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Reproductive Abnormalities in Human IGF Binding Protein-1 Transgenic Female Mice

Physiologie de la Reproduction et des Comportements (P.F., S.H., C.P., D.M., P.M.), Unité Mixte de Recherche 6073 Institut National de la Recherche Agronomique-Centre National de la Recherche Scientifique-Université F. Rabelais de Tours, 37380 Nouzilly; Institut National de la Santé et de la Recherche Médicale (D.S., M.B.), Unité 515, Croissance, différenciation et processus tumoraux, Hôpital Saint-Antoine, 75571 Paris Cedex 12; Department of Neurobiology and Physiology (J.E.L.), Northwestern University, Evanston, Illinois 60208

Address all correspondence and requests for reprints to: Philippe Monget, Physiologie de la Reproduction et des Comportements, Unité Mixte de Recherche 6073 Institut National de la Recherche Agronomique-Centre National de la Recherche Scientifique-Université F. Rabelais de Tours, 37380 Nouzilly, France. E-mail: . monget{at}tours inra.fr

	Abstract

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The mechanisms responsible for reproductive abnormalities in transgenic female mice overexpressing human IGF binding protein-1 (IGFBP-1) in the liver have been investigated. At 2 months of age, none of these transgenic mice exhibited ovarian cyclicity. Genital tract and ovary tissue weights were reduced in transgenic mice, this weight reduction being disproportionate with the reduction of body weight. Examination of ovarian follicular population revealed a marked decrease in the number of corpora lutea and gonadotropin-dependent follicles, suggesting an alteration of terminal follicular growth and ovulation. Stimulation of ovaries by exogenous gonadotropins revealed that ovaries from transgenic mice ovulated less oocytes than nontransgenic mice. This lower responsiveness of ovaries from transgenic mice to gonadotropins was not associated with a decrease in FSH-, LH- or IGF-I receptor expression. Transgenic and nontransgenic mice have similar circulating LH and FSH concentrations at dioestrus, after castration, 46 h after equine CG administration, or 15 min after GnRH injection. However, LH concentrations were 8-fold higher in pituitaries from transgenic vs. nontransgenic mice. Moreover, the size of LH-immunoreactive cells was reduced and their number was increased, suggesting a subtle alteration of LH secretion.

Overall, these data indicate that reduced fertility in transgenic female mice overexpressing human IGFBP-1 are mainly due to an alteration of terminal follicular growth leading to a decrease in natural and induced ovulation rate, likely due to an impairment of IGF-I action on follicular cells. Increased circulating IGFBP-1 concentrations may additionally lead to altered GnRH and LH pulsatility and thereby exacerbate the ovulation defect.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
GONADOTROPINS PLAY a major role in ovarian follicular development and ovulation, their secretion and action being modulated by IGFs and their binding proteins (IGFBPs). In hypothalamo-pituitary axis, IGF-I might be a major signal that integrates somatic development to the GnRH-releasing system during sexual maturation. Indeed in vitro, IGF-I stimulates GnRH secretion (1) and potentiates the GnRH-stimulating effect on LH secretion by pituitary cells (2). High concentrations of type I IGF receptors are present in the hypothalamus and the median eminence, which contain the nerve terminals of GnRH neurons (3). In vivo, serum IGF-I concentrations increase during puberty and intraventricular administration of IGF-I is able to induce LH release in peripubertal female rats and to advance the onset of their puberty (4).

It has been established that the ovary is also a direct target of IGF-I action. Indeed, several in vitro experiments have shown that IGF-I stimulates proliferation and steroidogenesis of granulosa and theca cells, mostly by potentiating gonadotropin actions (5). In vivo, inactivation of the IGF-I gene leads to the sterility of female mice, the arrest of follicular development at the preantral stage and the loss of ovarian responsiveness to exogenous gonadotropins suggesting alterations in both hypothalamo-pituitary and ovarian functions (6).

In biological fluids, more than 95% of IGFs are bound to IGFBPs that control their bioavailability and modulate their interaction with type I IGF receptor. IGFBP-1 is mainly produced in the liver, in kidney, and in human decidua. Secretion of IGFBP-1 in the mouse liver is high during fetal life and decreases after birth, its expression being mostly dependent on nutritional status. In adult, hepatic IGFBP-1 gene expression is stimulated by glucocorticoids, glucagon, glucose and amino acids deprivation, and inhibited by insulin and GH (7, 8). In vitro (9, 10) and in vivo studies (11, 12) have shown an inhibiting role of IGFBP-1 in cell proliferation, differentiation and glucoregulation, mainly by inhibiting IGF-I action. Moreover, growth retardation observed in cases of diabetes or negative energy balance, in particular during chronic undernutrition or anorexia, was shown to be associated with increased IGFBP-1 concentration in serum. Such increase in IGFBP-1 concentration is also observed during an intense and chronic training, in particular in athletes like marathon runners. In all these cases, metabolic disturbances were shown to be associated with menstrual cycle alterations or amenorrhoea in women.

A model of transgenic mice overexpressing human (h)IGFBP-I in the liver have been established in two mouse lines as previously described (13). In this model, peripheral IGFBP-1 concentration is increased approximately 4-fold and serum IGF-I concentration is reduced approximatively 2-fold, mimicking negative energy balance situations. Reproductive function was also shown to be markedly affected in homozygous hIGFBP-1 transgenic females mice (13). In particular, homozygous transgenic females crossed with nontransgenic males exhibited a marked alteration of fertility and a reduction of litter size (4.8 vs. 8.4 pups/litter in transgenic and nontransgenic mice, respectively).

The aim of the present study was to identify the causes of the alteration of fertility and fecundity observed in hIGFBP-1 transgenic mice. For this purpose, we have studied the functionality of the hypothalamo-pituitary-ovarian axis of these mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
GnRH and equine CG (eCG) were purchased from Sigma (L’Isle d’Albeau Chesnes, France) and Intervet (Angers, France), respectively. The primary antibodies raised against rat ß-LH and rat ß-FSH were kindly provided by Dr. A. F. Parlow, (NIDDK, Bethesda, MD). Goat peroxidase-labeled antibody raised against rabbit IgG was obtained from Interchim (Montlucon, France). 3,3'-Diaminobenzidine tetrahydrochloride dihydrate was purchased from Aldrich Chimie (L’Isle d’Albeau Chesnes, France). Rat (r)FSH was kindly provided by Dr. J. Closset (Liège, Belgium). Concentrations of LH in serum and pituitary extracts were determined by RIA using reagents generously supplied by Dr. A. F. Parlow. hCG, rLH, rFSH, and IGF-I were iodinated by the Iodogen method and purified by Sephadex G-50 chromatography with a 10 mM Tris buffer containing 0.1% BSA (pH 7) for hCG, rLH, and rFSH and with a 0.1 M acetate ammonium buffer containing 0.1% BSA for IGF-I. Schiff reagent for Feulgen staining was purchased from Merck \|[amp ]\| Co., Inc. (Schuchardt, Germany). NTB2 emulsion for autoradiography was obtained from Integra Bioscience (Cergy Pontoise, France).

Animals
The generation and characterization of the transgenic mice have been previously described in details (13). The transgene consisted of a human cDNA of the IGFBP-1 gene inserted downstream of the human 1-antitrypsine (700 bp) promoter. Homozygous transgenic female mice and nontransgenic female mice of the 149 strain were used for all studies. Mice were maintained under controlled conditions of light (12 h light, 12 h darkness) and temperature with ad libitum access to food and water.

All procedures were approved by scientific research agency (approval no. A37801) and conducted in accordance with the guidelines for care and use of laboratory animals.

Blood collection and tissue removal
Blood samples (500 µl) were recovered by intraorbital puncture in adults, collected in EDTA and centrifuged for 15 min at 3000 x g. Plasma samples were stored at -20 C. For histological studies, animals were killed by cervical dislocation and ovaries and pituitaries were immediately recovered, fixed in a Bouin’s fluid, then dehydrated in alcohol baths, and embedded in paraffin. For autoradiographic studies, ovaries were embedded in cryoembedding compound (Tissue-Tek, Miles Laboratories, Elkhart, IN) and immediately frozen in liquid nitrogen vapors.

Assessment of the functionality of the hypothalamo-pituitary-ovarian axis
To assess female cyclicity, vaginal smears were performed daily during 15 d. Smears were stained with methylen blue and examined under light microscope. To study responsiveness of ovaries to exogenous gonadotropins, mice were injected ip with 5 IU eCG during dioestrus, and blood samples were collected 46 h later, during the induced proestrus, for LH and FSH measurements. Blood samples were also collected 4 wk after castration. Finally, to study the response of pituitary gland to exogenous GnRH, 30 ng of GnRH were injected ip, and blood samples were collected 15 min later for LH and FSH measurements.

Southern blot analysis
Briefly, the genotype of mice was determined by Southern Blot analysis of DNA extracted from tails. Ten micrograms mouse genomic DNA were digested with the restriction endonuclease NcoI at 37 C overnight. The digested DNA were fractionated on 1% agarose gel, transferred to Hybond-N⁺ membrane (Amersham Pharmacia Biotech, Saclay, France) and hybridized with hIGFBP-1 cDNA fragment labeled by random priming (1 x 10⁶ cpm/ml) as described previously (13).

Histological analysis of ovaries
Ovaries included in paraffin were serially sectioned at a thickness of 10 µm, then sections were stained with Feulgen. Five classes of follicles were selected according to their diameter: 50–99 µm, 100–199 µm, 200–299 µm, and 300 µm or greater. The quality of ovarian follicle was estimated by histological examination of sections, follicles being judged normal (frequent mitosis, no pycnosis in granulosa), or atretic (rare normal granulosa cells and a great majority of pycnotic bodies).

Autoradiography of [¹²⁵I]-IGF-I, [¹²⁵I]-hCG and [¹²⁵I]-rFSH binding sites on histological sections
The binding of [¹²⁵I]-IGF-I, [¹²⁵I]-hCG and [¹²⁵I]-rFSH to ovarian frozen sections was performed as described previously (14, 15). Briefly, ovaries were serially sectioned at a thickness of 10 µm with a cryostat. After fixation for 10 min at 4 C in picric acid-formaldehyde and three subsequent washings in cold PBS (pH 7.4), sections were stored at -20 C overnight. To assess the quality of ovarian follicles, some adjacent sections were fixed in methanol-formaldehyde-acetic acid (80:15:5) and stained with Feulgen.

For IGF-I binding sites, sections were incubated at room temperature during 7 h in duplicate in a drop of PBS (0.1% BSA; pH 8) containing [¹²⁵I]-IGF-I alone (200,000 cpm/section), or with unlabeled IGF-I (500 ng/section) or insulin (5 µg/section) to check for the specificity of the binding. Nonspecific binding was obtained after incubation of [¹²⁵I]-IGF-I together with an excess of unlabeled IGF-I and binding corresponding to IGFBPs was estimated by incubating the [¹²⁵I]-IGF-I in the presence of an excess of insulin, as previously described (14). The hCG and rFSH binding assay used a protocol described previously (15). Sections were incubated in a drop of PBS (0.1% BSA; pH 7.4) containing [¹²⁵I]-hCG or [¹²⁵I]-rFSH alone (400,000 cpm/section), or with unlabeled hCG or rFSH (500 ng/section) to measure the nonspecific binding.

For these three experiments, at the end of the incubation period, sections were washed twice in PBS, postfixed in 3% glutaraldehyde-PBS, washed in PBS and air dried. For autoradiography, sections were then dipped in emulsion, air dried, exposed for 2 wk at 4 C, then developed, fixed by classical procedure, and stained with hematoxyline.

Microscopic analysis of autoradiography
Quantitative autoradiographic analysis of [¹²⁵I]-hCG or [¹²⁵I]-rFSH binding sites was performed using a microscope linked PC-based image analyzer (SAMBA TM 2005, Alcatel TITN, Meylan, France). Each section was analyzed with an objective x40. Quantification of labeling was performed by measuring the area occupied by silver grains present in a constant area (250 µm²). This quantification has been performed on granulosa cells from 10–14 follicles of HM mice, and 12–22 follicles of NT mice for FSH and LH receptors, respectively. Labeling was estimated from 20 measurements on each antral follicle (>200 µm in diameter). Specific binding was obtained by subtracting the values of labeling associated with nonspecific binding from the total binding values.

Immunohistochemistry
Pituitaries (homozygotes, n = 5; nontrangenic, n = 4) embedded in paraffin were serially sectioned at a thickness of 10 µm. Sections were incubated for 30 min in PBS containing 0.3% H₂O₂ to remove endogenous peroxidase activity and were then incubated for 15 min in PBS containing 7% sheep serum to saturate nonspecific binding sites. Sections were incubated overnight at 4 C with PBS containing 0.1% BSA and rabbit primary antibody raised against rat ß-LH (1:40,000) and rat ß-FSH (1:1000). After washing, sections were incubated for 6 h at room temperature with a peroxidase-labeled antibody raised against rabbit IgG (1:20,000). Staining was obtained by incubating sections in Tris-HCl (20 mM, pH 7.8) containing 0.2 mg/ml diaminobenzidine and 0.0036% H₂O₂ (vol:vol). Negative controls were performed by replacing primary antibodies by a nonimmune serum. The number and the size of cells immunoreactive for FSH and LH were assessed by using a 100x objective lens within an ocular micrometer.

LH assay
Concentrations of LH in serum and pituitary extracts were determined by RIA. LH concentration in pituitary samples was measured in three different dilutions 1, 1/2, 1/4 in duplicate; plasma samples were assayed without dilution. All samples were incubated overnight at 4 C with [¹²⁵I]-rLH (20,000 cpm/tube) and rabbit polyclonal antiserum against rLH (1:75,000). Volumes were adjusted with a solution of 0.03 M NaH₂PO₄, 3.72 g/liter of EDTA, 500 µl/liter Tween 20, 200 mg/liter of protamine sulfate, and 200 mg/liter of azide (pH 7.4) for a final volume of 500 µl. After incubation, samples were incubated with sheep serum raised against rabbit IgG (6 µl/tube), polyethylene glycol (PEG 6000, 0.06 g/tube) in PBS (2 ml/tube) overnight at 4 C. Then samples were subjected to centrifugation, and radioactivity was counted in the pellet. LH concentrations were calculated on the basis of a range standard from 5 pg to 5 ng of mouse LH. Concentration of proteins in pituitary extracts was determined by the Bradford method.

The sensitivity of LH RIA was 20 pg/tube. The interassay coefficient of variation was 18% for serum and 15% for pituitary LH concentrations.

FSH assay
FSH assay was performed by using FSH standard and RP-2 provided by NIDDK as described previously (16). The sensitivity of FSH RIA was 40 pg/tube, and the intraassay coefficients of variation was 12.7%.

Statistical analysis
All data are presented as the mean ± SEM. A t test, or in the case of heterogeneity of variance, the Mann and Whitney U test were used to compare means between two groups. In the case of multiple comparisons of means, statistical analysis was performed by ANOVA followed by the Newman-Keuls test, or Kruskal-Wallis ANOVA as appropriate. Comparison with P > 0.05 were not considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
As previously reported (13), the average body weight as well as the weight of ovaries and genital tract were 1.5- to 3-fold lower in hIGFBP-1 homozygotes (HM) than in hemizygotes (HT) and than in nontransgenic (NT) adult mice at 2–5 months of age (Table 1), ovaries and genital tract being disproportionately smaller. At 2 months of age, none of the HM mice were cyclic as assessed by examination of vaginal smears and none were plugged by wild-type vasectomized males, in contrast to 95% of NT mice. At 3–5 months of age, 50% of HM mice remained acyclic and were never plugged by wild-type vasectomized males, the other HM mice presenting irregular cyclicity.

View larger version (85K): [in this window] [in a new window]   Figure 1. Histology of ovaries of hIGFBP-1 female transgenic mice. Histological sections of ovaries from 4- to 5-month-old nontransgenic (left) and homozygotes mice (right). Bar, 150 µm.

View larger version (24K): [in this window] [in a new window]   Figure 2. Population of follicles in hIGFBP-1 female transgenic mice. Follicles were ranked by size [50–99 µm], [100–199 µm], [200–299 µm], [300 µm] and degree of atresia (healthy or atretic follicles). The morphometric studies were performed on ovaries from 2- to 5-month-old homozygotes (n = 4), hemizygotes (n = 3), and nontransgenic (n = 3) mice except for follicles 300 µm diameter and corpora lutea, which were counted on ovaries from 9 homozygotes, 3 hemizygotes, and 7 nontransgenic mice. Experimental data are expressed as the mean ± SEM. *, P < 0.05; ***, P < 0.001.

Binding experiments performed on ovarian sections showed similar levels of LH receptors in thecal cells of preantral and early antral follicles, as well as in both thecal and granulosa cells of preovulatory follicles from HM and NT mice, in both basal and eCG-treated conditions (Fig. 3, A and B). The levels of LH receptors in corpora lutea were also similar in HM and NT mice. FSH receptors were also present at similar levels in granulosa of healthy growing follicles from both HM and NT mice (Fig. 3, A and B).

View larger version (88K): [in this window] [in a new window]   Figure 3. FSH and LH receptors in ovarian follicles of transgenic and nontransgenic mice. A, Follicles from homozygous (1–5) and nontransgenic (6–10) mice (2 months old) 46 h after 5 UI eCG administration. 1, 6: Sections stained with Feulgen. 2, 7 and 3, 8: [125I]-FSH and [125I]-hCG binding sites on a large antral follicle, respectively. 4, 9 and 5, 10: Nonspecific labeling. Original magnification, x200. Bar, 100 µm. Similar results were obtained on mice not treated with eCG. B, Quantification of autoradiography of [125I]-hCG (left) and [125I]-rFSH (right) binding sites.

The dramatic decrease in the number of corpora lutea in ovaries of HM mice suggested an alteration of ovulation due to an alteration of gonadotropic secretion. We therefore tested the ability of pituitary to release LH and FSH. LH plasma concentrations were similar at dioestrus in HM and NT mice (Fig. 4A). Moreover, plasma LH and FSH concentrations were similar in both HM and NT mice 46 h after eCG administration, 4 wk after castration or 15 min after GnRH administration (Fig. 4, A and B). The pituitary FSH content was the same in HM and NT mice (Fig. 5B). Interestingly, pituitary LH concentration was more than 8-fold higher in HM mice than in NT mice (P = 0.02) (Fig. 5A). Histological analysis showed an increase in the number of LH-immunoreactive cells per surface unit in HM mice (HM, 5.05 ± 0.26 LH-cells vs. NT, 3.89 ± 0.21 LH-cells/10,000 µm surface, P = 0.01) (Fig. 6), a reduction in the size of LH-immunoreactive cells (HM, 222 ± 6 µm vs. NT, 246 ± 6 µm cell surface, P < 0.05) and of FSH-immunoreactive cells (HM, 203 ± 11 µm vs. NT, 241 ± 9 µm cell surface, P < 0.05). In each genotype, the size of LH- and FSH-immunoreactive cells was similar, as expected.

View larger version (21K): [in this window] [in a new window]   Figure 4. Plasma LH and FSH concentrations in hIGFBP-1 female transgenic mice. A, Levels of LH were measured in plasma during dioestrus (HM, n = 2; HT/NT, n = 7), 46 h after a eCG stimulation (HM, n = 13; HT, n = 9; NT, n = 8), 15 min after GnRH stimulation (HM, n = 4; HT/NT, n = 4) and 4 wk after castration (HM, n = 3; NT, n = 3). B, Levels of FSH were measured in plasma 46 h after a eCG stimulation (HM, n = 6; HT, n = 4; NT, n = 5), 15 min after GnRH stimulation (HM, n = 3; HT/NT, n = 4) and 4 wk after castration (HM, n = 3; NT, n = 3). All these LH and FSH assays were performed on 2- to 7-month-old females. Experimental data are expressed as the mean ± SEM.

View larger version (15K): [in this window] [in a new window]   Figure 5. Pituitary LH and FSH content in hIGFBP-1 female transgenic mice. Concentration of LH (A) and FSH (B) were measured in pituitaries from 2- to 10-month-old HM mice (n = 3), HT mice (n = 8), and NT mice (n = 4). Experimental data are expressed as the mean ± SEM. *, P < 0.05.

View larger version (41K): [in this window] [in a new window]   Figure 6. LH-immunoreactive cells in adult pituitary in hIGFBP-1 female transgenic mice. LH-immunoreactive cells in the pituitary of nontransgenic (left) and homozygous (right) adult mice. Original magnification, x400.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Transgenic female mice overexpressing ubiquitously rIGFBP-1 [particularly in the ovary (17)] as well as GH-Receptor knock-out female mice (Laron mice, 18) (19 and Bachelot, A., P. Monget, J. J. Kopchick, P. A. Kelly, and N. Binart, submitted), were also shown to present a decrease in seric IGF-I levels and bioavailability, associated with a reduction of natural and induced ovulation rate. Moreover, IGF-I -/- female mice are characterized by a blockage of follicular growth before antrum formation and a complete loss of ovarian responsiveness to gonadotropins (6). Overall, these results strongly suggest that IGF-I plays a key role in the responsiveness of the ovary to FSH action. Interestingly, we did not find any changes in the number of atretic follicles >200 µm diameter in HM ovaries, whereas there was no increase in the number of healthy follicles <200 µm. Baker et al. (6) also reported that ovaries from IGF-I -/- mice were almost totally devoid of large antral follicles, without any changes in the number of smaller (secondary and primary) follicles, nor any increase in the percentage of atretic follicles whatever the size class . Of note in the ovary, the number of follicles per size class is the result of 1) the number of smaller follicles that reach this size class, 2) the growth rate of follicles in this size class, which determines the transit time of follicles in the class, 3) the rate of atresia in this size class, and 4) the number of follicles that reach the upper size class. Then in the case of IGFBP-1 transgenic mice as well as IGF-I -/- mice, the main alteration of ovarian functionality is likely a decrease in the growth rate of follicles rather than an increase in their rate of atresia.

In contrast to IGF-I -/- females and to transgenic females overexpressing ubiquitously rIGFBP-1 (17), the decrease in IGF-I levels in hIGFBP-1 mice is strictly of peripheral origin, without clear evidence of accumulation of hIGFBP-1 at the level of follicular cells. In the ovary, several arguments play in favor of a seric origin (at least in part) of IGF-I in large antral follicles. First in several species, IGF-I concentrations in large normal follicles are lower and positively correlated to serum levels (see Ref. 20). Secondly, the systemic 150-kDa IGFBP-IGF complex, that carries more than 90% of hepatically produced IGFs in serum, has been identified in ovine (21) and human follicular fluid (22), proteins with molecular weights below 500 kDa being able to cross the basal membrane of the follicle (23). Thirdly, immunization of cattle against GHRH leads to a decrease and an increase in IGF-I and IGFBP-2 levels, respectively, in both serum and follicular fluid of large follicles (24). In contrast, treatment of cattle with GH leads to an increase in IGF-I levels in both compartments. So, in hIGFBP-1 transgenic mice, the decrease in IGF-I levels in serum may lead to a decrease in intrafollicular IGF-I concentration, and a subsequent decrease in FSH biological effects. Of note, and as in GH-Receptor -/- females (19 and Bachelot, A., P. Monget, J. J. Kopchick, P. A. Kelly, and N. Binart, submitted), but in contrast to IGF-I -/- females, FSH receptor levels were not clearly altered in granulosa cells of hIGFBP-1 (present work), suggesting that the increase in hIGFBP-1 levels may impair the enhancement effect of IGF-I on FSH signaling pathway (25) at the level of granulosa cells, rather than FSH receptor expression.

Even if single serum assay likely prevented us from seeing changes in the pattern of FSH and LH secretion, several arguments suggest that hIGFBP-1 transgenic mice presented an alteration of LH secretion. First, LH concentration in pituitaries from HM mice was more than 8-fold higher than in NT mice. Furthermore, the number of immunoreactive LH gonadotrophs per surface area of pituitary section was shown to be 1.3-fold higher in HM compared with NT mice, and their size was 0.8-fold reduced. As pituitaries of HM mice were able to respond to exogenous GnRH treatment in vivo as well as on pituitary explant in vitro (Froment, P., C. Staub, S. Hembert, C. Pisselet, M. Magistrini, J. E. Levine, D. Monniaux, B. Binoux, and P. Monget, in preparation), we could hypothesize that hIGFBP-1 overexpression in the liver of HM mice have led to an alteration of endogenous GnRH pulsatility. Recently, we have shown that this probable alteration of the functionality of the hypothalamo-pituitary axis of hIGFBP-1 transgenic mice was not limited to GnRH and LH secretion. Indeed, in these mice, we have observed a 30% decrease in the number of somatotrophs and lactotrophs (Seurin, D., P. Froment, M. T. Bluet-Pajot, J. Epelbaum, P. Monget, and M. Binoux, submitted) with a strong diminution of GH content in pituitaries (Seurin, D., P. Froment, M. T. Bluet-Pajot, J. Epelbaum, P. Monget, and M. Binoux, submitted), suggesting an alteration of the somato-lactotroph lineage. IGF-I-/- mice also presented a 50% reduction of the number of lactotroph cells and the size of somatotroph cells was reduced (26). Interestingly, streptozotocin-induced diabetic rats, which also exhibit an increase in IGFBP-1 concentrations and a decrease in IGF-I concentrations in serum associated with an insulin deficiency (27, 28). These rats present a severe reduction of pulsatile LH secretion, an increase of LH pituitary content and of the number of LH-gonadotrophs associated with a reduction of their size, as well as a decrease in plasma GH and PRL concentrations (29, 30, 31, 32). A decrease of GnRH pulse secretion (33) and an increase of degenerate GnRH axons in median eminence were also observed (34). The similarity between this model and our hIGFBP-1 transgenic mice reinforces the hypothesis of an alteration of the hypothalamo-pituitary axis in hIGFBP-1 transgenic mice.

IGF-I is known to play a pivotal role in the development of the central nervous system and in protecting the brain from the consequences of a nutritional deprivation (35). In particular, ectopic brain expression of IGFBP-1 reduced brain weight and induced abnormality in the formation of neurons and axons, likely by inhibiting IGF actions (36). In our model, expression of hIGFBP-1 transgene was only restricted to the liver, but expression was particular high during pre- and perinatal period, when the blood brain barrier is not functional (13). The presence of significant concentrations of hIGFBP-1 in cerebrospinal fluid from homozygous animals (Gay, E., and M. Binoux, unpublished results) would be responsible for an alteration of brain development (37) and functionality in fetus and young pups. Moreover, several studies (4, 38) suggest that IGF-I and insulin play a role in sexual maturation in peripubertal animals. In adults, both factors could be key links between metabolism, growth status, and reproduction in certain hypothalamic areas that have been located outside the blood brain barrier (39). In particular, IGF-I and/or insulin are able to stimulate GnRH release in the median eminence (1, 3, 40). Intraventricular administration of IGF-I to peripubertal female rats and in GHRH knockout mice is able to induce dose dependently LH release and to advance puberty (4, 41). In contrast, intraventricular infusion of IGF-I antiserum to male rats delays pubertal development (42). More recently, it has been shown that conditional invalidation of insulin receptor in the brain leads to an infertility associated with a strong reduction of serum LH concentrations (43). According to these data, the alteration of hypothalamic functionality in hIGFBP-1 transgenic mice as well as pituitary disorders, which might be partly responsible for the delayed puberty and the irregular cyclicity of adult homozygous females, could be due to a decrease in IGF-I action.

Overall, data obtained on IGF-I -/- mice, different IGFBP-1 transgenic mice, as well as Laron mice, strongly suggest that IGF-I is a key factor necessary for ovarian folliculogenesis. IGF effects on females might occur in the ovary through sensitization of the follicles to gonadotropins, as well as through regulation of gonadotropins secretion at the hypothalamus-pituitary level. Moreover, in the present model, it is possible that part of the alteration of fertility in the IGFBP-1 transgenic mice could be explained by an IGF-independent effect of IGFBP-1, in particular via binding to 5ß1 integrin receptors, as previously shown (44, 45). However, none of the present results showing an alteration of fertility could be explained by independent actions of IGFBP-1. Such a conclusion could only be drawn after treatment of mice with IGFBP-1 in comparison with RGD- mutated IGFBP-1, in the presence or absence of IGF-I.

In conclusion, we have shown that overexpression of hIGFBP-1 reduced fertility and fecundity, with a significant decrease in the number of growing antral follicles and corpora lutea, and a strong decrease in ovarian responsiveness to gonadotropin treatments. Moreover, an augmentation of the LH content and of the number of LH-immunoreactive cells in pituitary, as observed in diabetic rats, also suggests an alteration of GnRH pulse generator and LH secretion. Overall, all these results show that the somatotropic axis is necessary for the full development and functionality of the hypothamo-pituitary-ovary axis.

	Acknowledgments

 
We thank Claude Cahier, Michel Vigneau and Jean-Claude Braguer for expert animal care and Thierry Delpuech for excellent technical assistance. We wish also acknowledge Dr. J. Closset for donating rFSH. We are grateful to Dr. A. F. Parlow for hold the FSH and LH reagents and to Alain Beguey for the photographic work.

	Footnotes

 
This work was supported by the FARO (Fonds d’Aide à la Recherche Organon) institution, by Institut National de la Recherche Agronomique and by Institut National de la Santé et de la Recherche Médicale. Pascal Froment was supported by a fellowship from Institut National de la Recherche Agronomique and Région Centre.

Abbreviations: eCG, Equine CG; HM, homozygotes; HT, hemizygotes; h, human; IGFBP, IGF binding protein; NT, nontransgenic; r, rat.

Received September 5, 2001.

Accepted for publication February 1, 2002.

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