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Endocrinology Vol. 143, No. 10 4104-4112
Copyright © 2002 by The Endocrine Society

ARTICLE

Growth Hormone Is Required for Ovarian Follicular Growth

Anne Bachelot, Philippe Monget, Prune Imbert-Bolloré, Karen Coshigano, John J. Kopchick, Paul A. Kelly and Nadine Binart

Molecular Endocrinology, INSERM U-44 (A.B., P.I.B., P.A.K., N.B.), 75730 Paris, France; UMR 6073, INRA (P.M.), 37380 Nouzilly, France; and Edison Biotechnology Institute, Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University (K.C., J.J.K.), Athens, Ohio 45701

Address all correspondence and requests for reprints to: Dr. Nadine Binart, INSERM, U-344, Endocrinologie Moléculaire, Facultéde Médecine Necker, 156 rue de Vaugirard, 75730 Paris, France. E-mail: binart{at}necker.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To analyze the consequences of the absence of GH receptor (GHR) and GH-binding protein (GHBP) on female reproductive function, we used a mouse model in which the GHR/GHBP gene has been disrupted by homologous recombination. The major effect on reproductive function seen in GHR/GHBP knockout (KO) compared with wild-type animals is a dramatic decrease in litter size; this defect is due to a reduction of the ovulation rate. The ovulatory response to exogenous gonadotropin treatment is also 3-fold reduced in GHR/GHBP KO compared with the wild-type ovaries. These results establish that the reduced rate of ovulation is essentially due to an ovarian defect rather than a deficiency in pituitary gonadotropins. The number of follicles per ovary is markedly reduced, although all categories of follicles are represented. Interestingly, the number of healthy follicles from antral and preovulatory stages is dramatically decreased in GHR/GHBP KO in comparison with wild-type follicles. The capacity of follicles to bind LH, FSH, and IGF-I was not diminished. IGF-I treatment using micropumps is not able to rescue either fertility or ovarian responsiveness to exogenous gonadotropins, suggesting that the effect of GH is independent of IGF-I. In conclusion, these results indicate that the reduction of litter size in GHR/GHBP KO mice is the consequence of an alteration of the growth of follicles and suggest that the effects of GH effects on follicular growth are independent of IGF-I.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH EXERTS its action via the GH receptor (GHR) expressed in target tissues and by stimulating IGF-I expression both locally and systemically. In the circulation, IGFs are bound to binding proteins (IGFBPs) that are able to prolong the half-life and modulate the bioavailability of IGFs. There is evidence that both GH and IGF-I play a role in reproductive function, particularly during sexual maturation. In mice, deletion of the IGF-I gene leads to failure of ovarian follicles to ovulate because of a block in folliculogenesis at a late preantral or early antral stage (1). The study of IGF-I knockout (KO) mice demonstrated that IGF-I is required for acquisition of FSH responsiveness, follicular development beyond the antral stage, and completion of oocyte growth and maturation (2). Moreover, in vitro studies have shown that IGF-I is able to enhance FSH action on granulosa cells (3). All of these observations have led to the suggestion that the ultimate fate of the follicle, i.e. growth or atresia, depends on the ability of IGF-I within the follicle to interact with its cognate receptor, the IGF type I receptor (IGF-IR).

Although IGF-I ovarian actions are well established, little is known about the direct action of GH in ovarian function. Some in vitro data suggest that GH plays a role in follicular growth in the early gonadotropin-independent stage and could have a direct inhibitory action on follicle apoptosis (4, 5, 6). Moreover, using a bovine GH transgenic mouse model, it has been shown that GH overexpression decreases follicle apoptosis and thus atresia in the mouse ovary (7). Nevertheless, there is no evidence to our knowledge that GH exerts a direct effect on ovarian folliculogenesis.

In humans, Laron syndrome (GH insensitivity syndrome) is a hereditary dwarfism resulting from a variety of GHR mutations (8, 9, 10). Affected patients are characterized by short stature, truncal obesity, delayed puberty, low serum IGF-I, and elevated serum GH and GH resistance (11), and growth can be partially restored by IGF-I treatment (12, 13). As three pregnancies have been reported, the fertility of the Laron patient seems to be normal, although folliculogenesis has not been extensively examined (14, 15).

To analyze the consequences of the absence of GH signaling, GH-binding proteins (GHBP), and IGF-I on female reproductive function, we used a mouse model in which the GHR/GHBP gene has been disrupted by homologous recombination, thus representing the Laron phenotype (16). The mutant mice exhibit a severe postnatal growth retardation, proportionate dwarfism, absence of GHR and GHBP, greatly decreased serum levels of IGF-I, and elevated serum GH concentrations. GHR/GHBP KO females are fertile, but their age at first conception is delayed, and litter size is decreased compared with wild-type females (17). The present study was designed to examine precisely the reproductive phenotype in GHR/GHBP KO females and to determine the extent to which any alterations due to GH deficiency could be rescued by IGF-I treatment or were due to a direct effect of GH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse generation and care
GHR/GHBP KO mice were produced as described previously (16) and were subsequently established on a pure Sv129Ola background. Adult GHR/GHBP KO females were mated with either GHR/GHBP KO or wild-type males. Mice were housed in a room with a control photoperiod of 12 h of light/d (lights on from 0700–1900 h) and a temperature of 20 C. Mice were given free access to a nutritionally balanced diet and tap water and were handled in accordance with the institutional animal care policies.

Estrous cycle
The duration of estrous cycles in wild-type and GHR/GHBP KO mice (6 and 28 wk of age) was determined by inspection of daily vaginal smears over 3 consecutive wk.

Analysis of implantation and periimplantation stages
Wild-type and GHR/GHBP KO females (6–8 wk of age) were mated with fertile males of the same strain to induce pregnancy. The morning a vaginal plug was found was designated d 0.5 of pregnancy. On d 5.5 the mice were killed, and the number of implantation sites was recorded by monitoring the localized uterine vascular permeability at the sites of blastocysts after iv injection of Chicago blue dye solution in saline (Sigma, St. Louis, MO). On d 0.5, 1.5, and 2.5, oviducts were flushed with Whitten’s medium (18) to recover eggs or embryos and counted under a microscope. Ovaries were fixed in Bouin’s fluid for histology. The corpora lutea were counted in parallel on histological sections.

Superovulation
Wild-type and GHR/GHBP KO females (6–8 wk of age) were superovulated by ip injection of 10 IU pregnant mare’s serum gonadotropin (PMSG; Folligon, Intervet Inc., Millsboro, DE) at 1800 h, followed by ip injection of 5 IU hCG (Chorulon-Intervet) 48 h later. They were killed 17 h after the hCG injection. Oocytes were extracted from ampulla and counted after enzymatic dissociation from the surrounding cumulus with hyaluronidase (Sigma; 100 µl type IV-S; 10 mg/ml). Ovaries were fixed in Bouin’s fluid for histology.

Morphological characterization of ovarian follicles
Ovaries fixed in 4% buffered formaldehyde and included in paraffin were serially sectioned at a thickness of 5 µm, then sections were stained with hematoxylin and eosin and examined by light microscopy. Five classes of follicles were selected according to their diameter: 50–99, 100–199, 200–299, and 300 µm or more. The quality of ovarian follicle was estimated by histological examination of sections; follicles were judged normal (frequent mitosis, no pycnosis in granulosa) or atretic (rare normal granulosa cells and a great majority of pycnotic bodies).

Binding assays on histological sections
Ovaries were collected on diestrus or 48 h after the injection of 10 IU PMSG and were coated with cryoprotectant embedding medium (Tissue-Tek, Miles, Elkhart, IN), frozen in cold isopentane, and then stored at -20 C. The binding of [125I]IGF-I, [125I]LH, and [125I]FSH to ovarian frozen sections was studied by an autoradiographic method, as described previously (19, 20). Briefly, ovaries were serially sectioned at a thickness of 5 µm with a cryostat. After fixation for 10 min at 4 C in picric-acid formaldehyde, sections were stored at –20 C and then circled with Depex (Gurr, BDH, Poole, UK). Recombinant IGF-I was iodinated by the Iodogen method (Sigma, St Quentin, France) and purified by Sephadex G-50 chromatography. Sections were incubated in a drop of PBS (0.1% BSA, pH 8) containing [125I]IGF-I (2 x105 cpm/50 µl) or in a drop of PBS (0.1% BSA, pH 7.4) containing [125I]LH (4 x105 cpm/50 µl) or [125I]FSH (4 x 105 cpm/50 µl). To determine nonspecific binding, for each tested ligand an adjacent serial section of ovary was incubated with an excess of unlabeled ligand (500 ng/50 µl for IGF-I, LH, and FSH). To discriminate IGF-I binding to IGFBPs from binding to type I receptor, competition was performed with a large excess of insulin (5 µg/50 µl). At the end of the incubation period, the sections were washed twice in PBS, postfixed in 3% glutaraldehyde-PBS, washed in PBS, air-dried, and stained with Feulgen. For autoradiography, they were then dipped in Kodak NTB2 emulsion diluted 1:1 with distilled water, air-dried, exposed for 2 wk at 4 C, then developed and fixed by classical procedures.

IGF-I treatment
GHR/GHBP KO mice were treated with recombinant human IGF-I (Genentech, Inc., South San Francisco, CA) by micropumps (Alzet, Palo Alto, CA) releasing 6 mg/kg·d for 14 d. Micropumps were inserted dorsally at 4 wk of age and were removed at 6 wk of age (4–6 wk) or were inserted at 10 wk of age (10–12 wk) (21).

RT-PCR
For detection of both class 1 and class 1-del of IGF-I, primers were prepared as described previously (22). Total RNA was prepared from both ovaries and liver of wild-type or GHR/GHBP KO mice using TRIzol reagent (Life Technologies, Inc., Grand Island, NY). The cDNA synthesized was used as a template for PCR in a reaction mixture containing 2.5 mM MgCl2, 5% dimethylsulfoxide, 5 mM of each of the two primers, and 0.5 mM deoxy-NTP. After 25 cycles (1 min at 95 C, 1 min at 55 C, and 2 min at 72 C) of PCR, PCR products were electrophorised on a 1% agarose gel.

Semiquantitative RT-PCR analysis
Total RNA was extracted from both ovaries by using TRIzol reagent (Life Technologies, Inc.) of GHR/GHBP KO and IGF-I-treated GHR/GHBP KO, and wild-type female. Two micrograms of total RNA were reverse transcribed using oligo poly(deoxythymidine) and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). cDNA sequences of each IGFBP available from GenBank were aligned and compared. Specifics primers of each IGFBP (IGFBP-2, -3, -4, and -5) were chosen in nonconserved regions to generate different size fragments. Primers corresponding to IGFBP-2, -3, -4, and -5 were, respectively: 5'-GAC CAT GAA CAT GTT GGG AG-3'(sense) and 5'-CCC GCG CTG TCC GTT CAG-3' (antisense); 5'-CGC GAA GGC GAC GCG TGC (sense) and 5'-CAT TGA GGA ACT TCA GAT GAT TC-3'(antisense); 5'-CCA GGG TTC CTG CCA GAG-3' (sense) and 5'-CCC TTG GGT TCC AAA CCC-3' (antisense); 5'-CCT TAA TCC TCG CAA TTG GGAC-3' (sense) and 5'-GTC AGC TTC TTT CTG CGG TC-3' (antisense). The expected sizes were, respectively, 357 bp for IGFBP-2, 471 bp for IGFBP-3, 241 bp for IGFBP-4, and 692 bp for IGFBP-5. One microliter of each RT reaction was used in 20 µl PCR mixture containing 2.5 mM MgCl2 (except for 1.5 mM IGFBP-3 and 5% dimethylsulfoxide), 5 mM of the two specific primers, and 0.5 mM deoxy-NTP. Increasing numbers of cycles were tested to assess best conditions and perform linear amplification. After 20 cycles (45 sec at 94 C, 1 min at 61 C, and 45 sec at 72 C) of amplification, products were separated on a 1% agarose gel stained with ethidium bromide and analyzed with a DC digital camera (Kodak, Rochester, NY) coupled with Kodak ID 2.02 software. In parallel, primers corresponding to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were included in each experiment as an internal control. The intensity of each IGFBP band was reported to the intensity of GAPDH.

Statistical analysis
Statistical analysis was performed by ANOVA, followed by the Student-Newman-Keuls test. A t test was used to compared the values of two groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reproductive phenotype of GHR/GHBP KO female
GHR/GHBP-KO female mice were previously shown to produce a reduced number of pups per litter (mean litter size, 2.7 vs. 6.9 in wild-type females) (16). The average age of first pregnancy was delayed, implying delayed sexual maturation, as has also been reported in Laron syndrome patients (7). We backcrossed animals to a pure 129Ola background and still observed a statistical difference in litter size (3.9 ± 2.1 vs. 8.6 ± 2.6; P < 0.001). To determine whether this striking difference in litter size can be explained by an early embryonic loss or a defective ovulation rate, we examined the number of implantation sites in 8- or 12-wk-old GHR/GHBP KO or wild-type female on d 5.5 using the blue dye method. Although 8.2 ± 0.6 (n =9) implantation sites were recorded in wild-type mice, only 4.7 ± 0.6 (n = 7) were seen in GHR/GHBP KO mice (P < 0.05; Fig. 1AGo). These results suggest that the implantation process proceeds normally in GHR/GHBP KO mice and that the defect in embryo number occurs before the implantation step.



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  		Figure 1. Number of implantation sites on d 5.5 of gestation and preimplantatory egg development. A, The number of implantation sites recorded on d 5.5 after blue dye injection is reduced in GHR/GHBP KO ({square}) compared with wild-type ({blacksquare}) females. Values are the mean ± SEM (n = 7 mice/group). *, P < 0.05 vs. GHR/GHBP KO. B, The number of eggs recorded in GHR/GHBP KO ({square}) is reduced compared with that in wild-type ({diamondsuit}) females on d 0.5, 1.5, and 2.5 postcoitum (d.p.c.). C, Number of corpora lutea decreased in parallel in GHR/GHBP KO mice ({square}) on d 0.5, 1.5, and 2.5 compared with that in wild-type mice ({diamondsuit}). Values are the mean ± SEM (n = 5 mice/group). ***, P < 0.001; **, P < 0.01; *, P < 0.05 (vs. GHR/GHBP KO mice).

 
To analyze the preimplantatory development of fertilized eggs, mice were killed on d 0.5, 1.5, or 2.5 after observation of the vaginal plug. The number of eggs recorded was significantly diminished in GHR/GHBP KO mice compared with wild-type mice [respectively, 3.0 ± 1.0 vs. 9.1 ± 0.3 (P < 0.001) on d 0.5, 3.0 ± 0.6 vs. 6.5 ± 1.2 (P < 0.05) on d 1.5, and 2.5 ± 1.5 vs. 6.5 ± 0.5 (P < 0.05) on d 2.5; Fig. 1BGo]. Concomitantly, the number of corpora lutea counted in ovarian sections of these mice was also decreased in GHR/GHBP KO mice [3.0 ± 0.6 vs. 9.3 ± 0.8 in wild-type mice (P < 0.05) on d 0.5, 3.0 ± 0 vs. 8.6 ± 1.3 in wild-type mice on d 1.5 (P < 0.05), and 5.2 ± 0.6 vs. 10.7 ± 0.9 in wild-type mice on d 2.5 (P <0.001); Fig. 1CGo]. The reduced litter size in GHR/GHBP KO mice was thus probably due to a reduced rate of ovulation, as the values observed in Fig. 1Go, B and C, were in accord.

Response to superovulation
To assess whether this defect could be due to a deficiency of ovarian responsiveness to gonadotropins, GHR/GHBP KO females were superovulated by exogenous gonadotropin treatment. An average of 35.3 ± 2.3 eggs/mouse was recorded from all examined wild-type animals (n = 8), whereas only 11.8 ± 2.3 eggs/mouse (n = 11) were seen in GHR/GHBP KO mice (P < 0.001; Fig. 2Go). These results establish that although a minor central defect could not be excluded in GHR/GHBP KO females, the reduced rate of ovulation is primarily due to an ovarian defect rather a deficiency of pituitary gonadotropins.



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  		Figure 2. Response to superovulation. The rate of ovulation (oocyte number) after injection of PMSG at 1800 h, followed by the injection of hCG 48 h later, is diminished in GHR/GHBP KO ({square}) vs. wild-type ({blacksquare}) mice. Values are the mean ±SEM (n = 8 mice/group). ***, P < 0.001.

 
Estrous cycle and ovarian morphology
To dissect the primary ovarian defect in GHR/GHBP KO mice, we then evaluated the population of ovarian follicles and the follicular development. Estrous cycles were recorded in 6- or 28-wk-old mice by inspection of daily vaginal smears for 3 consecutive wk. Pubescent GHR/GHBP KO females had regular estrous cycles and normal mating behavior. Ovaries and genital tracts from at least 10 GHR/GHBP KO and wild-type age-matched mice were analyzed. Whereas the length of the oviducts, uterine horns, and vagina were significantly reduced in GHR/GHBP KO females, sexual maturation of the genital tracts was similar in GHR/GHBP KO and WT females (data not shown).

Despite their reduced weight (40%), light microscopic examination of GHR/GHBP KO ovaries revealed that their structure was well organized (Fig. 3Go, A and B). Whereas a reduced number of growing follicles appeared, all developmental stages were represented, as shown in Fig. 3Go, C and D. To better understand this defect, all classes of follicles were counted according to their diameter in ovarian sections of 8-wk-old GHR/GHBP KO and wild-type females (Fig. 4Go). The number of healthy follicles per ovary was markedly reduced in GHR/GHBP KO compared with wild-type animals, with a statistically significant difference in follicles reaching a diameter from 200 µm (Fig. 4AGo), with no change in the number of atretic follicles (Fig. 4BGo). Then, the ratio of atretic/healthy follicles from 200 µm was increased in GHR/GHBP KO females compared with wild-type mice. Interestingly, despite a reduced number of growing ovarian follicles, all females more than 18 months of age were able to bear litters, suggesting that there was no premature ovarian failure in GHR/GHBP KO females.



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  		Figure 3. All follicular stages are present within GHR/GHBP KO ovaries in adult mice. Representative view of ovaries of wild-type (A) and GHR/GHBP KO mice (B–D). Ovaries of GHR/GHBP KO mice are reduced in size by 40% compared with those of wild-type mice, but display all classes of follicles. Details are shown in C and D. Scale bar. 400 µm in A and B; 145 µm in C and D. PA, Preantral follicle; SA, small antral follicle; LA, large antral follicle.

 


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  		Figure 4. Classes of healthy and atretic follicles according to diameter. Follicles were ranked by size (50–99, 100–199, 200–299, and 300 µm or larger) and degree of atresia. A, Healthy follicles; B, atretic follicles. The morphometric studies were performed on ovaries from wild-type (n = 4; {square}) and GHR/GHBP KO (n = 8; {blacksquare}) mice. Experimental data are expressed as the mean ± SEM. *, P < 0.05.

 
Ovarian receptivity to IGF-I and gonadotropins
IGF-I KO females are infertile and do not appear to undergo puberty. Ovarian follicular development is arrested at a preantral/early antral stage, with no mature Graafian or luteinized follicles. Study of these mice has shown that the role of IGF-I is to entrain murine follicular development in response to gonadotropin regulation (2). In preantral follicles some unknown signal initiates granulosa cell proliferation and IGF-I production. IGF-I increases FSH receptor expression, and FSH augments both IGF-IR and FSH receptor expression. Maximal FSH action leads to mature antral formation, granulosa cell aromatase, and LHR expression. We examined the gonadotropin responsiveness in term of IGF-I, LH, and FSH binding in ovaries from GHR/GHBP KO and wild-type mice during diestrus or after injection of 10 IU PMSG. Binding sites for IGF-I and IGFBPs were widespread in the ovary (Fig. 5Go, A and C). Binding was detected in all oocytes examined and in granulosa and thecal cells of healthy follicles as well as in antrum, corpora lutea, and stroma. The binding to IGF-IR in granulosa cells could for the most part be competed by an excess of insulin, then the labeling in Fig. 5Go, B and D, correspond to IGFBPs. Specific LH binding was seen in thecal cells of healthy antral follicles, and in granulosa and thecal cells of preovulatory follicles as well as in corpora lutea (Fig. 6Go, A and C). Specific FSH binding was seen in granulosa cells of healthy preantral and antral follicles (Fig. 6Go, B and D). There were no clear differences between binding for both genotypes and follicular stages (diestrus or PMSG treatment) in terms of LH, FSH, and IGF-I receptivity. Thus, in contrast to the IGF-I KO model, the loss of GH signaling had no clear effect on follicular responsiveness to gonadotropins or on terminal follicular development.



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  		Figure 5. Ovarian expression and localization of binding sites for IGF-I and IGFBPs. Autoradiography of IGF-I binding was performed on sections from wild-type (A and B), GHR/GHBP KO (C and D), and IGF-I-treated GHR/GHBP KO (E and F) mice ovaries on diestrus. Binding of radiolabeled IGF-I to IGF-IR or IGFBPs appeared in different compartments of the wild-type (A), GHR/GHBP KO (C), and IGF-I-treated GHR/GHBP KO (E) ovaries. Binding of IGF-I to granulosa cells was partially competed by insulin (B, D, and F). PA, Preantral follicle;SA, small antral follicle; LA, large antral follicle; CL, corpus luteum. Arrows represent specific labeling.

 


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  		Figure 6. Ovarian expression and localization of binding sites for LH and FSH. Autoradiography of LH and FSH binding was performed on sections from wild-type (A and B), GHR/GHBP KO (C and D), and IGF-I-treated GHR/GHBP KO (E and F) mice ovaries after stimulation by PMSG. Specific LH binding was seen in healthy follicles in granulosa, thecal, and stroma cells as well as in corpora lutea (A, C, and E), whereas specific FSH binding is mostly seen in granulosa cells (B, D, and F). SA, Small antral follicle; LA, large antral follicle;CL, corpus luteum; Th, theca; St, stroma cells. Arrows represent specific labeling.

 
Effect of IGF-I treatment in GHR-/- mice: IGF/GH axis and IGFBP levels
Micropumps releasing recombinant human IGF-I placed in 4- and 10-wk-old GHR/GHBP KO mice were sufficient to increase circulating IGF-I 5.6-fold compared with that in GHR/GHBP KO mice, reaching levels only 25% less than those in wild-type mice, as described previously (21). Reduced IGF-I binding by IGFBP-3 in GHR/GHBP KO mice was partially restored by IGF-I treatment. IGF-I binding to the IGFBP-1, -2, or -5 was also increased (21), suggesting that IGF-I alone can partially restore the IGFBP profile. We examined the number of implantation sites in these mice on d 5.5 using the blue dye method and found no increased number of implantation sites compared with that in GHR/GHBP KO mice. After superovulation of IGF-I-treated GHR/GHBP KO mice, the number of oocytes ovulated never reached that observed in wild-type mice (see Fig. 3Go; 14.5 and 11.8 eggs/mouse in IGF-I-treated or untreated GHR/GHBP-KO mice, respectively). In addition, IGF-I, LH, and binding were not different in IGF-I-treated GHR/GHBP KO mice (Figs. 5Go and 6Go, E and F). Thus, the ovulation rate was not improved in 4- or 10-wk-old GHR/GHBP KO mice treated with IGF-I.

Analysis of ovarian gene expression
Ovarian expression of IGF-I mRNA has been reported to be independent of GH secretion, as hypophysectomy had no effect (2). We examined liver and ovarian IGF-I mRNA expression by RT-PCR in GHR/GHBP KO mice. As expected, there was a dramatic reduction of IGF-I mRNA liver expression in GHR/GHBP KO mice compared with wild-type mice. However, ovarian mRNA expression was similar in both genotypes, confirming that IGF-I expression in the ovary is independent of GH signaling (Fig. 7AGo). These results suggest GH-independent IGF-I expression in the ovary.



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  		Figure 7. Analysis of IGF-I and IGFBP expression in wild-type (+/+), GHR/GHBP KO (-/-), and IGF-I-treated GHR/GHBP KO (IGF-I) females. A, RT-PCR products of control and GHR/GHBP KO hepatic and ovarian RNA amplified for 25 cycles with primers spanning the alternatively spliced exon, which distinguishes both class 1 and class 1-del of IGF-I. B, RT-PCR analysis of ovarian expression of IGFBPs in wild-type (+/+), untreated (-/-), and IGF-I-treated (IGF-I) GHR/GHBP KO mice. RNA was amplified for 20 cycles. IGFBP expression was normalized to the expression of GAPDH, which was used as an internal control. Comparison of band intensities normalized to GAPDH (GHR/GHBP KO, control) yields a ratio of 1:1 for all IGFBPs.

 
The bioavailability of IGF-I in the ovary is regulated by several IGF-binding proteins (IGFBP-2 to -6) (23, 24). We previously reported, using Western ligand blot analysis, a reduction of IGFBP-3 levels in the serum of GHR/GHBP KO mice that could be restored by IGF-I treatment (21). IGFBPs are known to play a role in follicular development and atresia in mice (25). We thus examined the pattern of mRNA expression of different IGFBPs in ovaries using semiquantitative RT-PCR (Fig. 7BGo). We were unable to find any difference in ovarian expression of IGFBP-2, -3, -4, and -5 mRNA in GHR/GHBP KO females compared with wild-type females even after IGF-I treatment. These results suggest that IGF-I bioavailability may not be modified in GHR/GHBP KO ovaries.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that GH deficiency in female mice is responsible for reduced litter size due to reduced ovulation rate, a reduced number of antral follicles, and increased ratio of atretic/healthy follicles from 200 µm, whereas the implantation process occurs normally. These effects cannot be reversed by IGF-I treatment, suggesting that the main defect in GHR/GHBP KO mice may be related to the absence of GHR itself and not to reduced plasma IGF-I levels. These data show an effect of GH on follicular survival and development.

In contrast to IGF-I, which is known to play a major role in sexual maturation and ovarian folliculogenesis, GH is not classically considered a reproductive hormone. GHR has been detected in granulosa cells, thecal cells, and luteal cells in many species (26, 27, 28, 29). However, although a large body of literature attests that this hormone plays a role in reproductive function, no clear demonstration of an IGF-independent action of GH in the ovary has been reported. Although several in vitro studies demonstrated certain actions (4, 5), the direct consequence of an absence of GH on the reproductive axis in vivo has not been examined. Infertility is often observed in mice with a dwarf phenotype, but none of these models reproduces GH deficiency; Snell-dwarf mice are sterile, but they show a combined hypopituitarism (30, 31). Hepatocyte nuclear factor 1{alpha}-null mice are sterile and exhibit a phenotype reminiscent of Laron-type dwarfism due to the loss of expression of IGF-I (32). GHRH-deficient lit/lit mice are fertile, probably because they may still have sufficient levels of GH and IGF-I to permit ovulation (33). Thus, our model is unique, as it allows the study of direct effects of GH and IGF-I on reproductive functions.

GHR/GHBP KO females are fertile, suggesting that GH is not a limiting factor for reproduction and that GH action is not detrimental for fertility. It has been previously reported that these mice exhibit later vaginal opening, suggesting delayed puberty, as seen in patients with Laron syndrome (34). However, we report that the pattern of estrous cyclicity was unchanged, indicating that the ovulation process occurs in GHR/GHBP KO females. We also show that the reduction of prolificacy was primarily due to an alteration of ovulation rate rather than a perturbation of embryonic development or of the implantation process. Recently, it was demonstrated that transgenic mice expressing the rat inhibin {alpha}-subunit gene exhibited small litter size due to a reduced ovulation rate reversible by superovulation, suggesting a central defect (35). We demonstrate that the reduction of ovulation rate in the absence of GHR is primarily due to an ovarian defect, as GHR/GHBP KO mice produced fewer eggs in response to superovulation than wild-type mice. Moreover, the fact that GHR/GHBP KO mice were able to ovulate suggests that GH is not essential for terminal follicular development. In bovine GH transgenic mice there is a significant decrease in follicle cell apoptosis and thus atresia in the ovary (7). Nevertheless, nothing is known about the propensity for ovulation in these animals. Histological evaluation of GHR/GHBP KO ovaries revealed major depletion of healthy follicles from 200 µm. This was accompanied by an increase in the atretic/healthy follicle ratio, rather than a decrease in sensitivity to LH, FSH, and IGF-I. This was in accordance with the detection of the number of follicles exhibiting apoptotic cells, as evidenced by terminal deoxynucleotidyltransferase deoxy-UTP nick end labeling assay (data not shown). Overall, these results suggest that the reduction in ovulation rate in GHR/GHBP KO mice is essentially due to a reduction in the number of recruitable follicles and a deficit in terminal follicular growth, rather than to central gonadotropin secretion.

This oocyte deficit is similar to that observed in Zfx mutant mice, where it occurs before sexual maturation (36). Such mice represent a potential model for ovarian failure, as a shortage of oocytes results in diminished fertility and shortened reproductive life span. In contrast, oocyte depletion may not be the cause of the reduced ovulation rate in GHR/GHBP KO mice, as several females older than 18 months continued to bear litters. Thus, there appears to be no premature ovarian failure in our model.

The phenotype of GHR/GHBP KO mice differs from that of IGF-I KO mice, where there was no effect on the number of atretic follicles and no apparent increase in programmed death of granulosa cells (1). Follicles in IGF-I KO mice are arrested in a late preantral or early antral stage of development. These ovaries have a normal complement of granulosa cells, at least up to preantral or early antral stage, and appear morphologically very similar to those in the FSH KO ovary (1). These ovaries fail to respond to gonadotropin treatment for ovulation induction. This is thought to be due to diminished FSHR expression, and thus inadequate FSH activity, i.e. no amplification of FSH activity to induce the formation of mature Graafian follicles (2). In an attempt to understand this difference between the two KO models, we treated GHR/GHBP KO mice with IGF-I to return the systemic levels of IGF-I to near-normal values, as serum IGF-I in GHR/GHBP KO mice is reduced to 10% of wild-type levels. Treatment failed to rescue the ovulation rate, suggesting that the defects in GHR/GHBP KO ovarian function could be ascribed not to an action of IGF-I, but, more precisely, to an action of GH on follicles. Furthermore, we demonstrated that endogenous expression of ovarian IGF-I mRNA was not modified, confirming that IGF-I is not important for the reproductive phenotype of GHR/GHBP KO mice. This observation is supported by studies demonstrating that gonadotropin treatment had no effect on the levels of ovarian IGF-I mRNA expression in hypophysectomized mice (2). It is impossible at present to determine whether it is peripherally or locally produced IGF-I (or both) that is important. Taken together, these data suggest that ovarian IGF-I production is not strictly under the control of GH. Other local factors, such as growth differentiation factor-9, may have a role in the induction of ovarian IGF-I expression (37).

In GHR/GHBP KO mice, serum IGFBP-3 is virtually absent, resulting in an altered ratio between the different binding proteins, as occurs in Laron syndrome (11, 21), but is restored after IGF-I treatment to near-normal values. Previous studies showed that these binding proteins play an important role in the survival or atresia of mouse ovarian follicles (25). We found the same level of expression of ovarian IGFBPs and no binding difference in IGFBPs in GHR/GHBP KO and wild-type mice, suggesting that ovarian IGF-I expression partially restores IGFBP profiles, although more detailed studies of the individual expression of IGFBP in each class of follicles remain to be performed. We suggest that GH does not act through intraovarian IGF-I, but we cannot rule out that it is the bioavailable IGF-I that is important.

In conclusion, our results provide in vivo evidence that GH itself is required for normal follicular development and ovulation rate. GH probably acts on the recruitment of follicles proceeding to terminal growth, and seems to be one of the cofactors necessary for survival and growth of follicles. IGF-I expression in the ovary is not modified, demonstrating that it is not responsible to the reproductive defect in GHR/GHBP KO mice. The mechanism of follicular recruitment and growth differs between mice and women; the latter belongs to monoovulated species. Nevertheless, it would be interesting to study folliculogenesis in women with Laron syndrome to determine whether GH plays a role in human follicular growth as it does in mice.

Finally, GHR/GHBP KO mice offer a useful model to further understand the effect of a lack of GH action on ovarian function. Further studies will be required to determine which mediators and cofactors are responsible for GH actions in follicular cells.


   Acknowledgments
 
We thank Carine Géry and Gérard Pivert for excellent technical assistance, and Athanassia Sotiropoulos for helpful discussions. The generous supply of recombinant human IGF-I from Genentech, Inc. (South San Francisco, CA), is gratefully appreciated. We thank C. Coridun for secretarial assistance.


   Footnotes
 
This work was supported in part by grants from INSERM and in part by the State of Ohio’s Eminent Scholars’ Program (to J.J.K.) that includes a gift from Milton and Lawrence Goll and by Sensus Corp. (to J.J.K.).

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GHBP, GH-binding protein; GHR, GH receptor; IGFBP, IGF-binding protein; IGF-IR, IGF type I receptor; KO, knockout; PMSG, pregnant mare’s serum gonadotropin.

Received January 23, 2002.

Accepted for publication June 12, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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