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Endocrinology, doi:10.1210/en.2003-0956
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Endocrinology Vol. 145, No. 4 2080-2091
Copyright © 2004 by The Endocrine Society

Reproductive Abnormalities in Human Insulin-Like Growth Factor-Binding Protein-1 Transgenic Male Mice

Pascal Froment, Christophe Staub, Stéphanie Hembert, Claudine Pisselet, Michèle Magistrini, Bernadette Delaleu, Danielle Seurin, Jon E. Levine, Larry Johnson, Michel Binoux and 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 (P.F., S.H., C.P., M.M., B.D., P.M.), 37380 Nouzilly, France; Department of Veterinary Anatomy and Public Health, Texas A&M University (C.S., L.J.), College Station, Texas 77843-4458; Institut National de la Santé et de la Recherche Médicale, Unité 515, Croissance, Différenciation et Processus Tumoraux, Hôpital Saint-Antoine (D.S., M.B.), 75571 Paris, France; and Department of Neurobiology and Physiology, Northwestern University (J.E.L.), Evanston, Illinois 60208

Address all correspondence and requests for reprints to: Dr. 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
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adult transgenic mice overexpressing human insulin-like growth factor-binding protein-1 in the liver present reproductive abnormalities in both sexes. In the present work, we have investigated the mechanisms responsible for limiting breeding capacity in these transgenic male mice. Homozygous adult transgenic male mice (3–6 months old) exhibited irregular copulatory behavior and a reduction of the number of pregnancies per female as well as of litter size per pregnancy. Genital tract weight, more specifically epididymal and seminal vesicle weights, were reduced by 45% in homozygous transgenic vs. nontransgenic mice. Homozygous transgenic mice exhibited a 30% reduction of the length of seminiferous tubules (P = 0.007), a 30% decrease in daily sperm production per testis (P = 0.019), and a 50% decrease in the number of spermatozoa in testis (P = 0.037), associated with morphological abnormalities of the sperm heads leading to an approximately 50% reduction of fertilized two-cell eggs (P = 0.002) and of implanted embryos on d 5.5 after mating (P = 0.004). The round spermatids also appeared altered in their morphology. In addition, Leydig cells in homozygous transgenic mice exhibited an altered appearance, with a 1.8-fold increase in lipid droplets in their cytoplasm (P < 0.001). Moreover, the concentration of 3ß-hydroxysteroid dehydrogenase was 66% lower in testis from transgenics compared with those from normal mice (P = 0.01), leading to a tendency toward lower plasma testosterone levels (P = 0.1). Interestingly, LH concentrations were increased by 40% in transgenic pituitary extracts (P = 0.02), and basal LH secretion by pituitary explants in vitro was increased by 60% in homozygous transgenic vs. normal mice (P = 0.04), suggesting an alteration of LH pulsatile secretion in vivo. In conclusion, these data suggest that the breeding impairment of human insulin-like growth factor-binding protein-1 transgenic males is due at least in part to an alteration of the process of spermatogenesis, leading to a diminution of sperm production and of its quality. Minor impairment of steroidogenesis may also contribute to the reduced reproductive capacity of these animals. Our observations are consistent with the idea that normal spermatogenesis and perhaps also steroidogenesis are dependent on the actions of sufficient concentrations of unbound IGF-I.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SOMATOTROPIC axis is known to play a key role in male reproduction. In particular, in hypothalamo-pituitary axis, IGF-I might be a major signal that connects somatic development to the GnRH-releasing system during sexual maturation (1). Moreover, it has been established by in vitro and in vivo studies in rodent models that the testis is a target of IGF-I action, mainly through the regulation of steroidogenesis and spermatogenesis (2). Different elements of the IGF system are expressed in the testis (3, 4). In particular, IGF-I has been shown to be expressed by Leydig and Sertoli cells. Type I IGF receptor expression has been observed in germ cells and somatic cells in several species (5, 6, 7). Interestingly, GH or IGF-I administration to dwarf rats in vivo has been shown to increase the concentration and motility of spermatozoa in epididymis (8). IGF-I is also capable of up-regulating steroidogenesis by stimulating LH receptor expression in pig Leydig cells in vitro (9) and by enhancing the activity of steroidogenesis enzyme (5, 10, 11). Finally, gonadotropins have been shown to increase the local production of IGF-I in testicular somatic cells in vivo and in vitro (12, 13, 14).

The bioavailability of IGFs and their interaction with type I IGF receptor are known to be modulated by IGF-binding proteins 1–6 (IGFBP-1 to -6). One of them, IGFBP-1, is known to be subject to major regulation by nutritional status. IGFBP-1 is mainly produced in the liver as well as in kidney. Its expression is stimulated by glucose, glucagon, and glucocorticoids and is inhibited by insulin and GH (15, 16). The secretion of IGFBP-1 in mouse liver is high during fetal life and decreases after birth. Moreover, it has been established by in vitro (17, 18) and in vivo (19, 20) studies that IGFBP-1 plays a major role in inhibiting the actions of IGF-I. Of note, the growth retardation observed in cases of diabetes or negative energy balance was shown to be associated with an increase in IGFBP-1 concentrations in serum.

In our previous work (21), we studied the mechanisms responsible for reproductive abnormalities in female transgenic mice overexpressing human (h) IGFBP-I in the liver. In preliminary experiments (22), homozygous transgenic males exhibited a reduction of fertility as well as a reduction of litter size (5.5 vs. 8.4 pups/litter in transgenic and nontransgenic mice, respectively). In the present work we have studied the functionality of the hypothalamo-pituitary-gonadal axis of these transgenic male mice to determine the causes of this alteration in fertility.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The generation and characterization of the transgenic mice have been previously described in detail (22). The transgene consisted of a human cDNA of the IGFBP-1 gene inserted downstream of the human 1-antitrypsin (700 bp) promoter. Homozygous (HM), heterozygous (HT), and nontransgenic (NT) male mice of the 149 strain were used for all studies. Mice were maintained under controlled conditions of light (12 h of light, 12 h of darkness) and temperature, with ad libitum access to food and water.

For the study of fertility, each male was place in an individual cage in the presence of four control females. Male sexual behavior was assessed by examining for vaginal plugs every day.

All procedures were approved by scientific research agency and were conducted in accordance with the guidelines for the care and use of laboratory animals.

Southern analysis
Briefly, the genotype of mice was determined by Southern analysis of DNA extracted from tails. Ten micrograms of mouse genomic DNA were digested overnight with the restriction endonuclease NcoI at 37 C. The digested DNA were fractionated on 1% agarose gel, transferred to a Hybond-N+ membrane (Amersham Pharmacia Biotech, Arlington Heights, IL), and hybridized with the hIGFBP-1 cDNA fragment labeled by random priming (1 x 106 cpm/ml) as described previously (22).

Blood collection and tissue removal
In adult mice, total body, carefully dissected testes, epididymis, and seminal vesicles were weighed in mice of both genotypes. Blood samples (~500 µl) were recovered by intraorbital puncture in adults, collected in 1% 0.13 M 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 testis, epididymis, and pituitaries were immediately recovered, fixed in Bouin’s fluid, and imbedded in paraffin. The testis and epididymis weights were averaged before fixation.

Embryo implantation sites
Adult wild-type females were mated with transgenic males. The morning of vaginal plug identification was designated d 0.5 of pregnancy. On d 5.5 of pregnancy, implantation sites were visualized after iv injections of 1% Chicago Blue in saline 5 min before death (23).

Culture of eggs in vitro
Four or five wild-type females were mated with HM, HT, and NT male mice. Females that presented a vaginal plug were killed on d 0.5 of pregnancy, eggs were removed from oviducts and collected in M2 medium (Sigma-Chemie, l’Isle d’Abeau Chesnes, France) containing 4% BSA and 300 µg/ml hyaluronidase. Eggs were washed through several droplets of 4% BSA/M2 medium, and cultured overnight in M16 medium (Sigma-Chemie) at 37 C with 5% CO2. Eggs were scored as successfully fertilized if they progressed to a two-cell embryo stage.

Sperm heads counts
Testis previously frozen at -20 C were thawed at 4 C, cut into 2-mm pieces, suspended in 1 ml water, homogenized with an Ultraturax homogenizer, and sonicated for 30 sec to dissociate somatic cells and sperm heads and tails. Only sperm nuclei that remained were counted by hemocytometry from several aliquots, and their concentration in the testis was determined after correction for sample volume and testis weight as previously described (24).

Morphometry of testis
Testis included in paraffin were serially sectioned at a thickness of 10 µm, and sections were stained with Feulgen (Schiff reagent for Feulgen staining was purchased from Merck & Co., Schuchardt, Germany). At least 30 measurements of transverse sections of seminiferous tubules diameters for each testis (testes from three animals per genotype) were measured using an ocular measuring device. The tubular diameter was measured in round or nearly round seminiferous tubules from each testis using a x100 objective lens within an ocular micrometer.

The seminiferous tubule length was assessed by using the following formula: tubule length = [seminiferous tubule volume/(3.1416 x seminiferous tubule ray2)]. The seminiferous tubule volume represents the product of the fresh weight of the testis and the relative volume of the seminiferous tubule, as previously described (25).

Stereology of testis and calculation of daily sperm production
Basic stereological principles were employed to assess alterations in spermiogenesis (26). Testes from four different animals per genotype were fixed in 4% glutaraldehyde and 0.1 M sodium cacodylate buffer, postfixed in 1% osmium tetroxide, embedded in Eponaraldite resin, and sectioned at 0.5 µm for stereological study or at 20 µm for cellular diameter. The number of spermatids with spherical nuclei was determined by stereology of Epon sections involving the measurement of nuclear volume density, parenchymal volume, and volume of a single nucleus for this cell type (26). Stereological procedures included Chalkley’s point-counting method (27) of 0.5-µm sections stained with toluidine blue and observed by brightfield light microscopy at x1,000 magnification to determine the nuclear volume density of round spermatids (percentage of the parenchyma occupied by nuclei of that cell type). A total of 10,000 points on eight sections of tissue per animal were counted. The nuclear volume of individual cells was determined in 20-µm sections observed unstained with Nomarski optics, by measurement of the maximum nuclear diameter and the formula for the volume of a sphere (28). The number of round spermatids was determined by dividing the product of the nuclear volume density and a histological correction factor for section thickness and nuclear diameter (29) by the mean volume of a single round spermatid nucleus (26, 28, 30). The daily sperm production per gram was calculated by dividing the number of round spermatids (steps 1–7 of the spermatogenic cycle) by their life span or the duration of that phase of development (4.9 d) (31).

Quantification of the surface area occupied by lipid droplets in Leydig cells
Quantitative analysis of the lipid droplet surface in Leydig cells was performed using a microscope-linked personal computer-based image analyzer (SAMBA TM 2005, Alcatel TITN Meylan, France). The lipid surface was analyzed by measuring the area occupied by lipid droplets present in a constant area (100 µm2). This quantification has been performed with an objective of x63 on Epon semithin sections (0.5 µm) stained with toluidine blue from testes of four different animals per genotype. Lipid surface was estimated from an average of 50 measurements on each testis.

Transmission electron microscopy
Testes from four different animals per genotype were fixed in 4% glutaraldehyde, 0.1 M sodium cacodylate buffer, postfixed in 1% osmium tetroxide, and embedded in Eponaraldite resin. Sections 70 nm thick were placed on 200-mesh copper grids, stained with uranyl acetate followed by lead citrate, and examined using an electron microscope (CM 10 Philips, Eindhoven, The Netherlands).

Immunohistochemistry
Testis included in paraffin were serially sectioned at a thickness of 10 µm. Sections were deparaffinized, hydrated, and microwaved for 5 min in antigen unmasking solution (Vector Laboratories, Inc., AbCys, Paris, France), then left to cool to room temperature. After washing in a PBS bath for 5 min, sections were immersed in 0.3% hydrogen peroxide for 20 min at 4 C to quench endogenous peroxidase activity. After washing in a PBS bath for 5 min, nonspecific background was eliminated by blocking with 1.5% normal horse serum in PBS for 30 min, followed by incubation overnight at 4 C with PBS containing rabbit primary antibody raised against p21 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), cleaved caspase-3 (1:500; Cell Signaling Technology, Beverly, MA) and mouse antibody raised against proliferating cell nuclear antigen [PCNA (PC10); 1:500; Santa Cruz Biotechnology]. Sections were washed twice for 5 min each time in a PBS bath and were incubated for 30 min at room temperature with the biotinylated secondary antibody raised against mouse and rabbit IgG and 1.5% normal horse serum. Then sections were incubated for 30 min at room temperature with the avidin and biotinylated horseradish peroxidase macromolecular complex as indicated in the kit instructions (Vectastain Elite ABC reagent, Vector Laboratories, Inc., AbCys). Visualization was achieved by incubation in a peroxidase substrate solution (Vector DAB, Vector Laboratories, Inc., AbCys). Negative controls were performed by replacing primary antibodies with rabbit or mouse IgG.

Pituitaries (HM, n = 3; NT, n = 3) embedded in paraffin were serially sectioned at a thickness of 10 µm. The immunoreactivity for FSH and LH was performed as previously described (21). The number and the size of the cells immunoreactive for FSH and LH were assessed using a x100 objective lens within an ocular micrometer.

In situ cell detection
Deparaffinized and hydrated 10-µm sections of testis were microwaved for 5 min in antigen unmasking solution (Vector Laboratories, Inc., AbCys) for permeabilization and left to cool to room temperature. After washing in a PBS bath, sections were directly labeled by the addition of fluorescein dexoy-UTP at the 3'-OH DNA ends by terminal deoxynucleotidyltransferase for 1 h at 37 C in a humidified atmosphere in the dark as described in the instruction manual (In Situ Cell Death Detection Kit, Fluorescein, Roche, Meylan, France). Sections were counterstained with Hoechst 33258 (Calbiochem, La Jolla, CA). Negative controls were treated identically, except that the incubation with terminal deoxynucleotidyltransferase was omitted.

Western immunoblotting
Lysates of testes were prepared on ice with an Ultraturax homogenizer in lysis buffer [10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 0.5% Igepal] containing various protease inhibitors (2 mM phenylmethylsulfonylfluoride, 10 mg/ml leupeptin, and 10 mg/ml aprotinin) and phosphatase inhibitors (100 mM sodium fluoride, 10 mM sodium pyrophosphate, and 2 mM sodium orthovanadate; Sigma-Chemie). Lysates were centrifuged at 15,000 x g for 20 min at 4 C, and the protein concentration in the supernatants was determined using a colorimetric assay (kit BC Assay, Uptima Interchim, Montluçon, France).

Equal amounts of proteins were submitted to electrophoresis on 12% (wt/vol) SDS-PAGE under reducing conditions. The proteins were then electrotransferred onto nitrocellulose membranes (Schleicher & Schuell, Ecquevilly, France) for 2 h. Membranes were incubated for 1 h at room temperature with Tris-buffered saline [TBS; 2 mM Tris-HCl (pH 8.0) and 15 mM NaCl (pH 7.6)] containing 5% nonfat dry milk powder (NFDMP) and 0.1% Tween 20 to saturate nonspecific sites. Thereafter, membranes were incubated overnight at 4 C with anti-cytochrome P450 side-chain cleavage enzyme (anti-P450scc; final dilution, 1:2,000), anti-3ß-hydroxysteroid dehydrogenase (anti-3ßHSD; final dilution, 1:500), or antiactin (final dilution, 1:1,000; Sigma-Chemie) antibodies in TBS containing 0.1% Tween 20 and 5% NFDMP. Rabbit polyclonal antibody raised against bovine P450scc and against human placental 3ßHSD were provided by Dr. D. B. Hales (University of Illinois, Chicago, IL) and Dr. V. Luu-The (Centre de Recherche en Endocrinologie Moléculaire, Québec, Canada), respectively. After washing in TBS/0.1% Tween 20, nitrocellulose membranes were incubated for 2 h at room temperature with horseradish peroxidase-conjugated antirabbit or antimouse antibody (final dilution, 1:10,000; Diagnostic Pasteur, Marnes-la-Coquette, France) in TBS/0.1% Tween 20/5% NFDMP. After washing in TBS/0.1% Tween 20, the signal was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Orsay, France). The signals quantified with Mac Bas software (version 2.52, Fuji Photo Film, Tokyo, Japan). The results are expressed as the intensity signal in arbitrary units and correspond to the average of three cell lysates signals per genotype after normalization by visualization of actin as an internal standard.

Assessment of the functionality of the pituitary axis
To study the functionality of the pituitary gland in vivo, blood samples were collected under basal conditions, 4 wk after castration, or 15 min after an ip injection of 30 ng GnRH (Sigma-Chemie) for LH and FSH measurements. For pituitary explants in vitro, five pituitaries per genotype were removed after cervical dislocation and individually collected in DMEM (Sigma-Chemie) containing transferrin (10 µg/ml), ascorbic acid (17.6 µg/ml), and antibiotics. Cultures of explants were established according to the method described by Watanabe (32) with minor modifications. Each pituitary was placed on a round piece of cellulose acetate (13-mm diameter; Millipore Corp., St. Quentin en Yvelines, France) on stainless mesh platforms in a culture dish containing DMEM with 10% fetal ovine serum. The culture dishes were placed at 37 C in a 5% CO2/95% atmosphere. Explants were incubated in the absence of GnRH for 2 h, then in the presence of 10 nM GnRH for 2 h. Medium was removed and stored at -20 C before LH and FSH assay.

Hormone assay
Concentrations of LH in serum, pituitary extracts, and culture medium were determined by RIA using rar LH-RP-3 for standard calibration, rabbit polyclonal antiserum against rat LH, and reagents supplied by Dr. A. F. Parlow (NIDDK, Bethesda, MD) as previously described (21). The sensitivity of the LH RIA was 20 pg/tube. The interassay coefficient of variation was 18% for serum and 15% for pituitary LH concentrations. FSH assay was performed using the FSH standard, RP-2, provided by NIDDK as described previously (33). The sensitivity of the FSH RIA was 40 pg/tube, and the intraassay coefficient of variation was 7.2%. The testosterone concentration was determined by RIA after solvent extraction (34). The sensitivity of the assay was 15 pg/tube, and the intraassay coefficient of variation was 5.3%.

IGF-I assay
Plasma samples (25 µl) were incubated in acid medium (0.01 M HCl) for 30 min at room temperature to dissociate IGFs from IGFBPs, then ultrafiltered on Centricon 30 (Amicon, Epernon, France) to separate IGFs from IGFBPs. The ultrafiltrate containing IGFs was lyophilized, then taken up in 0.1 M phosphate buffer and 1 mg/ml BSA (pH 7.4) and incubated for 2–3 d in a final volume of 400 µl with a specific polyclonal anti-hIGF-I antibody (1:120,000 dilution) that cross-reacts with murine IGF-I (gift from F. Frankenne, Centre Hospitalo-Universitaire de Liege, Liege, Belgium) and [125I]hIGF-I (10,000 cpm/tube). Iodination was performed using the Iodogen method. Unknown samples were tested at three concentrations plus one blank (without antibody), each in duplicate, so as to confirm parallelism with the standard curve. After incubation, free and bound IGFs were separated using albumin-coated charcoal. The threshold sensitivity of the assay was 1–2 ng/ml plasma. Intraassay variation was close to 5%, and interassay variation was 10%.

Statistical analysis
All data are presented as the mean ± SEM. A t test, or in the case of heterogeneity of variance, Mann-Whitney U test was 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. Differences among mean daily sperm production values were analyzed by one-way ANOVA, with mean separation by the Student-Newman-Keuls test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-I levels in IGFBP-1 transgenic males
In a previous study by Gay et al. (22) no sex difference in the secretion of IGFBP-1 was reported. In the present study we measured IGF-I levels in serum of males and females. Surprisingly, whereas the circulating IGF-I levels were 4-fold lower in HM than NT females, no differences were observed between HM and NT males (Fig. 1Go).



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  		FIG. 1. Plasma IGF-I levels in hIGFBP-1 mice. Levels of IGF-I were measured in plasma from 4- to 6-month-old males (HM, n = 8; NT, n = 6) and females (HM, n = 4; NT, n = 7) by RIA as described in Materials and Methods. *, P < 0.05

 
Alteration of fertility in IGFBP-1 transgenic males
The weight of the testes in HM transgenic mice was reduced approximately 1.6-fold compared with that in NT adult mice. This decrease was proportional to the reduction of body weight. The weights of the epididymis and seminal vesicles were reduced 1.8-fold in HM compared with NT mice. This reduction was slightly disproportionate compared with the reduction of body weight (Table 1Go).


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  		TABLE 1. Weights of the body, testis, and genital tract in adult hIGFBP-1 transgenic males

 
Approximately 30% of HM male mice were sterile, exhibiting no aggressive behavior against other males, a complete absence of sexual behavior, and a complete failure to produce offspring after 3 months in the presence of control females.

The other adult HM males irregularly mated wild-type females, and litter size per pregnancy was reduced about 50%, confirming the previous study (22). After mating HM males with wild-type females, the number of embryos implanted on d 5.5 of pregnancy was severely reduced compared with NT mice [HM, 3.8 ± 0.9 (n = 20); NT, 7.9 ± 0.7 (n = 12); P < 0.001; Fig. 2AGo]. We have tested the hypothesis that this decrease was due to an alteration of fertilization of oocytes rather than a defect of implantation. After mating males with wild-type females, eggs were recovered on d 0.5 of pregnancy and submitted to microscopic examination. The percentage of eggs morphologically normal dividing into two-cell embryos was 50% lower when females were mated with HM compared with NT males [34.9 ± 13.7 embryos/ova (n = 14 females mated with five HM males); 79.0 ± 2.3 embryos/ova (n = 12 females mated with four HT males); P < 0.05; or vs. 83.0 ± 3.9 embryos/ova (n = 25 females mated with seven NT males); P < 0.01; Fig. 2BGo), suggesting a failure in the production and/or the quality of spermatozoa.



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  		FIG. 2. Number of sperm heads in testis from hIGFBP-1 mice and capacity of the sperm to fertilize wild-type oocytes. A, Adult wild-type females were mated with HM (n = 20), HT (n = 4), or NT (n = 12) transgenic males. The number of implantation sites was visualized by staining with Chicago blue on d 5.5 of pregnancy as described in Materials and Methods. B, Adult wild-type females were mated with HM (n = 5), HT (n = 4), or NT (n = 7) transgenic males. Ova were recovered on d 0.5 of pregnancy, cultured in vitro, and monitored for progression to two-cell stage embryos as described in Materials and Methods. C, Number of sperm heads present in the testis from HM (n = 3) or NT (n = 3) transgenic males. D, Determination of the daily sperm production per gram based on the number of round spermatids and their life span in NT and HM mice. *, P < 0.05; **, P < 0.01.

 
Alteration of production of spermatozoa in male IGFBP-1 mice
The number of heads of spermatids and spermatozoa in testes was 2-fold diminished in HM compared with NT mice [HM, 19.03 x 106 ± 6.15 (n = 3); NT, 40.38 x 106 ± 3.145 (n = 3); P < 0.05; Fig. 2CGo]. In addition, spermatozoa from HM mice tended to exhibit more head abnormalities than NT counterparts [HM, 32.04 ± 4.85% of abnormal sperm heads (n = 3); NT, 11.75 ± 1.0% (n = 3); P = 0.1; Table 2Go]. No abnormalities of flagella were observed (P > 0.3; Table 2Go). These results indicate that the number, but clearly not the quality, of spermatozoa was altered in HM mice.


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  		TABLE 2. Histological characteristics of testis of hIGFBP-1 transgenic mice

 
To assess the production of spermatozoa in the testis of HM mice, histological examination and stereological studies were performed. Histological examination of the epididymis and testis showed no clear alteration of morphology (Fig. 3Go, A and B). In particular, the diameters of the seminiferous tubules were similar in HM and NT mice (P > 0.9; Table 2Go and Fig. 3Go, A and B). The reduction of testis weight in HM mice was associated with a 28% reduction of the length of seminiferous tubules [HM, 2.07 ± 0.12 m (n = 3); NT, 2.87 ± 0.11 m (n = 3); P < 0.01]. Moreover, in three of the four HM mice analyzed, several seminiferous tubules were totally devoid of germ cells (Sertoli cell-only tubules; Fig. 3Go, C and D). The daily sperm production per gram of testis, based on the number of round spermatids, was also lower in HM mice than in NT mice [HM, 27,180 ± 2,842 x 106 number of cells (n = 4); NT, 37,012 ± 1,297 x 106 number of cells (n = 4); P < 0.05], suggesting an alteration before or during meiosis in transgenic mice (Fig. 2DGo). The decrease in tubule length was proportional to the general reduction in mouse size. However, based on the 1.6-fold decrease in testis weight and the 1.4-fold decrease in daily sperm production per gram of testis, the overall decrease in daily sperm production per HM animals was not proportional to the general reduction in mouse size. Finally, the mean nuclear volume of round spermatids was unaffected in HM mice (Fig. 4AGo), but elongated spermatids in stage IX (middle stage of spermiogenesis) presented clear morphological abnormalities of their heads (Fig. 4BGo).



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  		FIG. 3. Histology of the testis from 9-month-old hIGFBP-1 transgenic mice. Sections [10 µm (A and B) and 0.5 µm (C and D)] of testes from 9-month-old HM (B and D) and NT (A and C) hIGFBP-1 mice are shown. All stages of spermatogenesis were visible, and no clear alteration of tubule diameter was observed (A and B). On 0.5-µm sections (C and D), some tubules from homozygous mice were devoid of germ cells (D, see arrow). Scale bar, 100 µm.

 


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  		FIG. 4. Stereological study of hIGFBP-1 transgenic testis. A, Nuclear volume of spermatid nucleus in HM and NT mice. B, Brightfield photomicrographs of 0.5-µm testis sections stained with toluidine blue. Elongated spermatids from HM mice presented morphological abnormalities (see arrows). Scale bar, 10 µm.

 
Increase in the number of apoptotic spermatocytes in male IGFBP-1 mice
Terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling analysis of testis sections revealed that the number of seminiferous tubules containing apoptotic germ cells was about 2-fold higher in testes from HM than in those from NT mice [HM, 13.2 ± 2.8% (n = 4); NT, 4.9 ± 1.4% (n = 5); P < 0.05; Fig. 5Go, A and B]. Spermatocytes were the most predominant apoptotic cells, suggesting that apoptosis essentially occurred during meiosis. Moreover, approximately 3% of seminiferous tubules from HM mice exhibited cleaved caspase-3-immunoreactive spermatocytes, and this labeling was completely absent in seminiferous tubules from NT mice (Fig. 5Go, C–F). In addition, the percentage of seminiferous tubules containing p21-immunoreactive spermatocytes was 3-fold higher in HM than in NT mice [HM, 7.38 ± 0.94% (n = 6); NT, 2.13 ± 0.44% (n = 4); P < 0.01; Fig. 6Go, A–C]. Finally, the percentage of seminiferous tubules containing a double layer of PCNA-positive spermatogonia was similar in both genotypes [HM, 26.77 ± 2.52% (n = 4); NT/HT, 30.35 ± 2.22% (n = 5); P > 0.3; Fig. 6Go, D–G].



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  		FIG. 5. Apoptosis in testes from hIGFBP-1 transgenic mice. Detection of apoptotic DNA fragmentation in germ cells of HM mice (see arrows, A and B) and quantification of the number of seminiferous tubules containing apoptotic cells from five NT and four HM mice. Testicular sections immunostained for caspase-3-cleaved proteins in hIGFBP-1 NT (C) and HM (D and F) adult mice. Note that caspase-3-cleaved proteins are immunodetected in spermatocytes, in approximately two seminiferous tubules per 70 tubules/section (F). E, Negative control, section incubated with nonimmune serum. Scale bar, 100 µm

 


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  		FIG. 6. Proliferation in testes from hIGFBP-1 transgenic mice. Testicular sections immunostained for p21 (A and B) and PCNA (D–G) in seminiferous tubules of NT (A, D, and F) and HM (B, E, and G) adult mice. Cellular proliferation is restricted to a single or a double layer of premeiotic germ cells on the periphery of the seminiferous tubules (see arrows). Scale bar, 100 µm. C, Quantification of seminiferous tubules containing p21-immunoreactive cells.

 
Alteration of testosterone production in male IGFBP-1 mice
Plasma testosterone levels tended to be decreased (60%) in HM mice compared with those in NT mice [HM, 1.4 ± 0.2 ng/ml (n = 8); NT, 3.2 ± 0.9 ng/ml (n = 7); P = 0.1; Fig. 7Go]. Obviously, Leydig cells from HM mice presented an accumulation of lipid droplets (Fig. 8Go); their surface was 1.8-fold increased in HM testis vs. NT testis (P < 0.001).



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  		FIG. 7. Concentration of testosterone in serum of hIGFBP-1 transgenic mice. Testosterone assays were performed on 3- to 8-month-old males (HM, n = 8; HT, n = 9; NT, n = 7).

 


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  		FIG. 8. Surface occupied by lipid droplet in hIGFBP-1 transgenic mice. A, Brightfield photomicrographs of Epon sections of the interstitial compartment stained with toluidine blue. Lipid droplets appeared as white vesicles. Scale bar, 20 µm. B, Quantification of the surface occupied by lipid droplets.

 
At the ultrastructural level, Leydig cells from transgenic hIGFBP-1 mice exhibited a strong reduction of the area occupied by smooth endoplasmic reticulum (SER), which appeared with granular structure in HM mice (Fig. 9Go). A depletion of myelin-like structure around lipid droplets was observed in HM mice, with no clear alteration of the mitochondria (Fig. 9Go). Finally, the level of 3ßHSD was reduced about 3-fold in HM testis, as shown by immunoblot analysis (P < 0.01). Levels of P450scc enzyme in testis of HM mice were not altered (P > 0.5; Fig. 10Go). All of these observations suggested an alteration of lipid metabolism and steroidogenic activity.



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  		FIG. 9. Electron micrographs of Leydig cells from hIGFBP-1 transgenic mice. Note the reduction of the SER that appeared with granule-like structures in the Leydig cells from HM mice. Leydig cells from NT, but not HM, mice presented myelin-like structure associated with lipid droplets. No clear alteration of the mitochondria was observed in HM mice. Ld, Lipid droplet; M, mitochondria; M St, myelin-like structure; nu, nucleus; RER, rough endoplasmic reticulum.

 


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  		FIG. 10. Expression of P450scc and 3ßHSD in testes from hIGFBP-1 transgenic mice. A, Proteins extracts from three testes of NT and HM hIGFBP-1 transgenic mice were submitted to SDS-PAGE as described in Materials and Methods. The membrane was incubated with antibodies raised against P450scc and 3ßHSD. Each sample contained an equal level of protein, as confirmed by reprobing membrane with an anti-{alpha}-actin antibody. B, Quantification of the expression of P450scc and 3ßHSD in both genotypes.

 
Subtle alteration of the functionality of gonadotrope cells in male IGFBP-1 mice
We have investigated a possible alteration of pituitary functionality in hIGFBP-1 male transgenic mice. In basal conditions, FSH and LH plasma levels were at the limit of detection of our assay and seemed similar in HM and NT mice (data not shown). Four weeks after castration or 15 min after GnRH administration, plasma LH and FSH levels were similar in HM and NT mice (Fig. 11Go, A and B). Interestingly, the pituitary LH, but not FSH, concentration was more than 1.4-fold higher in HM than in NT mice [HM, 11.83 ± 0.8749 (n = 7); NT, 8.299 ± 0.7581 (n = 4); P < 0.05; Fig. 11Go, C and D]. In addition, in vitro, pituitary explants from HM mice exhibited 1.6- and 1.4-fold increases in basal LH and FSH secretion, respectively, compared with NT pituitaries (LH, P = 0.039; FSH, P < 0.05; n = 5 pituitaries of each genotype). However, LH and FSH secretion after GnRH stimulation of pituitary explants in vitro was not significantly different in the two genotypes (Fig. 12Go). Histological analysis showed no clear alteration in the number of LH- and FSH-immunoreactive cells per surface unit [HM, 10.17 ± 0.24 LH cells/19,000 µm2 (n = 3); NT, 10.82 ± 1.30 LH cells/19,000 µm2 (n = 3); P > 0.6; and HM, 7.04 ± 0.42 FSH cells/19,000 µm2 (n = 3); NT, 8.19 ± 0.52 FSH-cells/19,000 µm2 (n = 3); P > 0.16]. The size of LH-immunoreactive cells [HM, 222.7 ± 14.2 µm2 (n = 3); NT, 206.9 ± 5.1 µm2 (n = 3); P > 0.3] and that of FSH-immunoreactive cells [HM, 225.7 ± 22.8 µm2 (n = 3); NT, 221.1 ± 4.5 µm2 (n = 3); P > 0.8] were similar in the two genotypes.



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  		FIG. 11. Plasma and pituitary LH and FSH concentrations in hIGFBP-1 transgenic mice. Levels of LH (A) and FSH (B) were measured in plasma from 3- to 8-month-old males 15 min after GnRH stimulation (LH: HM, n = 8; NT, n = 7; FSH: HM, n = 4; NT, n = 6) and 2 wk after castration (LH: HM, n = 6; HT/NT, n = 3; FSH: HM, n = 4; HT/NT, n = 3). Concentrations of LH (C) and FSH (D) were measured in pituitaries from 4- to 10-month-old HM mice (LH, n = 7; FSH, n = 8), HT mice (LH and FSH, n = 20), and NT mice (LH and FSH, n = 4). *, P < 0.05.

 


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  		FIG. 12. LH and FSH secretion by hIGFBP-1 pituitary explants in culture. Concentrations of LH (A) and FSH (B) were measured in pituitary explant-conditioned medium. Pituitaries from 3- to 10-month-old transgenic (n = 5) or nontransgenic male mice (n = 5) were cultured for 2 h in the absence of GnRH, then treated for 2 h in the presence of 10 nM GnRH. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have shown that reproductive impairments of hIGFBP-1 transgenic males were associated with an alteration of spermatogenesis, leading to a diminution of the production and quality of spermatozoa as well as a probable alteration of testosterone secretion and pulsatile LH secretion.

Several models of dwarf mice, such as pigmy, Snell, and Ames dwarf mice (35, 36), as well as IGF-I-deficient (37) and GH receptor-deficient male mice (38, 39) exhibit small testis, alteration of testosterone secretion, and alteration of spermatogenesis. A diminution of sperm quality and production occurs in dwarf rats. Sperm concentration and motility in the cauda epididymis are also lowered in dwarf rats, and spermatozoa exhibit more abnormalities than control males (40). Moreover, IGF-I administration in dwarf rats in vivo increases sperm concentration and the number of morphologically normal spermatozoa (8). Overall, these data suggest that the GH-IGF axis plays a key role in male spermatogenesis in vivo.

However, the severity of fertility problems due to any alteration of the somatotropic axis is not strictly correlated with the intensity of the decrease in serum IGF-I levels. In particular, IGF-I-/- male mice are infertile, with marked defects of spermatogenesis, but mice for which the IGF-I gene has been specifically deleted in the liver, that exhibited a 80% decrease in IGF-I levels, did not show any alteration of male fertility (41). Moreover, GH receptor-/- male mice had undetectable IGF-I levels in plasma, with a 50% decrease in body weight and exhibited only mild defects of fertility. In particular, these mice presented a delay in puberty and a decrease in FSH levels, with no alteration of basal testosterone and LH serum levels (38, 39). In the present model, in contrast to females, we did not observe any difference in IGF-I concentrations in HM compared with NT male mice (data not shown). Thus, the alteration in homozygous male mouse fertility in this study is probably due to a decrease in IGF-I bioavailability caused by the increase in IGFBP-1 levels rather than to a decrease in IGF-I levels.

To our knowledge, this is the first model for which an alteration of expression/secretion of one IGFBP leads to a clear alteration of male fertility. In particular, targeted disruption or overexpression of IGFBP-2, -3, -4, -5, and -6 (for review, see Ref. 42), IGFBP-6 (43), or acid-labile subunit (44, 45) did not alter male fertility. Very recently, Leu et al. (46) also inactivated the IGFBP-1 gene and did not observe any effect on male reproduction. Finally, ubiquitous overexpression of IGFBP-1 leads to some reproductive abnormalities in females, but not males (47). Of note, the phenotype observed in our transgenic line is not due to an insertion effect: alteration of male (and female) fertility was observed in two other lines overexpressing IGFBP-1 under the same promoter (22) (data not shown).

The alteration of testis functionality in hIGFBP-1 mice could be due to an alteration of IGF action at the level of somatic cells in gonads. In particular, in vitro studies have shown that IGF-I is able to stimulate steroidogenesis as well as LH responsiveness of Leydig cells (5), probably by stimulating the expression of gonadotropin receptors and several steroidogenic enzymes (9, 48). Moreover, IGF-I antiserum was able to inhibit the LH-induced secretion of androgen by Leydig cells in vitro, alone or in coculture with Sertoli cells (49). In vivo, a 1-wk treatment with GH or IGF-I of premature rats provoked an increase in the responsiveness to gonadotropins (50). Responsiveness to exogenous gonadotropin was also shown to be reduced in GH receptor knockout mice (38, 39). Thus, the reduction of testosterone production, the decrease in 3ßHSD expression, the reduction of weight of the male genital tract, and the alteration of spermatogenesis in hIGFBP-1 mice could be explained by the impairment of IGF-I action in testis. Interestingly, deletion of the IGF-I gene in male mice leads to a reduction of the weight of the reproductive organs, a delay in maturation of Leydig cells, and a drastic reduction in testosterone concentration. These males also present a complete absence of mating behavior (37). In addition, the accumulation of lipid droplets, which could be a storage site for steroid precursor, and the reduction of SER reinforced the hypothesis of a Leydig cell failure. Interestingly, the association between an increase in Leydig cell lipid droplet content, a decrease in SER content, a decrease in steroidogenic enzyme activity (mainly 3ßHSD), and a lowering of testosterone production has been previously described in the spontaneously diabetic BB rat as well as in streptozotocin-diabetic rats models (51, 52, 53), both models that exhibit an increase in IGFBP-1 levels.

In the present work the reduced sperm production in transgenic animals could be partly explained by an increase in apoptosis rather than a decrease in proliferation. Indeed, no clear difference in the number of immunoreactive PCNA spermatogonia was observed between HM and NT mice. In contrast, an increase in apoptotic spermatocytes during meiosis, as assessed by terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling analysis and immunodetection of cleaved capase 3, was observed in HM compared with NT mice. Moreover, an increase in p21-immunoreactive spermatocytes was observed in HM vs. NT mice. Interestingly, the expression of p21 was previously shown to be increased in spermatocytes after x-irradiation, suggesting that this factor could be important for DNA repair mechanisms during the meiotic prophase in spermatocytes (54).

We have recently shown that transgenic IGFBP-1 mice also exhibit a 30% reduction of the lactotroph population (25), suggesting an alteration of prolactin (PRL) secretion that would amplify the spermatogenesis defects. Indeed, prolactin receptor was shown to be expressed in Leydig cells, Sertoli cells, and germ cells (55, 56). Despite the fact that PRL does not seem to be required in male fertility, PRL-/- male mice presented a reduction of pituitary LH release and a reduction of the weight of accessory reproductive glands (57). Moreover, PRL treatment was shown to partially restore the fertility of dwarf mice (for review, see Ref. 58).

In hIGFBP-1 transgenic mice, the Leydig cell hypofunction and the increases in LH levels in pituitary extracts and in basal LH levels in pituitary explant cultures suggested an alteration of endogenous GnRH and/or LH secretion, as previously observed in females (21). In vitro, a pituitary explant from an hIGFBP-1 male presented an increase in LH secretion under basal conditions compared with NT counterparts. One could hypothesize that this secretion would be due to a passive release of the excess LH secreted in vivo by pituitaries of HM males. Interestingly, an alteration of LH release was also observed in GH receptor knockout mice (38). These alterations in LH secretion could be due to an impairment of IGF-I action at the hypothalamo-pituitary level. In particular, in vitro, IGF-I is able to stimulate GnRH secretion (59) and to potentiate the GnRH-stimulating effect on LH secretion by pituitary cells (60). Moreover, 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 (1).

Finally, a similar alteration of fertility has been observed in streptozotocin-induced diabetic rats (61, 62), which also exhibit an increase in plasma IGFBP-1 levels and a decrease in plasma IGF-I levels (63, 64). In particular, diabetic male rats were shown to present a reduction in the number of pregnancies per mating, a decrease in litter size, and a reduction of the number of eggs implanted per pregnancy. Reductions of sperm concentration and plasma testosterone concentrations were also observed in diabetic rats (65). A diminution of GnRH pulse secretion associated with an increased LH pituitary content (66) as well as decreases in plasma GH and PRL concentrations were also observed in these rats (67, 68, 69), suggesting that overexpression of IGFBP-1 in vivo might lead to an overall alteration of functionality of the pituitary-testis axis.

In conclusion, we have shown that overexpression of hIGFBP-1 altered spermatogenesis by reducing sperm production, increasing germ cell apoptosis, and increasing abnormalities of the head of spermatozoa, leading to male infertility. The apparent minor reduction in testosterone secretion by Leydig cells would be due to the alteration of GH/IGF-I actions in the testis as well.


   Acknowledgments
 
We thank Claude Cahier, Michel Vigneau, Jean-Claude Braguer, and Eric Jean-Pierre for expert animal care, and Martine Plat for excellent technical assistance. We thank Bernard Jegou for technical assistance and helpful discussion. We acknowledge Christophe Gauthier for the testosterone assay, Dr. Valerie Chabot for pituitary explant culture, and Rebecca S. Heck and Vince B. Hardy for technical assistance with the stereology. We are grateful to Dr. A. F. Parlow for the FSH and LH reagents. We thank Drs. Dale Buchanan Hales and Van Luu-The for generously providing the CYP11A and 3ß-HSD antibodies. We are grateful to Alain Beguey for the photographic work.


   Footnotes
 
This work was supported by the Fonds d’Aide à la Recherche Organon, Institut National de la Recherche Agronomique, and Institut National de la Santé et de la Recherche Médicale. P.F. was supported by a fellowship from Institut National de la Recherche Agronomique and Région Centre.

Abbreviations: h, Human; HM, homozygous; 3ßHSD, 3ß-hydroxysteroid dehydrogenase; HT, heterozygous; IGFBP, IGF-binding protein; NFDMP, nonfat dry milk powder; NT, nontransgenic; P450scc, cytochrome P450 side-chain cleavage enzyme; PCNA, proliferating cell nuclear antigen; PRL, prolactin; SER, smooth endoplasmic reticulum; TBS, Tris-buffered saline.

Received July 29, 2003.

Accepted for publication December 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hiney JK, Srivastava V, Nyberg CL, Ojeda SR, Dees WL 1996 Insulin-like growth factor I of peripheral origin acts centrally to accelerate the initiation of female puberty. Endocrinology 137:3717–3728[Abstract]
  2. Spiteri-Grech J, Nieschlag E 1992 The role of growth hormone and insulin-like growth factor I in the regulation of male reproductive function. Horm Res 38(Suppl 1):22–27
  3. Zhou J, Bondy C 1993 Anatomy of the insulin-like growth factor system in the human testis. Fertil Steril 60:897–904[Medline]
  4. Hansson HA, Billig H, Isgaard J 1989 Insulin-like growth factor I in the developing and mature rat testis: immunohistochemical aspects. Biol Reprod 40:1321–1328[Abstract]
  5. Lin T, Haskell J, Vinson N, Terracio L 1986 Characterization of insulin and insulin-like growth factor I receptors of purified Leydig cells and their role in steroidogenesis in primary culture: a comparative study. Endocrinology 119:1641–1647[Abstract/Free Full Text]
  6. Tres LL, Smith EP, Van Wyk JJ, Kierszenbaum AL 1986 Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures. Exp Cell Res 162:33–50[CrossRef][Medline]
  7. Vannelli BG, Barni T, Orlando C, Natali A, Serio M, Balboni GC 1988 Insulin-like growth factor-I (IGF-I) and IGF-I receptor in human testis: an immunohistochemical study. Fertil Steril 49:666–669[Medline]
  8. Vickers MH, Casey PJ, Champion ZJ, Gravance CG, Breier BH 1999 IGF-I treatment increases motility and improves morphology of immature spermatozoa in the GH-deficient dwarf (dw/dw) rat. Growth Horm IGF Res 9:236–240[CrossRef][Medline]
  9. Chuzel F, Clark AM, Avallet O, Saez JM 1996 Transcriptional regulation of the lutropin/human choriogonadotropin receptor and three enzymes of steroidogenesis by growth factors in cultured pig Leydig cells. Eur J Biochem 239:8–16[Medline]
  10. Bernier M, Chatelain P, Mather JP, Saez JM 1986 Regulation of gonadotropin receptors, gonadotropin responsiveness, and cell multiplication by somatomedin-C and insulin in cultured pig Leydig cells. J Cell Physiol 129:257–263[CrossRef][Medline]
  11. Perrard-Sapori MH, Chatelain PG, Jaillard C, Saez JM 1987 Characterization and regulation of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) receptors on cultured pig Leydig cells. Effects of Sm-C/IGF-I on luteotropin receptors and steroidogenesis. Eur J Biochem 165:209–214[Medline]
  12. Closset J, Gothot A, Sente B, Scippo ML, Igout A, Vandenbroeck M, Dombrowicz D, Hennen G 1989 Pituitary hormones dependent expression of insulin-like growth factors I and II in the immature hypophysectomized rat testis. Mol Endocrinol 3:1125–1131[Abstract/Free Full Text]
  13. Cailleau J, Vermeire S, Verhoeven G 1990 Independent control of the production of insulin-like growth factor I and its binding protein by cultured testicular cells. Mol Cell Endocrinol 69:79–89[CrossRef][Medline]
  14. Naville D, Chatelain PG, Avallet O, Saez JM 1990 Control of production of insulin-like growth factor I by pig Leydig and Sertoli cells cultured alone or together. Cell-cell interactions. Mol Cell Endocrinol 70:217–224[CrossRef][Medline]
  15. Lee PD, Giudice LC, Conover CA, Powell DR 1997 Insulin-like growth factor binding protein-1: recent findings and new directions. Proc Soc Exp Biol Med 216:319–357[CrossRef][Medline]
  16. Takenaka A, Komori K, Morishita T, Takahashi SI, Hidaka T, Noguchi T 2000 Amino acid regulation of gene transcription of rat insulin-like growth factor-binding protein-1. J Endocrinol 164:R11–R16
  17. Ritvos O, Ranta T, Jalkanen J, Suikkari AM, Voutilainen R, Bohn H, Rutanen EM 1988 Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells. Endocrinology 122:2150–2157[Abstract/Free Full Text]
  18. Angervo M, Koistinen R, Suikkari AM, Seppala M 1991 Insulin-like growth factor binding protein-1 inhibits the DNA amplification induced by insulin-like growth factor I in human granulosa-luteal cells. Hum Reprod 6:770–773[Abstract/Free Full Text]
  19. Cox GN, McDermott MJ, Merkel E, Stroh CA, Ko SC, Squires CH, Gleason TM, Russell D 1994 Recombinant human insulin-like growth factor (IGF)-binding protein-1 inhibits somatic growth stimulated by IGF-I and growth hormone in hypophysectomized rats. Endocrinology 135:1913–1920[Abstract]
  20. VanBuul-Offers SC, VanKleffens M, Koster JG, Lindenbergh-Kortleve DJ, Gresnigt MG, Drop SL, Hoogerbrugge CM, Bloemen RJ, Koedam JA, Van Neck JW 2000 Human insulin-like growth factor (IGF) binding protein-1 inhibits IGF-I-stimulated body growth but stimulates growth of the kidney in snell dwarf mice. Endocrinology 141:1493–1499[Abstract/Free Full Text]
  21. Froment P, Seurin D, Hembert S, Levine JE, Pisselet C, Monniaux D, Binoux M, Monget P 2002 Reproductive abnormalities in human IGF binding protein-1 transgenic female mice. Endocrinology 143:1801–1808[Abstract/Free Full Text]
  22. Gay E, Seurin D, Babajko S, Doublier S, Cazillis M, Binoux M 1997 Liver-specific expression of human insulin-like growth factor binding protein-1 in transgenic mice: repercussions on reproduction, ante- and perinatal mortality and postnatal growth. Endocrinology 138:2937–2947[Abstract/Free Full Text]
  23. Paria BC, Huet-Hudson YM, Dey SK 1993 Blastocyst’s state of activity determines the "window" of implantation in the receptive mouse uterus. Proc Natl Acad Sci USA 90:10159–10162[Abstract/Free Full Text]
  24. Jegou B, Velez de la CJ, Bauche F 1991 Protective effect of medroxyprogesterone acetate plus testosterone against radiation-induced damage to the reproductive function of male rats and their offspring. Proc Natl Acad Sci USA 88:8710–8714[Abstract/Free Full Text]
  25. Courot M 1967 Endocrine control of the supporting and germ cells of the impubertal testis. J Reprod Fertil 2(Suppl):89–101
  26. Johnson L 1985 Increased daily sperm production in the breeding season of stallions is explained by an elevated population of spermatogonia. Biol Reprod 32:1181–1190[Abstract]
  27. Chalkley HW 1943 Method for the quantitative morphologic analysis of tissues. J Natl Cancer Inst 4:47–53
  28. Johnson L, Neaves WB 1981 Age-related changes in the Leydig cell population, seminiferous tubules, and sperm production in stallions. Biol Reprod 24:703–712[Abstract]
  29. Weibel ER, Paumgartner D 1978 Integrated stereological and biochemical studies on hepatocytic membranes. II. Correction of section thickness effect on volume and surface density estimates. J Cell Biol 77:584–597[Abstract/Free Full Text]
  30. Johnson L, May MR, Busbee DL, Williams JD 1992 Effect of age and dietary restriction on daily sperm production and number and transit time of epididymal spermatozoa in the mouse. Age 15:65–71
  31. Oakberg EF 1956 Duration of spermatogenesis in the mouse and timing of stages of the cycle of seminiferous epithelium. Am J Anat 99:507–516[CrossRef][Medline]
  32. Watanabe YG, Matsumura H, Daikoku S 1973 Electron microscopic study of rat pituitary primordium in organ culture. Z Zellforsch Mikrosk Anat 146:453–461[CrossRef][Medline]
  33. Chappell PE, Schneider JS, Kim P, Xu M, Lydon JP, O’Malley BW, Levine JE 1999 Absence of gonadotropin surges and gonadotropin-releasing hormone self-priming in ovariectomized (OVX), estrogen (E2)-treated, progesterone receptor knockout (PRKO) mice. Endocrinology 140:3653–3658[Abstract/Free Full Text]
  34. Hochereau-de Reviers MT, Copin M, Seck M, Monet-Kuntz C, Cornu C, Fontaine I, Perreau C, Elsen JM, Boomarov 1990 Stimulation of testosterone production by PMSG injection in the ovine male: effect of breed and age and application to males carrying or not carrying the ‘F’ Booroola gene. Anim Reprod Sci 23:21–32[CrossRef]
  35. Chubb C, Nolan C 1985 Animal models of male infertility: mice bearing single-gene mutations that induce infertility. Endocrinology 117:338–346[Abstract/Free Full Text]
  36. Chubb C 1989 Genetically defined mouse models of male infertility. J Androl 10:77–88[Abstract/Free Full Text]
  37. Baker J, Hardy MP, Zhou J, Bondy C, Lupu F, Bellve AR, Efstratiadis A 1996 Effects of an Igf1 gene null mutation on mouse reproduction. Mol Endocrinol 10:903–918[Abstract/Free Full Text]
  38. Chandrashekar V, Bartke A, Coschigano KT, Kopchick JJ 1999 Pituitary and testicular function in growth hormone receptor gene knockout mice. Endocrinology 140:1082–1088[Abstract/Free Full Text]
  39. Chandrashekar V, Bartke A, Awoniyi CA, Tsai-Morris CH, Dufau ML, Russell LD, Kopchick JJ 2001 Testicular endocrine function in GH receptor gene disrupted mice. Endocrinology 142:3443–3450[Abstract/Free Full Text]
  40. Gravance CG, Breier BH, Vickers MH, Casey PJ 1997 Impaired sperm characteristics in postpubertal growth-hormone-deficient dwarf (dw/dw) rats. Anim Reprod Sci 49:71–76[CrossRef][Medline]
  41. Yakar S, Liu JL, Stannard B, Butler A, Accili D, Sauer B, LeRoith D 1999 Normal growth and development in the absence of hepatic insulin-like growth factor I. Proc Natl Acad Sci USA 96:7324–7329[Abstract/Free Full Text]
  42. Schneider MR, Lahm H, Wu M, Hoeflich A, Wolf E 2000 Transgenic mouse models for studying the functions of insulin-like growth factor-binding proteins. FASEB J 14:629–640[Abstract/Free Full Text]
  43. Bradshaw SL, Schuller AGP, Grewal A, Pintar JE, Analysis of reproductive capability in IGFBP-6 deficient mice. Program of the 81st Annual Meeting of The Endocrine Society, San Diego, CA, 1999, p 406 (Abstract P2-594)
  44. Ueki I, Ooi GT, Tremblay ML, Hurst KR, Bach LA, Boisclair YR 2000 Inactivation of the acid labile subunit gene in mice results in mild retardation of postnatal growth despite profound disruptions in the circulating insulin-like growth factor system. Proc Natl Acad Sci USA 97:6868–6873[Abstract/Free Full Text]
  45. Silha JV, Gui Y, Modric T, Suwanichkul A, Durham SK, Powell DR, Murphy LJ 2001 Overexpression of the acid-labile subunit of the IGF ternary complex in transgenic mice. Endocrinology 142:4305–4313[Abstract/Free Full Text]
  46. Leu JI, Crissey MA, Craig LE, Taub R 2003 Impaired hepatocyte DNA synthetic response posthepatectomy in insulin-like growth factor binding protein 1-deficient mice with defects in C/EBP beta and mitogen-activated protein kinase/extracellular signal-regulated kinase regulation. Mol Cell Biol 23:1251–1259[Abstract/Free Full Text]
  47. Huang H, Rajkumar K, Murphy LJ 1997 Reduced fecundity in insulin-like growth factor-binding protein-1 transgenic mice. Biol Reprod 56:284–289[Abstract]
  48. Gelber SJ, Hardy MP, Mendis-Handagama SM, Casella SJ 1992 Effects of insulin-like growth factor-I on androgen production by highly purified pubertal and adult rat Leydig cells. J Androl 13:125–130[Abstract/Free Full Text]
  49. Verhoeven G, Cailleau J 1990 Influence of coculture with Sertoli cells on steroidogenesis in immature rat Leydig cells. Mol Cell Endocrinol 71:239–251[CrossRef][Medline]
  50. Ohyama K, Ohta M, Nakagomi Y, Yamori T, Sano T, Shimura Y, Sato K, Nakazawa S 1995 Effects of growth hormone and insulin-like growth factor I on testosterone secretion in premature male rats. Endocr J 42:817–820[Medline]
  51. Anderson JE, Thliveris JA 1987 Morphometry and cytochemistry of Leydig cells in experimental diabetes. Am J Anat 180:41–48[CrossRef][Medline]
  52. Murray FT, Cameron DF, Orth JM, Katovich MJ 1985 Gonadal dysfunction in the spontaneously diabetic BB rat: alterations of testes morphology, serum testosterone and LH. Horm Metab Res 17:495–501[Medline]
  53. Orth JM, Murray FT, Bardin CW 1979 Ultrastructural changes in Leydig cells of streptozotocin-induced diabetic rats. Anat Rec 195:415–430[CrossRef][Medline]
  54. Beumer TL, Roepers-Gajadien HL, Gademan LS, Rutgers DH, de Rooij DG 1997 P21(Cip1/WAF1) expression in the mouse testis before and after X irradiation. Mol Reprod Dev 47:240–247[CrossRef][Medline]
  55. Hondo E, Kurohmaru M, Sakai S, Ogawa K, Hayashi Y 1995 Prolactin receptor expression in rat spermatogenic cells. Biol Reprod 52:1284–1290[Abstract]
  56. Guillaumot P, Tabone E, Benahmed M 1996 Sertoli cells as potential targets of prolactin action in the testis. Mol Cell Endocrinol 122:199–206[CrossRef][Medline]
  57. Steger RW, Chandrashekar V, Zhao W, Bartke A, Horseman ND 1998 Neuroendocrine and reproductive functions in male mice with targeted disruption of the prolactin gene. Endocrinology 139:3691–3695[Abstract/Free Full Text]
  58. Bartke A 1999 Role of growth hormone and prolactin in the control of reproduction: what are we learning from transgenic and knock-out animals? Steroids 64:598–604[CrossRef][Medline]
  59. Anderson RA, Zwain IH, Arroyo A, Mellon PL, Yen SS 1999 The insulin-like growth factor system in the GT1–7 GnRH neuronal cell line. Neuroendocrinology 70:353–359[CrossRef][Medline]
  60. Soldani R, Cagnacci A, Paoletti AM, Yen SS, Melis GB 1995 Modulation of anterior pituitary luteinizing hormone response to gonadotropin-releasing hormone by insulin-like growth factor I in vitro. Fertil Steril 64:634–637[Medline]
  61. Perez Diaz J, Benitez A, Fernandez Galaz C 1982 Effect of streptozotocin diabetes on the pituitary-testicular axis in the rat. Horm Metab Res 14:479–482[Medline]
  62. Sudha S, Valli G, Julie PM, Arunakaran J, Govindarajulu P, Balasubramanian K 2000 Influence of streptozotocin-induced diabetes and insulin treatment on the pituitary-testicular axis during sexual maturation in rats. Exp Clin Endocrinol Diabetes 108:14–20[Medline]
  63. Pao CI, Farmer PK, Begovic S, Goldstein S, Wu GJ, Phillips LS 1992 Expression of hepatic insulin-like growth factor-I and insulin-like growth factor-binding protein-1 genes is transcriptionally regulated in streptozotocin-diabetic rats. Mol Endocrinol 6:969–977[Abstract/Free Full Text]
  64. Ooi GT, Tseng LY, Tran MQ, Rechler MM 1992 Insulin rapidly decreases insulin-like growth factor-binding protein-1 gene transcription in streptozotocin-diabetic rats. Mol Endocrinol 6:2219–2228[Abstract/Free Full Text]
  65. Cameron DF, Rountree J, Schultz RE, Repetta D, Murray FT 1990 Sustained hyperglycemia results in testicular dysfunction and reduced fertility potential in BBWOR diabetic rats. Am J Physiol 259:E881–E889
  66. Bestetti GE, Boujon CE, Reymond MJ, Rossi GL 1989 Functional and morphological changes in mediobasal hypothalamus of streptozocin-induced diabetic rats. In vitro study of LHRH release. Diabetes 38:471–476[Abstract]
  67. Bestetti G, Locatelli V, Tirone F, Rossi GL, Muller EE 1985 One month of streptozotocin-diabetes induces different neuroendocrine and morphological alterations in the hypothalamo-pituitary axis of male and female rats. Endocrinology 117:208–216[Abstract/Free Full Text]
  68. Dong Q, Lazarus RM, Wong LS, Vellios M, Handelsman DJ 1991 Pulsatile LH secretion in streptozotocin-induced diabetes in the rat. J Endocrinol 131:49–55[Abstract/Free Full Text]
  69. Boujon CE, Bestetti GE, Abramo F, Locatelli V, Rossi GL 1995 The reduction of circulating growth hormone and prolactin in streptozotocin-induced diabetic male rats is possibly caused by hypothalamic rather than pituitary changes. J Endocrinol 145:19–26[Abstract/Free Full Text]



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