IGFBP-4 degradation by direct interaction of its HBD with the protease. The involvement of other HBD-containing proteins in the modulation of intrafollicular proteases degrading IGFBP-4 remains to be investigated.     

Expression profile of IGF system during lung injury and recovery in rats exposed to hyperoxia: a possible role of IGF-1 in alveolar epithelial cell proliferation and differentiation

Although several studies have shown that an induction of insulin-like growth factor (IGF) components occurs during hyperoxia-mediated lung injury, the role of these components in tissue repair is not well known. The present study aimed to elucidate the role of IGF system components in normal tissue remodeling. We used a rat model of lung injury and remodeling by exposing rats to > 95% oxygen for 48 h and allowing them to recover in room air for up to 7 days. The mRNA expression of IGF-I, IGF-II, and IGF-1 receptor (IGF-1R) increased during injury. However, the protein levels of these components remained elevated until day 3 of the recovery and were highly abundant in alveolar type II cells. Among IGF binding proteins (IGFBPs), IGFBP-5 mRNA expression increased during injury and at all the recovery time points. IGFBP-2 and -3 mRNA were also elevated during injury phase. In an in vitro model of cell differentiation, the expression of IGF-I and IGF-II increased during trans-differentiation of alveolar epithelial type II cells into type-I like cells. The addition of anti-IGF-1R and anti-IGF-I antibodies inhibited the cell proliferation and trans-differentiation to some extent, as evident by cell morphology and the expression of type I and type II cell markers. These findings demonstrate that the IGF signaling pathway plays a critical role in proliferation and differentiation of alveolar epithelium during tissue remodeling.     

Insulin-like growth factor (IGF)-independent effects of IGF binding protein-4 on human granulosa cell steroidogenesis

The ovarian insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system operates to permit maximal stimulation of steroidogenesis in the dominant follicle. In atretic follicles, the predominant IGFBPs are IGFBP-2 and IGFBP-4, which appear to be selectively cleaved in healthy follicles. We have recently demonstrated potent inhibition by IGFBP-4 of both theca and granulosa cell steroid production. The degree to which the inhibition occurred suggested that it was greater than might be expected by sequestration of IGF alone. Our study was designed to test this idea. Granulosa cells were harvested from follicles dissected intact from patients undergoing total abdominal hysterectomy and bilateral salpingoophorectomy. Granulosa cells were incubated with or without gonadotropins and IGFBP-4 in the presence or absence of either the IGF type I receptor blocker alphaIR3 or excess IGFBP-3 to remove the effects of endogenous IGF action. Steroid accumulation in the medium was assessed. IGFBP-4 continued to exert potent inhibitory effects when the action of endogenous IGF was removed from the system, demonstrating that its actions are independent of IGF binding. There was no effect on cell metabolism, and the effects on steroidogenesis were reversible after IGFBP-4 removal from the culture medium. No similar effects were seen with IGFBP-2. These reasults are the first evidence of IGF-independent IGFBP-4 actions and the first evidence of IGF-independent actions of any IGFBPs in the ovary.