Human Reproduction, Vol. 15, No. 6, 1241-1246,
June 2000
© 2000 European Society of Human Reproduction and Embryology
Growth hormone does not increase the expression of insulin-like growth factors and their receptor genes in the pre-menopausal human ovary
1 Institut Clinic of Gynecology, Obstetrics and Neonatolgy and 2 Hormonal Laboratory, Faculty of Medicine–University of Barcelona, Hospital Clínic–Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, 3 Instituto de Bioquímica, CSIC, Facultad de Farmacia, Madrid and 4 Clínica de Reproducción Asistida FIV–Madrid, Madrid, Spain
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Abstract |
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A growing body of information now supports the existence of a complete intraovarian insulin-like growth factor I (IGF-I) system. Although the precise role of IGF-I in the context of ovarian physiology remains to be determined, it is likely that IGF-I may engage in the amplification of gonadotrophin hormonal action. These facts and experiments with animals establishing the ovaries of multiple species as a site of growth hormone (GH) reception and action have led to the use of recombinant GH (rGH) as an adjunctive agent to potentiate ovulation induction by exogenous gonadotrophins. Whether intraovarian IGF-I plays an intermediary role in GH hormonal action at the ovarian level remains uncertain at present. The aim of this study was to evaluate whether rGH administration to pre-menopausal women could modify the expression of the IGF-I gene in the ovary. The expression of the IGF-I gene was examined in a time-dependent manner in normal pre-menopausal ovaries obtained from nine women treated with rGH and nine control women treated with placebo, using solution hybridization/RNase protection assays. Ovarian tissue samples were obtained 24 h (six women) and 7 days (12 women) following rGH/placebo injection. Total RNA (20 µg) from whole pre-menopausal ovaries (with or without rGH treatment) as well as from human granulosa cells was hybridized with a human IGF-I antisense RNA. IGF-I peptide, but not oestradiol, serum concentrations increased significantly 24 h after rGH injection. IGF-I gene, however, was not expressed in the luteinized granulosa cells and whole pre-menopausal ovaries irrespectively of rGH treatment in ovarian samples analysed both 1 and 7 days following rGH injection. On the contrary, IGF-II mRNA transcribed from the fetal or fetal–neonatal IGF-II promoter and IGF-I receptor mRNA (both used as hybridization control) were both found in whole pre-menopausal ovary and luteinized granulosa cells. Nevertheless, no changes in the hybridization patterns were seen in the absence or presence of rGH. These studies demonstrate that rGH administration to normal premenopausal women does not change the expression of insulin-like growth factors and their receptor genes in the pre-menopausal human ovary. Furthermore, these results provide further evidence against locally produced IGF-I as responsible for any ovarian effects seen in systemic rGH administration.
Key words: growth hormone/human ovary/insulin-like growth factors/IGF-1 receptor/ovulation induction
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Introduction |
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During recent years there has been a growing interest in the potential use of exogenous recombinant growth hormone (rGH) as an adjunctive agent to potentiate ovulation induction by exogenous gonadotrophins both in anovulatory patients and normally cycling women subjected to ovarian stimulation for IVF (Homburg et al., 1990a
; Blumenfeld, 1991; Jacobs et al., 1991; Christman and Halme, 1992; Katz, 1993; Katz et al., 1993; Adashi, 1994; Homburg and Ben-Rafael, 1996; Howles et al., 1999). This has been based on experiments with animals establishing the ovaries of multiple species as a site of GH reception and action (Jia et al., 1986; Manson et al., 1990; Katz et al., 1993; Adashi et al., 1997).
The actions of GH in the ovary could be manifested interaction with putative GH receptors localized in the human granulosa but not theca cells (Sharara and Nieman, 1994) resulting in direct modulation of the actions of gonadotrophins. However, the main mechanism by which GH acts in vivo on different tissues is a secondary one; rather than directly eliciting a tissue response, GH causes the production of insulin-like growth factor-I (IGF-I) (Davoren and Hsueh, 1986). Although traditionally viewed as a hepatic product, it is now well established that IGF-I may also be synthesized in a variety of extra-hepatic sites wherein it may play auto/paracrine roles (Daughaday and Rotwein, 1989). A large body of experimental evidence exists for an intraovarian IGF system, and direct effects of IGF-I have been described on ovarian somatic cells, mainly in the rat (Adashi et al., 1985, 1991; Katz et al., 1993). IGF-I is capable of substantial amplification of gonadotrophin hormonal action when examined in vitro at the level of either the granulosa or the thecal interstitial cell (Adashi et al., 1985). In addition, it has previously been reported that GH (in concert with oestrogens) may up-regulate the generation of IGF-I by the murine granulosa cells (Hernandez et al., 1989). Therefore, GH may be acting indirectly by stimulating ovarian IGF gene expression which in turn could modulate (augment) gonadotrophin action on somatic cells.
Whether intraovarian IGF-I plays an intermediary role in the action of GH at the ovarian level, however, remains uncertain at present and the need for additional studies to clarify this central issue has recently been stressed (Katz et al., 1993; Homburg and Ben-Rafael, 1996). Furthermore, previous studies (Ramasharma and Li, 1987; Voutilainen and Miller, 1987; Geisthoevel et al., 1989; Hernandez et al., 1992) showed that at the level of the human ovary, the granulosa cell may be a site of IGF-II rather than IGF-I gene expression. The biological actions of IGF-II in the human ovary, however, are mediated through the type-I IGF receptor as recently demonstrated by Willis et al. (1998). These facts contrast sharply with the highly compartmentalized expression of the IGF-I and IGF-II genes in the murine ovary, wherein it was found that IGF-I is restricted to and acts upon granulosa cells (Hernandez et al., 1989), and IGF-II gene expression is solely found in thecal–interstital compartment (Hernandez et al., 1990).
To further clarify the role (if any) of the intraovarian IGF-I as a mediator of GH action in the human, the present study was undertaken to investigate whether rGH administration to pre-menopausal women could modify the expression of the IGF-I gene in the ovary. IGF-II and IGF-I receptor probes were used as hybridization control in this study.
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Materials and methods |
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Study protocol and tissues
Tissue samples of normal pre-menopausal ovaries were obtained from 18 women undergoing total abdominal hysterectomy for non-ovarian indications. Patients gave informed consent to be included in the study protocol which was approved by the Spanish Health Authorities and the Ethics Committee of our hospital. Women were aged between 38 and 45 years and all of them had regular menstrual cycles every 26–32 days.
Experimental data in rats indicate that gene expression in target tissues is increased shortly (within 24 h) after an acute administration of rGH (Murphy and Friesen, 1988; Hepler et al., 1990). Accordingly, IGF-I and oestradiol serum concentrations were investigated in four normal ovulatory women aged 29–34 years who were awaiting intracytoplasmic sperm injection in the IVF programme due to severe male infertility. Each woman received 24 IU of rGH s.c. (Genotonorm; Pharmacia Farmitalia, S.A., Madrid, Spain) on cycle day 5. This rGH dose was used on the basis of previous clinical studies showing that the single-dose regimen is virtually indistinguishable from the multiple-dose variety in terms of inducing ovarian differentiation and enhancement of gonadotrophin action by rGH (Homburg et al., 1990b). Blood sampling was performed immediately before rGH administration (time 0) and 2, 4, 6, 8, 12, 24, 30, 36 and 48 h after rGH injection. Whereas serum oestradiol concentrations were similar throughout the study period, IGF-I serum concentration showed a peak 24 h after rGH injection (Figure 1).
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The above notwithstanding, the rapid and transient increase in IGF-I but not oestradiol serum concentrations favours the hepatic origin of IGF-I increase. Furthermore, co-treatment with rGH and gonadotrophins for ovulation induction has been reported to be associated with an increase in serum IGF-I concentrations reaching a peak between 5 and 10 days of treatment (Homburg et al., 1990a
) and additional clinical studies have shown that the ovarian sensitizing effect of rGH to exogenous gonadotrophins persists over several subsequent treatment cycles without reinforcement, even for as long as 7 months in some patients (Burger et al., 1991). Therefore, a time course experiment was planned investigating the potential effect of rGH injection on ovarian IGF-I mRNA both at 24 h and 7 days following rGH administration. To this end, six women were randomly allocated to receive either 24 IU of rGH (n = 3) by s.c. injection or a placebo (n = 3) on the fifth day of their menstrual cycle (study day 0) and hysterectomy was scheduled 24 h after rGH/placebo injection. Blood samples for IGF-I and oestradiol serum measurements were obtained immediately before rGH administration and 24 h later in each one of these six women. The remaining 12 women were also randomized to receive rGH (n = 6) or placebo (n = 6) but these patients underwent hysterectomy on cycle day 12 (i.e. 7 days following rGH injection). All these 12 women underwent daily blood sampling for serum IGF-I and oestradiol determinations. Transvaginal ultrasound scanning to monitor ovarian follicular development was carried out from the day rGH/placebo injection was given (where blood sampling and ultrasonographic examination were performed before injections) to the day before surgery (study day 6). Ovarian tissue samples were obtained before starting surgical removal procedures. Human luteinized granulosa cells were collected at oocyte retrieval from women undergoing ovarian stimulation with urinary gonadotrophins as part of an IVF programme.
Hormones were measured using commercially available kits. Oestradiol concentrations in serum were estimated by direct radioimmunoassay (bioMérieux, Marcy l'Etoile, France). Intra- and inter-assay coefficients of variation (CV) were below 4.5 and 5.5% respectively. IGF-I concentrations were determined by radioimmunoassay (Nichols Institute, San Juan Campistrano, CA, USA) after acid–ethanol extraction; the intra- and inter-assay CV were 7 and 10% respectively. Serum samples of each woman were examined in one run. Ultrasonic scans were performed using a 5 MHz vaginal transducer attached to an Aloka sector scanner (Model SSD-620; Aloka Co. Ltd, Tokyo, Japan).
Extraction of RNA
RNA was extracted from tissues previously frozen on dry ice and stored at –70°C or from freshly isolated luteinized granulosa cells (selected by Percoll centrifugation), as previously described (Hernandez et al., 1992). Frozen pre-menopausal ovaries were homogenized in 4 mol/l guanidinium isothiocyanate using a polytron (Brinkmann; Westbury, NY, USA). Total RNA was isolated following the method of Chirgwin et al. (1979). The precipitated RNA was resuspended in sterile water and quantified by absorbance at 260 nm. The integrity of the RNA was assessed by visual inspection of the ethidium bromide-stained 28S and 18S rRNA bands after electrophoresis through 1.25%/2.2 mol/l formaldehyde gels as previously described (Hernandez et al., 1992).
Riboprobe construction
Human IGF-I, IGF-II and IGF-I receptor probes were generated as previously described (Hernandez et al., 1992). Briefly, human IGF-I riboprobe was constructed in order to measure total IGF-I mRNA by a solution hybridization/RNase protection assay. A 442-base pair (bp) PstI-RsaI fragment of a human IGF-I cDNA was subcloned into the plasmid vector pGEM-3 (Promega Biotech, Madison, WI, USA) which had previously been digested with PstI and SmaI. When the resulting construct was linearized with BstNI and gel-purified, the 362 base antisense RNA generated by T7 RNA polymerase contained 339 (coding region) bases complementary to all IGF-I mRNA species.
A 556 bp PstI-AccI fragment of a human IGF-II cDNA was subcloned into pGEM-3. The construct was then linearized with HindIII and gel-purified. Synthesis of antisense RNA using T7 RNA polymerase generated a 603 base transcript complementary to 251 bases of 5'-UTR and 283 bases of coding region. 534 bases of this antisense riboprobe are complementary to IGF-II mRNA transcribed from the adult promoter and 289 bases are complementary to IGF-II mRNA transcribed from either the fetal or fetal-neonate promoters.
A 379 bp EcoRI-XhoI fragment of a human type I IGF receptor cDNA was ligated into pGEM-3. After linearization of this construct with HindIII, an antisense riboprobe was generated with T7 RNA polymerase. The final riboprobe contained 32 bases of pGEM-3 plasmid sequence and 379 bases complementary to IGF-I receptor mRNA in the region encoding the 
-subunit. The second protected band may correspond to alternatively spliced variants of the human IGF-I receptor mRNA (Werner et al., 1993).
Solution hybridization/RNase protection assays
Solution hybridization/RNase protection assays were performed as described previously (Hernandez et al., 1992). Briefly, the riboprobes were synthesized with T7 or SP6 RNA polymerase and labelled using [32P]UTP according to the manufacturer's instructions (Promega Biotec). After transcription, 1 µg of DNase I (Cooper Biomedical, Malvern, PA, USA) was added and the mixture incubated for 15 min at 37°C. The labelled riboprobes were recovered by ethanol precipitation. 20 µg of total RNA were hybridized with 400 000 c.p.m. of each 32P-labelled riboprobe for 16 h at 45°C in 75% formamide–0.4 mol/l NaCl followed by digestion with 40 µg/ml RNase A and 2 µg/ml RNase T1. Protected hybrids were isolated by ethanol precipitation and separated on an 8% polyacrylamide/ 8 mol/l urea denaturing gel. The relative intensity of protected RNA bands was quantified by densitometric scanning.
Statistical analysis
The analysis of variance (ANOVA) test for repeated measures and the Wilcoxon signed rank test were used to compare hormonal concentrations in the groups studied. P < 0.05 was considered significant.
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Results |
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The mean age for patients in the rGH group (n = 9) was 42.3 years (range 39–45 years) and 41.9 years (range 39–45 years) for the placebo group (n = 9). Figure 2
shows basal and 24 h serum concentrations of IGF-I and oestradiol in patients receiving rGH or placebo and undergoing hysterectomy 24 h after rGH/placebo injection. IGF-I serum concentration increased at 24 h with respect to basal concentrations (P = 0.1) in women treated with rGH but not in those receiving placebo, whose absolute values were almost identical to those observed in Figure 1
. No significant changes were noted in oestradiol serum concentrations in both groups of women. In the 12 patients undergoing hysterectomy 7 days after rGH injection, transvaginal ultrasonography revealed the presence of only one growing follicle (13–17 mm in diameter) on study day 6 (i.e. the day before surgery). Figure 3 shows daily IGF-I and oestradiol serum concentrations in rGH-treated and untreated patients. As seen in Figure 3a, IGF-I serum concentrations increased significantly (P < 0.02) 24 h after rGH treatment. This acute but transient rise in IGF-I serum concentrations was not observed in the placebo group. Oestradiol concentrations were similar in both groups of patients throughout the study (Figure 3b)
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Ovarian IGF-I gene expression: rGH dependence
To evaluate rGH action on ovarian IGF-I gene expression, the relative abundance of IGF-I mRNA was determined in a time-dependent manner (24 h and 7 days after rGH treatment). To determine the acute (24 h) effect of rGH on ovarian IGF-I gene expression, total RNA (20 µg) from whole pre-menopausal ovaries (with or without rGH treatment) was hybridized with a human IGF-I antisense RNA. Given that the amounts of expression of the IGF-I gene in the human ovary is very low and sometimes undetectable, the IGF-I receptor probe was introduced as hybridization control in this experiment. As shown in Figure 4
, IGF-I was not expressed in whole pre-menopausal ovaries irrespective of rGH treatment. As expected, the IGF-I receptor riboprobe produced two distinct bands in the whole ovary; the larger band (379 bp) was the length predicted by the sequence of the construct to generate the riboprobe. Differences, not statistically significant, were found between the abundance of IGF-I receptor mRNA in the absence or presence of rGH when the hybridization pattern was normalized with the 18S riboprobe.
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Since clinical studies have suggested the persistence of the ovarian sensitizing effect of rGH (Homburg et al., 1990a
; Burger et al., 1991), the ovarian IGF-I gene expression 7 days after rGH administration was explored. To this end, total RNA (20 µg) from whole pre-menopausal ovaries (with or without rGH treatment) and luteinized granulosa cells (as IGF-II producers) was hybridized with a human IGF-I antisense RNA. IGF-II and IGF-I receptor probes were used as hybridization controls in this experiment. As expected, the IGF-I gene was not expressed in the luteinized granulosa cells. On the contrary, a protected band (289 bp) corresponding to IGF-II mRNA generated from either the fetal–neonatal or the fetal promoter was seen in whole ovary and luteinized granulosa cells (Figure 5). At this time period no IGF-I mRNA was seen in whole pre-menopausal ovaries irrespective of their rGH treatment status. IGF-I receptor gene was ubiquitously expressed in both tissues. rGH supplementation did not change the amounts of expression of the IGF-II and IGF-I receptor genes with respect to the untreated control (Figure 6).
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Discussion |
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IGF have been identified in virtually all tissues within the endocrine system and appear to be regulated by a variety of trophic hormones, particularly GH (Buyalos, 1995
). Experimental evidence has indicated that both IGF-I and IGF-II may play a contributory role in follicular development and steroidogenesis (Adashi et al., 1985, 1991; Buyalos, 1995). Furthermore, studies in the human ovary have shown that GH can have a synergistic local effect with gonadotrophins on oestradiol and progesterone production (Manson et al., 1990; Lanzone et al., 1992). On the basis of prior animal studies and of the evidence of the positive interaction of IGF-I and gonadotrophins in ovulation, the preliminary use of rGH seemed justified when first used in clinical trials (Homburg et al., 1990a; Blumenfeld, 1991; Jacobs et al., 1991; Christman and Halme, 1992; Katz, 1993; Katz et al., 1993; Adashi, 1994; Homburg and Ben-Rafael, 1996; Howles et al., 1999). However, with advances in molecular biology and increasingly sophisticated isolation and purification techniques, both the regulation and function of IGF peptides within the human ovary and other tissues are being clarified and not infrequently found to be contradictory to previous descriptive experimental evidence (Buyalos, 1995).
Prompted by the similarity between the observed ovarian effects of GH and IGF-I (Davoren and Hsueh, 1986), consideration was given to the possibility that at least some of the actions of GH at the level of the granulosa cell may be mediated by IGF-I (Katz et al., 1993). Implicit in this approach was the presumption that ovarian IGF gene expression is GH dependent (Katz et al., 1993). The observations reported in the present study demonstrate that rGH is not a stimulus for IGF-I gene, IGF-II gene and IGF-I receptor gene expression in the human ovary, thus adding further evidence against the intraovarian IGF system as a mediator of GH action.
The complex pattern of IGF system gene expression in the human ovary reveal significant differences from the IGF system in the rat ovary, which has been extensively studied (Buyalos, 1995). In the rat, IGF-I mRNA is expressed in the granulosa cells of growing but not atretic follicles. In postpubertal human granulosa, IGF-I is not detectable, and IGF-II is abundant. In the current study the IGF-I gene was not expressed in whole pre-menopausal ovaries (with or without rGH treatment), whereas IGF-II expression was high. This is in accordance with recent studies showing that IGF-II expression was high in human ovarian granulosa and thecal compartments, whereas IGF-I mRNA was only weakly detectable in thecal cells of small follicles by the very sensitive RT–PCR analysis but not Northern and dot blots (El-Roeiy et al., 1993; Voutilainen et al., 1996). Because IGF-I mRNA is very scarce in the normal cycling primate ovary, it may be detectable in specific cells populations only using in-situ hybridization (Vendola et al., 1999). Thus, there may have been changes in amounts of IGF-I mRNA in some cell types that were not detectable in the homogenized tissue samples. These observations lend support to significant species-specific differences as well as to the potential importance of IGF-II, rather IGF-I, in human folliculogenesis. This is further stressed by the fact that in Laron dwarfism, a natural model of IGF-I deficiency, normal follicular development and conception have been described (Dor et al., 1992).
In contrast to IGF-I and IGF-II mRNA, the pattern of IGF-I receptor gene expression in the human and rat ovary appears to be identical (Buyalos, 1995). In both species IGF-I receptor mRNA is most prominent in oocytes, followed by granulosa cells, and is lowest in theca–interstitial and stroma cells. The IGF-I receptor represents the final common pathway of IGF activity since recent evidence in the human indicates that the biological actions of the IGF-II ligand are mediated through the IGF-I receptor (Willis et al., 1998). Thus, an autocrine/paracrine mechanism is plausible but the current study failed to show any positive effect of rGH in this regard. It was found that rGH did not increase the expression of the IGF-I and IGF-II genes or the IGF-I receptor gene when analysed 24 h and 7 days after rGH administration. This is in agreememt with clinical studies failing to support the hypothesis that locally produced IGF-I is responsible for the effects seen after systemic rGH administration and indicating that hepatic production of IGF-I is the main source of follicular fluid concentrations of this substance (Rabinovici et al., 1990; Owen et al., 1991).
In conclusion, the present report demonstrates that rGH administration to normal pre-menopausal women does not change the expression of the genes encoding the IGF and their receptor in the human ovary.
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Acknowledgments |
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We are grateful to Pharmacia & Upjohn, Barcelona, Spain for the supply of Genotonorm, and to Dr Angels Ulied from Pharmacia & Upjohn for her assistance. We also thank Ms Luisa Boqué for her technical assistance. This work was supported in part by FIS Grant 95/0889 (to E.R.H.).
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Notes |
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5 To whom correspondence should be addressed at: Institut Clinic of Obstetrics and Gynecology, Hospital Clínic i Provincial;C/Casanova 143; 08036–Barcelona, Spain. E-mail: jbalasch{at}medicina.ub.es
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Submitted on October 22, 1999; accepted on February 18, 2000.
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