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BOR - Papers in Press, published online ahead of print October 29, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.022590
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BIOLOGY OF REPRODUCTION 70, 632–639 (2004)
DOI: 10.1095/biolreprod.103.022590
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Development of Leydig Cells in the Insulin-Like Growth Factor-I (IGF-I) Knockout Mouse: Effects of IGF-I Replacement and Gonadotropic Stimulation1

Guimin Wang, and Matthew P. Hardy2

Population Council and The Rockefeller University, New York, New York 10021


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Targeted gene deletion of insulin-like growth factor-I (IGF-I) results in diminished numbers of Leydig cells (LCs) and lower circulating testosterone (T) levels in adult males. The impact of endogenous IGF-I withdrawal on proliferation (labeling index, LI) and differentiation of LCs was investigated, testing for restorative effects of IGF-I replacement and/or LH stimulation. With IGF-I replacement in mutant mice, LIs increased more than 200% (P < 0.05). LC numbers were also increased by 200%, whereas the numbers of intermediate cell progenitors (PLCs) were unchanged compared to mutant vehicle controls. LIs of PLCs in wild-type males increased by 200% after LH stimulation, and LC numbers increased by 50% compared to vehicle-treated controls (P < 0.05). In contrast, there was no effect of LH on LI in mutant mice, but LC numbers still increased by 30% (P < 0.05). Additive effects on LI and cell numbers were observed in response to IGF-I plus LH in mutants, implying that the two hormones use separate signaling pathways. Serum T and LH levels in wild-type and mutant males were equivalent. Exogenous LH increased T production 8-fold in wild-type males (P < 0.01). In mutant mice, neither LH stimulation nor IGF-I alone affected serum T levels, but IGF-I plus LH stimulation increased serum T 2-fold (P < 0.05). These data support the conclusions that 1) IGF-I is a critical autocrine and/or paracrine factor in the control of adult LC numbers and function; and 2) LH is not a direct mitogenic factor for LCs, and acts in part through IGF-I to stimulate proliferative activity.

insulin-like growth factor receptor, Leydig cells, luteinizing hormone, puberty, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proliferation and differentiation of adult Leydig cells (ALCs) is a prerequisite for the onset of pubertal increases in circulating androgen levels. The ALC population is formed postnatally in three successive steps in rodents. First, there is a transformation of undifferentiated mesenchymal precursor cells (MPCs) into progenitor Leydig cells (PLCs) in the peritubular region of the interstitium starting on Postnatal Days 7 to 10. The fibroblast-like PLCs are overtly recognizable members of the Leydig cell (LC) lineage because they express steroidogenic enzymes such as 3ß-hydroxysteroid dehydrogenase (3ßHSD) and synthesize androgens in vitro. Second, there is a marked increase in LC number and change in the shape of the PLC, from spindle-like to round, and dispersion of the cells to the more central interstitial regions as PLCs develop into immature LCs (ILCs) between Days 10 and 20. Third, the ILCs mature into ALCs accompanied by further increases in cell numbers, cell size, and steroidogenic enzyme gene expression [1–3]. The ALC population is derived from differentiation of mesenchymal precursors and from the proliferation of the newly formed LCs [1, 3]. The ultimate number of LCs achieved at sexual maturity is a product of the rates of cell division and differentiation in their pool of precursor cells.

A complete accounting of factors regulating postnatal proliferation and differentiation of the ALC remains unavailable. LH appears not to be required for the onset of MPC differentiation into PLCs [4–6]. ALCs fail to develop in the absence of platelet-derived growth factor-A (PDGF-A) and desert hedgehog (Dhh), a nuclear transcription factor that stimulates differentiation of MPCs by up-regulating steroidogenic factor-1 (SF-1) [7, 8]. This indicates that PDGF-A and Dhh are involved in restricting pluripotent stem cells to the LC lineage. In this model, the action of LH occurs later, stimulating development of ALCs during prepubertal life. That LH plays a critical role in the development of LCs is apparent from studies of GnRHhpg mice, which are deficient in circulating LH. In these mice, LC numbers are about 10% of control [2]. Moreover, LCs are severely hypoplastic in LH receptor (LHR) knockout mice [9]. Increase of LC proliferative activity occurs following LH/hCG administration in vivo [10–12], but events subsequent to LHR binding leading to increased cell division have not been identified. In adult Snell dwarf mice, a deficiency in plasma gonadotropin prevents full differentiation of LCs without affecting their numbers [13]. In adult rats, neither long-term suppression of LH nor the return of LH to control values has a significant effect on LC numbers [14, 15]. In addition, although LH stimulates DNA synthesis in immature rat LCs in vitro, these increases are limited, and significant enhancement of the LH effect is achieved by coadministration of growth factors such as insulin-like growth factor-I (IGF-I) [16, 17]. These data raised the possibility that the action of LH on LC proliferation requires the participation of other factors.

The testis is a site of IGF-I biosynthesis and action. IGF-I mRNA, protein, and specific IGF-I receptors are present in the testis and have been identified in LCs, peritubular cells and spermatocytes [18–23]. Testicular levels of IGF-I peak during the fourth week postpartum, at the start of the pubertal rise in testosterone secretion [24, 25]. LH and hCG stimulate IGF-I secretion and upregulate type I IGF-I receptor gene expression in rodent LCs [25–28]. IGF-I stimulates the proliferation of LC precursors and pretreatment of these cells with LH augments the mitogenic effect [16, 17, 29].

We have shown previously that IGF-I null mutant mice have fewer and smaller LCs than normal, and lower serum T levels in adulthood [30]. Therefore, we hypothesized that the action of LH on LC proliferation requires IGF-I. Herein we assess: 1) the consequences of postnatal withdrawal of endogenous IGF-I for proliferation and differentiation of the LC lineage; and 2) the effects of IGF-I replacement and LH stimulation on restoration of the LC developmental process in immature IGF-I null mice. Bromodeoxyuridine (BrdU) labeling index and cell numbers were measured to address whether one or more of the intermediate stages of LCs were susceptible to the absence of IGF-I, and whether changes observed after IGF-I and LH treatment were the result of cell division, differentiation, or both. The results provide the first evidence that proliferation of LC precursors is not modulated directly by LH, but rather through induced secretion of IGF-I.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

IGF-I null mice were produced as described previously [31]. Adult heterozygous mice with a predominantly MF1 x 129/Sv background bearing the targeted IGF-I gene deletion (provided by Dr. Argiris Efstratiadis, Columbia University, New York, NY) were mated, and the genotypes of the resulting male IGF-I null mice and wild-type mice were ascertained by multiplex polymerase chain reaction (PCR) of DNA samples obtained from tail tips [31]. Mice were observed twice daily (morning and evening) for litters. The day of birth was considered as Day 1 of age. All animal procedures were approved by the Institutional Animal Care and Use Committee of Rockefeller University (Protocol 91200-R2).

Treatments

Twice daily (0700 h, 1900 h) s.c. injection of hormone or vehicle started from Postnatal Day 33 for 2 days. Mice were assigned to six groups (n = 5/group): 1) wild-type, vehicle control; 2) mutant, vehicle control; 3) wild-type + LH (oLH, NIH-26, Bethesda, MD; 0.5 µg/g body weight/day); 4) mutant + LH; 5) mutant + IGF-I (human recombinant Cat. No. 4101-80, Intergen Company, Purchase, NY; 0.8 µg/g body weight/day); and 6) mutant + IGF-I and LH. Previous studies have shown that these doses of LH and IGF-I effectively stimulate LC function [32, 33]. At 1 and 2 h before they were killed, all animals received an i.p. injection of BrdU (40 µg/g body weight, Cat. No. 280879; Boehringer Mannheim, GmbH, Mannheim, Germany) to label dividing cells.

Histological Procedures

The animals were anesthetized by i.p. injection of sodium pentobarbital (25 mg/100 g body weight; Abbott Laboratories, North Chicago, IL). Under deep anesthesia, the wild-type mice were fixed by whole-body perfusion through the left ventricle of the heart with Bouin solution [34]. The testes were then removed and stored in the fixative overnight. Testes from mutant mice were small enough to permit overnight immersion fixation. After dehydration, ethanol, and xylene, the testes were embedded in paraffin for immunocytochemical analysis.

LCs were identified through immunopositive staining for the marker enzyme 3ß-HSD. In brief, 6-µm-thick transverse sections were prepared and mounted on glass slides (Cat. No. 12-550-15; Fisher Scientific Company, Hampton, NH). Avidin-biotin immunostaining was performed using a kit (Cat. Nos. PK-2200 for BrdU and PK-6101 for 3ß-HSD; Vector Laboratories Inc., Burlingame, CA) according to the manufacturer's instructions. Antigen retrieval was carried out by microwave irradiation for 10 min in 10 mM (pH 6.0) citrate buffer, and endogenous peroxidase was blocked with 0.5% H2O2 in methanol for 30 min. The sections were then incubated with a monoclonal anti-BrdU antibody (RPN 202; Amersham Biosciences, Little Chalfont, UK) for 30 min at room temperature. The antibody bound to the nuclei was visualized with diaminobenzidine (Cat. No. sk-4100; Vector Laboratories Inc.) and the labeled nuclei were stained black by adding a nickel solution to the chromogen. After washing, the sections were double-labeled by incubation with a 3ß-HSD polyclonal antibody diluted 1:3000 (provided by Dr. Van Luu-The, Laval University, Quebec, Canada) for 1 h at room temperature. The antibody-antigen complexes were visualized with diaminobenzidine alone, resulting in brown cytoplasmic staining in positively labeled LCs. The sections were counterstained with Mayer hematoxylin, dehydrated in graded concentrations of alcohol, and cover-slipped with resin (Permount, SP15-100; Fisher Scientific). In control experiments, sections were incubated with nonimmune rabbit IgG (3ß-HSD) or mouse IgG (BrdU) using the same working dilution as the primary antibody.

Cell Counts and Computer Assisted Image Analysis

Twenty randomly selected fields in each of three nonadjacent sections per testis were captured using a Nikon Eclipse E800 microscope (Nikon, Inc., Melville, NY) equipped with a 40 x objective and a SPOT RT digital camera (model 2.3.0.; Diagnostic Instruments Inc. Michigan City, IN) interfaced to a computer. The images that were displayed on the monitor represented areas of 0.9 mm2 of testis. Interstitial cell numbers were estimated using image analysis software (Image-Pro Plus; Media Cybernetics, Silver Spring, MD). Identification of the different interstitial cell types (peritubular myoid cell, MPC, PLC, LC, and vascular or lymphatic endothelial cell) was based on their nuclear morphologies, locations, and cytoplasmic staining characteristics as described previously [1]. The BrdU labeling index for each cell type was determined by the number of BrdU-labeled cells divided by the total number of labeled plus unlabeled cells, multiplied by 100. More than 1000 cells of each type were counted in each testis. The numerical index of each of the enumerated cell types was defined as the average number counted per mm2 of cross-sectional area. The BrdU numerical index was the average number of BrdU-labeled cells per mm2 of cross-sectional area, measured as described for the cell counts.

LH and T Radioimmunoassay

Blood was collected intraorbitally and sera were stored at -80°C until RIA of T and LH. Three to five samples for each group were pooled from two or three mutant mice. Serum T concentrations were measured with a tritium-based radioimmunoassay (T-RIA) as previously described [35]. Interassay variation of the T-RIA was between 7 and 8%. Serum LH concentrations were measured using the method from Chandrashekar et al. [36]. LH standards and antibody were obtained through the National Hormone and Pituitary Program. Radioactive 125I-rat LH was obtained through Covance Laboratories (Vienna, VA), and IgG antiserum was obtained from ICN Pharmaceuticals (Costa Mesa, CA). The sensitivity and inter- and intra-assay coefficients of variation for the LH RIA were, respectively, 0.08 ng per ml and 5% and 6%. The overall experimental design was performed twice to ensure that the data were repeatable.

Statistical Analysis

Data are expressed as mean ± SEM. Significant differences between groups were identified using the General Linear Models of Statistical Analysis Systems (SAS Institute, Cary, NC). When a significant difference was observed among the groups, a Duncan multiple range test was employed to separate the means. Differences were regarded as significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Histology

The cell types in the testicular interstitium, as well as their proliferative status, were readily distinguished by the double labeling procedure, as shown in Figure 1. Peritubular myoid cells had elongated nuclei and were directly opposed to the basal lamina of the seminiferous tubules. Endothelial cells and pericytes were identifiable by their location in and adjacent to blood vessels. MPCs were located peritubularly and had oblong-shaped nuclei without 3ß-HSD cytoplasmic staining. PLCs were similar to MPCs but had 3ß-HSD positive cytoplasmic staining. LCs (including ILCs and ALCs) had spherical nuclei and 3ß-HSD positive staining. Dividing cells as marked by BrdU incorporation had black nuclei. Of the five distinguishable interstitial cell types, only those in the LC lineage (MPCs, PLCs and LCs) had significant responses in LI or cell numbers to IGF-I gene deletion and IGF-I replacement.



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  		FIG. 1. Testes from 35-day IGF-I null mice after double immunostaining for BrdU (black nuclei) and 3ß-HSD (brown cytoplasm). Representative mesenchymal precursor cells (MPCs), progenitor Leydig cells (PLCs), and Leydig cells (LC) are labeled with (+) or (-) to indicate the presence or absence of BrdU labeling. Bar = 20 µm

Effects of Endogenous IGF-I Deprivation and Replacement on Proliferation and Differentiation in the LC Lineage

The BrdU labeling index, BrdU numerical index, and numerical index for each interstitial cell type in wild-type control, IGF-I null mutant, and IGF-I null with IGF-I replacement are presented in Figure 2. The LIs of each interstitial cell type in mutant and wild-type mice were equivalent. However, in mutant testes, the BrdU numerical index was higher in MPCs, lower in LCs (P < 0.05), and unchanged in PLCs compared to wild-type. This implied either that the rate of proliferation of MPCs was increased or that an increased number of MPCs, with an unchanged rate of proliferation, were in the cell cycle. After two days of treatment with IGF-I, the LI in mutant mice increased more than 200% in PLCs and LCs compared to mutant vehicle controls (P < 0.05). Although the average LI of MPCs in treated mutant mice did not increase relative to mutant controls, it was significantly higher compared to wild-type levels (P < 0.05). The BrdU numerical index of all cell types was significantly increased after IGF-I replacement. This suggests that IGF-I is a mitogenic factor for LC precursors.



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  		FIG. 2. BrdU labeling index (A), BrdU numerical index (B), and Leydig cell numerical index (C) in the LC lineage on Day 35 in wild-type (black bars), IGF-I null mutant, null mutant after IGF-I replacement, and null mutant with LH stimulation (open and pattern-filled bars). Data are presented as mean ± SEM for five to seven animals per group. Different letters above bars indicate a significant difference in labeling index or cell number between the different treatment groups within a cell stage (P < 0.05)

The numbers of MPCs and PLCs did not differ between mutant and wild-type mice (Fig. 2B), whereas LC numbers per mm2 in mutant mice were only 25% of the wild-type value (P < 0.05). The relationship of LI and numerical index indicated that the rate of differentiation of MPCs and PLCs into LCs was slowed in the absence of IGF-I. Although IGF-I replacement increased the proliferative capacities in the LC lineage, only more mature LCs (designated LC), not MPCs or PLCs, were increased in number to over 200% compared to mutant vehicle control (P < 0.05). This indicated that IGF-I replacement in mutant mice accelerated the proliferation and differentiation of MPCs and PLCs, contributing to increased numbers of ALCs.

Effects of LH or IGF-I Plus LH Stimulation on Proliferation and Differentiation of the LC Lineage in Wild-type and Mutant Mice

The effects of LH stimulation on the BrdU labeling index, BrdU numerical index, and numerical index for each interstitial cell type in wild-type control and mutant mice are presented in Figure 3. Treatment with LH in wild-type animals increased the LI of PLCs by 200% compared to wild-type vehicle-treated controls (P < 0.05). LH also increased the BrdU numerical index of PLCs and LCs (P < 0.05) in wild-type animals. In contrast, there was no effect of LH on LI in mutant mice (Fig. 3A). Only LCs had an increased BrdU numerical index after LH stimulation. This indicated that LH is not directly mitogenic, and that LH stimulation of PLC proliferation in wild-type mice depends on the presence of IGF-I. In contrast, LH stimulated LC differentiation in both wild-type and mutant testes. An additive effect on LI in PLCs could be seen in the group that received IGF-I plus LH (Fig. 2A, P < 0.05 vs. IGF-I alone), indicating that LH potentiates the effect of IGF-I on LC proliferation and that the two hormones use separate signaling pathways. IGF-I action is mediated in part through phosphatidylinositol 3 kinase activity [37], whereas LH action, as measured by testosterone production, is not [38].



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  		FIG. 3. BrdU labeling index (A), numerical index (B), and LC numerical index (C) in the LC lineage on Day 35 in wild-type and IGF-I null mutants, untreated and after LH stimulation. Values presented are means ± SEM for five animals per group. *Indicates a significant difference in labeling index or cell number between control and LH stimulation groups within a cell stage and genotype (P < 0.05)

The numbers of MPCs and PLCs were unchanged by LH stimulation in wild-type control and mutant mice, whereas the numbers of mature LCs increased by 50% in wild-type, and 30% in mutant testes compared to their vehicle-treated controls (Fig. 3B, P < 0.05). The relationship between labeling index and numerical index suggested that increased LC numbers after LH stimulation in wild-type mice were derived from both proliferation and differentiation of PLCs, whereas in mutant mice, increased numbers were derived only from differentiation of PLCs. An additive effect on cell number could be seen in the IGF-I plus LH treatment group (Fig. 2B). In contrast, the numbers of PLCs were significantly reduced after IGF-I plus LH treatment in mutant mice (P < 0.05 vs IGF-I alone), indicating that LH potentiated the role of IGF-I, facilitating both division and differentiation of intermediates in the LC lineage.

Hormone Levels in Wild-Type and Mutant Mice

Basal serum LH levels in wild-type and mutant mice were similar on Day 35 (wild-type, 0.25 ± 0.05, vs. IGF-I null mutant, 0.21 ± 0.11 ng/ml, n = 9/group). The basal serum T concentrations were similar in 35-day mutant males and their normal siblings (Table 1). Administration of LH dramatically increased circulating T levels 8-fold in wild-type controls (P < 0.05). Neither LH stimulation nor IGF-I replacement affected serum T levels in mutant mice; however, when the two hormones were administered together, serum T levels increased 2-fold (P < 0.05), indicating that LH-responsive T production in LCs was greatly attenuated in the absence of endogenous IGF-I.


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  		TABLE 1. Serum T levels in wild-type and IGF-1 null mice after treatment with vehicle, LH, IGF-1, or LH plus IGF-1 from Days 33 to 35.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
LH is accepted as the main stimulus underlying the development of ALC populations during puberty [2, 9]. However, results from this study showed that mice with a targeted gene deletion of IGF-I had an abnormal pattern of LC proliferation and differentiation and attenuated testosterone production in response to LH stimulation. These data suggest that IGF-I is also required for establishment of normal numbers of ALCs and steroidogenic function. Most notably, the present data are the first to show that LH-stimulated proliferation of LCs occurs only in wild-type and not in IGF-I null testes in vivo. Previous studies have shown that LH stimulates IGF-I secretion in rodent testis [25, 26]. We and other investigators have also observed that testicular levels of IGF-I peak during the fourth week postpartum, concurrent with the pubertal development of LCs [23, 24]. Deprivation of LH by passive immunization in 21-day-old rats reduces PLC proliferative activity, with accompanying decreases in IGF-I and IGF-I receptor mRNA levels [39]. Taken together, the data support the hypothesis that the mitogenic effects of LH are mediated by local IGF-I production.

Proliferation was defined as an increase in BrdU incorporation relative to control at a given LC stage. Increases in the numbers of 3ß-HSD immunostained cells occurred through proliferation and differentiation of ALCs from MPCs and PLCs. We evaluated the BrdU labeling index, BrdU numerical index, and numerical index of the LC lineage, and found that basal proliferative activities were unaffected by IGF-I gene disruption in 35-day-old males. However, the normal prepubertal rise in LC numbers was significantly diminished, to 25% of control. While LI was unchanged in the mutant mice, the BrdU numerical index was higher in MPCs and lower in LCs. Thus, the results indicate that the absence of IGF-I stimulation delays and/or slows the differentiation of MPCs into PLCs. In this scenario, the subsequent wave of proliferation and differentiation of newly formed PLCs is inhibited, ultimately contributing to the reduction in LC numbers. A delay in puberty occurs in growth hormone receptor (GHR) knockout mice and is believed to be related to IGF-I deficiency [33].

The present results showed that IGF-I replacement, despite increases in proliferative activity at all stages of the LC lineage, elicited increases only in the numbers of mature LCs relative to untreated mutant controls. The data on labeling and numerical index of the LC lineage indicated that the rates of LC precursor proliferation and differentiation were accelerated by IGF-I replacement. This agrees with previous in vitro studies showing that IGF-I stimulates proliferation and differentiation of LC precursors [16, 17]. Other locally-acting growth factors, such as transforming growth factor {alpha}, [16] appear to greatly augment the proliferative effects of IGF-I and may be needed for full activity in vivo. The numbers of ALCs in IGF-I null mutants are only 30% of wild-type control [30], indicating that the early phase of the LC developmental process is IGF-I dependent.

LH action on the LC lineage was significantly attenuated in IGF-I null mice. Our results demonstrated that both IGF-I and LH caused an increase in the number of 3ßHSD-positive cells, but IGF-I alone increased the number of BrdU-labeled 3ßHSD-positive cells in mutant testes. LH had no effect on the proliferative activities of either MPCs or PLCs in IGF-I null testes, but stimulated these cells in wild-type controls. These results indicate that LH does not act directly as a LC mitogen, but rather promotes PLC division in wild-type mice through autocrine induction of IGF-I. Serum LH did not differ in wild-type and mutant animals, and the equivalence of the serum values was unexpected; a normal androgen feedback system in the null mutant males should have registered the decreased androgen production and responded with an increase in LH from the pituitary. The fact that there was no difference could imply that there are effects of IGF-I at the hypothalamic and hypophysial levels of the male reproductive axis. The results are consistent with the hypothesis that IGF-I stimulates the proliferation of newly formed LCs, with LH stimulating their further differentiation.

In the present study, the basal and LH-stimulated T levels on Day 35 were not affected in IGF-I null males (Table 1), which is consistent with results reported for the growth hormone knockout mouse [40]. LH increased T production in wild-type testes. In the mutant, however, neither IGF-I nor LH treatment stimulated T production beyond basal levels. The combination of LH plus IGF-I increased T production in mutant testes, but the response was significantly attenuated relative to wild-type. These results suggest that LH cannot produce a sufficient signal for adequate T production in the absence of IGF-I. It has been shown previously that circulating IGF-I levels are undetectable in IGF-I null mutant mice [41]. One possible explanation is that the absence of IGF-I stimulation results in a developmental delay, with the LC lineage arrested at the PLC stage of differentiation, which is relatively unresponsive to IGF-I or LH stimulation. In vitro studies have shown that ILCs [42, 43] are more responsive than progenitor LCs [44] to IGF-I1-stimulated steroidogenesis [40, 45, 46]. The absence of IGF-I action is also associated with a decrease in testicular LHR [47], which may explain the attenuated action of exogenously-administered LH on LCs. In growth hormone-deficient dwarf mice, IGF-I increases testicular LH receptors and LH-stimulated T production [47]. Conversely, LH upregulates the binding capacity of IGF-I receptors in LCs [28]. In murine LCs, IGF-I potentiates the action of LH by increasing the number of LH receptors and upregulating expression of steroidogenic gene products [18, 19, 48–51]. The proliferative effects of IGF-I on LCs are potentiated by LH at low concentrations [16, 23, 29]. Taken together, the evidence supports the conclusion that IGF-I and LH act in concert to facilitate LC proliferation and differentiation [9, 30].

Although circulating IGF-I is undetectable in GHR knockout mice [33, 52], a direct comparison with IGF-I null mutants shows that the genotypes have distinct consequences for LC development. In GHR null mutants, all reproductive organs are reduced allometrically with respect to body size (~50% of normal), and steroidogenesis, spermatogenesis and fertility are normal [52]. By comparison, the dwarfism is more severe in IGF-I null mutants, and although the reduction in testis size (~30% of normal) is proportional to body size, the seminal vesicles are disproportionately small (only 10% of normal), and spermatogenesis and serum testosterone are also only 18% of control. Moreover, the IGF-I null mouse is infertile [30]. GH deficiency appears to reduce serum IGF-I concentrations but not testicular IGF-I [53]. We therefore infer that the more severe reproductive phenotype of the IGF-I null mouse compared to the GHR knockout reveals the importance of local testicularly secreted IGF-I in LC development.

In summary, LC proliferation and differentiation from precursor cells were shown to be dependent on IGF-I signaling. LH alone was less effective in stimulating LC proliferation in vivo, and its stimulatory effects appeared to be mediated by IGF-I. We conclude that: 1) IGF-I regulates LC population size by promoting both proliferation and differentiation of precursors in the LC lineage; 2) LH is not directly mitogenic and potentiates LC proliferation through IGF-I; 3) Both IGF-I and LH are critical factors in determining LC numbers and steroidogenic capacity.


   ACKNOWLEDGMENTS
 
The technical assistance of Ms. Chantal Manon Sottas and Dr. Enmei Niu is gratefully acknowledged. We also thank Dr. Argiris Efstratiadis (Columbia University) for providing the IGF-I knockout mouse line; Dr. Renshan Ge for statistical analysis, graphics and manuscript preparation; and Dr. Van Luu-The (Laval University, Quebec) for the anti-3ß-HSD antibody.


   FOOTNOTES
 
1 Supported by NIH HD32588. A preliminary form of this study was presented at the 84th Annual Meeting of the Endocrine Society, San Francisco, California, and the 35th Annual Meeting of the Society for the Study of Reproduction, Baltimore, Maryland, 2002. Back

2 Correspondence: Matthew P. Hardy, Population Council, 1230 York Avenue, New York, NY 10021. FAX: 212 327 7678; mhardy{at}popcbr.rockefeller.edu Back

Received: 25 August 2003.

First decision: 12 September 2003.

Accepted: 21 October 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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