| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Male Reproductive Tract |
Molecular Biology Program,4 Memorial Sloan-Kettering Cancer Center and
Cornell University Graduate School of Medical Sciences,5
New York, New York 10021
Population Council and The Rockefeller University,6 New York, New York 10021
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
kinases, Leydig cells, signal transduction, spermatogenesis, testosterone
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In the seminiferous epithelium, Kit is expressed starting at Postnatal Day 5 (P5) and is restricted to differentiating type A spermatogonia, type B spermatogonia, primary spermatocytes, and interstitial Leydig cells [7]. Sertoli cells provide regulatory support and nourishment for maturing germ cells and are the sole source of Kitl in the testis. Two forms of Kitl exist, KL-1 and KL-2, which are produced from alternatively spliced Kitl mRNAs. KL-1 is processed quite readily to produce soluble Kitl. KL-2 lacks the major proteolytic cleavage site and is more membrane stable [8]. In the postnatal testis, KL-2 is the dominant form, implying a critical role for membrane signaling in spermatogenesis [6, 8].
Although Kit expression in Leydig cells has been known for some time, a role for Kit function in Leydig cells has not been established. The male sex hormone testosterone is produced in the testes by Leydig cells, and its production is controlled by a feedback mechanism involving the pituitary. The hypothalamus secretes GnRH, which causes the anterior pituitary to secrete LH. LH is released in a pulsatile manner and stimulates Leydig cells to synthesize and secrete testosterone (reviewed in [9]). The mechanism by which LH stimulates testosterone production involves the binding of LH to its receptor, LHR, and activation of Gs and adenylate cyclase, leading to cholesterol mobilization [10]. Although the cAMP-mediated signaling pathway is most likely the primary mechanism of testosterone biosynthesis in vivo, GnRH and epidermal growth factor both can stimulate Leydig cell steroidogenesis in vitro through non-cAMP-mediated pathways involving calcium.
Kit functions are mediated by receptor autophosphorylation and subsequent association with signaling molecules, including PI 3-kinase. To investigate the role of Kit-mediated PI 3-kinase signaling in vivo, a knockin mouse, KitY719F/KitY719F, was made containing a tyrosine-to-phenylalanine substitution mutation located in the canonical binding site for the p85 subunit of PI 3-kinase [11, 12]. Effects of this mutation on various cellular responses in bone marrow mast cells have been characterized previously [13–15]. In KitY719F/KitY719F mice, melanogenesis and hematopoiesis are not affected in major ways. Similarly, the KitY719F mutation does not affect PGCs during embryonic development. However, severe deficiencies in gametogenesis develop postnatally. In female mice, the defect occurs at the primary follicle stage with cuboidal granulosa cells. Male mice are sterile, and germ cell maturation and differentiation do not progress beyond the spermatogonial stages [11]. On P10, spermatogonia begin to undergo apoptosis, and by P21 the tubules are depleted of germ cells except for spermatogonial stem cells.
To investigate the role of Kitl and Kit signaling in Leydig cells, we performed experiments both in wild-type C57Bl/6J mice and in KitY719F/KitY719F mutant mice. In vitro, Kitl increased steroidogenic capacity of wild-type Leydig cells approximately 2-fold over basal levels in short-term cultures. Through the use of appropriate pathway inhibitors, Kitl-mediated steroidogenesis was found to be dependent on Kit-mediated PI 3-kinase signaling. However, Leydig cells from KitY719F/KitY719F mutant mice were steroidogenically more competent than wild-type littermate cells despite the interruption of the PI 3-kinase-mediated downstream signaling pathway. The mutant animals had elevated LH levels and apparently but not significantly lower serum testosterone levels than their wild-type littermates. We propose an endocrine compensation model to explain these observations.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The KitY719F/KitY719F mice have been described previously [11]. They have a mixed strain background derived from C57BL/6J, 129Sv, and FVB. Control mice in all experiments were age-matched homozygous littermates. The genotyping of all mice was performed as described previously[11]. The animal procedures were approved by the Institutional Animal Care and Use Committees of the Memorial Sloan-Kettering Cancer Center and Rockefeller University.
Immunohistochemistry
Bromodeoxyuridine labeling and staining Two injections of bromodeoxyuridine (BrdU; Sigma, St. Louis, MO) (50 µg/g body weight) were administered to the mice 1 h apart. Mice were killed 1 h after the second injection, and testes were harvested. Testes were fixed overnight in Bouin solution (Sigma) and embedded in paraffin, and 7-µm sections were obtained. The mouse monoclonal antibody detection kit (M.O.M.; Vector Laboratories, Burlingame, CA), was used for immunochemical localization. After treatment with proteinase K (5 µg/ml in 50 mM Tris, pH 7.5, 5 mM EDTA) for 10 min at 37°C followed by 1 N HCl for 10 min at 55°C, sections were incubated with avidin and biotin and M.O.M. blocking solutions. Incubation with 6 µg/ml anti-BrdU mouse monoclonal antibody (Roche Diagnostics, Indianapolis, IN) was performed overnight at 4°C. The biotinylated secondary antibody and avidin incubation steps were performed according to the manufacturer's instructions. Slides were incubated with diaminobenzidine, counterstained with Gill hematoxylin, dehydrated, and mounted with Permount resin.
TUNEL staining After tissue fixation, embedment in paraffin, and preparation of sections, apoptotic nuclei were detected by the TUNEL method as previously described [16].
Both BrdU incorporation and TUNEL staining were quantitated by counting 500 cells each in random sections.
Electron Microscopy
For electron microscopy, mice were vascularly perfused with a glutaraldehyde/acrolein-based fixative, 1% (v/v) sym-collidine, 3.5% (v/v) 1 N HCl, 0.9% (v/v) acrolein, 7.7% (v/v), and 2% glutaraldehyde, as described previously [17]. Testes were subsequently dissected, cut into cubes, and preserved in 2% glutaraldehyde. The samples were embedded in Epon-Araldite after 2 h of postfixation in 1% OsO4-1.5% K4[Fe(CN)6] as described previously [18]. Sections were mounted on grids and viewed with an electron microscope.
Serum and Intratesticular Testosterone and LH Determinations
Blood samples were obtained either intraorbitally or by cardiac puncture. For cardiac puncture, mice were anesthetized with methoxyflurane. RIA results were identical regardless of location from which blood was obtained. Blood was allowed to stand at room temperature for 15 min, and serum was obtained by centrifugation of clotted blood and then stored at -20°C until assayed. The RIA for testosterone was performed as described previously [19]. The RIA for LH was performed as described previously [20] using anti-rat LH antiserum and radioiodinated LH.
Intratesticular testosterone levels were determined using a previously published protocol [21]. Testes were homogenized on ice in 70% methanol. Tritiated testosterone (1000 cpm) was added to the homogenate, which was allowed to stand overnight at room temperature. The homogenate was then centrifuged, and the supernatant was aspirated and dried under air to remove the methanol. The water layer was extracted twice with diethyl ether, and the ether extract was dried under air. The extract was resuspended in 400 µl of RIA buffer (0.05 M Tris, pH 8.0, 0.1% gelatin), and the RIA was performed.
Stereology
The fractionator method of stereology was employed [22, 23]. Seven-week-old littermates and mutant mice were perfusion fixed with Bouin solution as described above. Testes were removed, subdivided, and embedded in paraffin after dehydration, all with systematic sampling. Transverse sections at 4 µm were prepared and mounted on glass slides. Randomly chosen slides were dewaxed, dehydrated, and stained with anti-3ß-hydroxysteroid dehydrogenase (3ß-HSD) antibody (rabbit monoclonal antibody; gift of Dr. Van Luu-The, University of Laval, Quebec, PQ, Canada) to identify Leydig cells. Sections were then counterstained with hematoxylin and eosin. Slides were examined with a light microscope and photographed electronically. Computer software (ImagePro Plus; Media Cybernetics, Silver Spring, MD) was employed to assist in the counting of Leydig cells. A grid was computer imposed over a systematically sampled section. Leydig cell nuclei were visually identified in the test area formed by the grid and in the corresponding reference area of the adjacent serial section. Nuclear profiles that were unique to the test area were counted. At least 60 test areas were scored for each animal.
Isolation of Leydig Cells
A multistep procedure including centrifugal elutriation was used for the isolation of Leydig cells from normal C57Bl6/J mice to remove contaminating germ cells. Because KitY719F/KitY719F mice have no germ cells, the isolation of their Leydig cells was performed without elutriation (as was the isolation of littermate Leydig cells for consistency within experiments). The purification of Leydig cells by elutriation was performed as previously described [24, 25]. The purity of purified Leydig cells was determined by 3ß-HSD staining as described and averaged 90–95%.
Testes of 8-wk-old mice were removed and decapsulated, and 6–12 decapsulated testes were placed in dissociation buffer supplemented with collagenase, dispase, and DNase. Tubes were incubated at 34°C in a shaking waterbath until the testes became dissociated. Dissociated testes were filtered through a nylon filter. The filtrate was then centrifuged, and the pellet was resuspended in Percoll diluted to 55% with Hanks buffered salt solution supplemented with sodium bicarbonate, Hepes, BSA, and soybean trypsin inhibitor. A density gradient was then generated in situ by centrifugation in a JA20 rotor for 30 min at 13.5K at 4°C with maximal acceleration and slow braking. The top layers of the gradient were aspirated with a pipette, and cells located at densities of 
1.070 g/ml were harvested and centrifuged. The pellet was resuspended in 1 ml Leydig cell culture medium, cells were counted with a hemacytometer, and the purity of the Leydig cell preparation was determined as described.
Tissue Culture
Short-term cultures of Leydig cells were grown in Leydig cell culture medium (2.2 g/L sodium bicarbonate, 2.4 g/L Hepes, 1 g/L BSA, 12 mg/L gentamycin, Dulbecco modified Eagle/Ham F12 medium, adjusted to pH 7.1–7.2) with added stimulatory or inhibitory factors. Aliquots of 5 x 105 cells in 1 ml were incubated for 3 h or as indicated in closed 1.5-ml centrifuge tubes in a shaking waterbath (70 rpm) at 34°C. Incubations longer than 3 h were done in tissue culture plates in a humidified incubator with 5% CO2 and 5% supplemental O2. All experiments were carried out in triplicate.
Statistics
The Student t-test was used to evaluate differences between the littermate and mutant treatment groups. The signed-rank test was used for determination of significance of enzymatic changes in the testosterone-biosynthesis pathway. Groups were judged to differ significantly at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In testes of KitY719F/KitY719F mice, the seminiferous tubules are depleted of germ cells, with the exception of occasional spermatogonia. The interstitial space is filled with Kit-expressing cells, as identified by in situ hybridization. These cells were further classified as Leydig cells based on their cytological features, including a large polyhedral cell profile and a prominent round nucleus. At first, the Leydig cells in mutant mice appeared to be hyperplastic based on their packing density in the interstitial spaces. Because the total number of Leydig cells and rate of steroidogenesis per cell are both critical factors in determining the overall level of steroidogenesis and regulation of spermatogenesis, the total numbers of Leydig cells in mutant and control littermate testes were measured stereologically [22, 23]. Whereas the numerical density of Leydig cells was higher in the mutant than in the control littermate (mutant: 22.03 ± 2.55 cells/field; littermate: 4.62 ± 0.96 cells/field; n = 3 animals/group), the total number of cells per testis was equivalent in both groups (mutant: 1.36 ± 0.03 x 106; littermate: 1.32 ± 0.18 x 106; n = 3 animals/group).
Having established that the total number of cells per testis in the mutant and control littermate animals was equivalent, it remained possible that the Kit mutation affected the cell cycle characteristics of Leydig cells. We therefore evaluated the proliferative index and the rate of apoptosis of Leydig cells in mutant mice. Animals were labeled with BrdU, and testes were processed for immunohistochemical characterization of BrdU incorporation. Leydig cells in littermate and mutant animals at ages P21, P30, and P60 were examined, but no significant differences in the frequencies of Leydig cells incorporating BrdU were observed; <0.5% of the Leydig cells were labeled in both littermate and mutant (Fig. 1A). To determine whether Leydig cells in mutant mice exhibited altered apoptotic characteristics, we processed testis sections for the TUNEL assay. Staining was performed on Day 11 and at 7 wk of age to assess the frequency of apoptosis (Fig. 1B). Increased frequencies of apoptotic cells were observed in seminiferous tubules of mutant testes, as reported previously [11], but significant differences between mutant and control were not detected in the frequency of TUNEL-positive Leydig cells; <0.5% of the Leydig cells were labeled in both littermate and mutant. This lack of increase in proliferative index and apoptotic index is consistent with the unchanged Leydig cell numbers in mutant animals.
|
Adult Leydig cells develop postnatally from mesenchymal progenitor cells. In the mouse, the onset of steroidogenic enzyme gene expression in the progenitor cells begins on Day 11, and Leydig cells reach maturity at 8 wk [26]. From the third to eighth week postpartum, immature Leydig cells primarily produce 5
-reduced androgens. When they reach maturity, the dominant steroidogenic product is testosterone. The different maturational stages of Leydig cells display different cytological, steroidogenic, and enzymatic characteristics [27]. Mature rat Leydig cells typically have a round nucleus, a prominent nucleolus, copious smooth endoplasmic reticulum (SER), lipid-containing droplets, and cytoplasmic whorls that are involved in steroidogenesis. Leydig cells in the testis of KitY719F/KitY719F mice develop in the absence of active spermatogonial development; therefore, we employed electron microscopy to examine their differentiation status (Fig. 2). The size of the Leydig cells and their organelle structure did not appear to differ between the mutant mice and the wild-type controls, which suggests that Leydig cells in the mutant mice differentiated normally.
|
KitY719F/KitY719F Mice Have Unchanged Serum Testosterone Concentrations but Higher Circulating LH Concentrations than Littermate Controls
In a previous study [11], serum LH concentrations in KitY719F/KitY719F mice were elevated despite unchanged concentrations of testosterone. To further test whether the mutant animals had a functional deficit in steroidogenesis, we extended the analysis of these hormones by 1) increasing the sample size, examining an additional 15 and 26 animals (for LH and testosterone, respectively), 2) including measurements of both serum and intratesticular testosterone concentrations in 8-wk-old mice, and 3) performing castration experiments on 8- to 11-wk-old mice and measuring LH concentrations both before and after castration.
Serum testosterone concentrations appeared to be lower in mutant animals but did not differ significantly relative to age-matched littermate controls (P = 0.21) (Fig. 3B). Intratesticular testosterone concentrations per testis also did not differ between mutant and control animals (Fig. 3, C and D). Serum LH concentrations were elevated (5-fold) in mutant mice compared with the controls (P < 0.05) (Fig. 3A). These observations suggest that the KitY719F mutation impaired testosterone production in Leydig cells in mutant animals, lowering feedback and thereby increasing LH concentrations.
|
To examine the possibility that the changes in the LH concentrations were a result of the KitY719F mutation operating on the pituitary and not solely on the gonads, 8-wk-old animals were castrated and then killed 48 h later. Castration increased circulating LH concentrations in both mutant littermate animals. The increase was of equal magnitude (14-fold in littermates and 17-fold in mutants) (Fig. 3E). Therefore, we infer that 1) the androgen negative feedback system functions normally in the KitY719F mutants and 2) increased gonadotropic stimulation compensated for a cell autonomous defect in steroidogenesis and increased testosterone production capacity in mutant Leydig cells without invoking an effect of the mutation in the pituitary.
There is substantial evidence for dependence of adult Leydig cells on Sertoli cells for proper growth, survival, and function. Therefore it seemed critical to determine whether this cellular interaction was normal and that the Leydig cell phenotype in the KitY719F/KitY719F mice was not secondary to a Sertoli cell defect. FSH is produced by the pituitary in response to inhibin produced by Sertoli cells. In turn, FSH is required for normal Sertoli cell function [28]. To determine whether Sertoli cells are compromised in KitY719F/KitY719F mice, we measured FSH serum concentrations in mutants and controls. FSH serum concentrations in mutant and control mice did not differ significantly (17.12 ± 8.07 ng/ml and 22.73 ± 7.07 ng/ml, respectively), indicating that the Sertoli cell-pituitary feedback system is not affected in mutant mice.
Kit Signaling Is Involved in the Regulation of Testosterone Production by Leydig Cells In Vitro
Although Kitl is produced by Sertoli cells and the Kit receptor is expressed on Leydig cells, no role has been demonstrated previously for Kitl and Kit in testosterone biosynthesis. Based on our finding of increased LH concentrations in KitY719F/KitY719F mice and the presence of unchanged testosterone concentrations, a deficit in androgen production was hypothesized. Therefore, we tested for a role of Kit signaling in testosterone biosynthesis by conducting short-term in vitro culture of Leydig cells. Whole testes from 8-wk-old C57Bl/6J mice were obtained, and Leydig cells were purified through elutriation and Percoll density gradient centrifugation. Stimulation of purified Leydig cells with Kitl (100 ng) increased testosterone production (2-fold) in a 3 h incubation at 34°C (Fig. 4A). A time-course analysis showed that the stimulation exerted by Kitl was most effective during the initial 3-h time period and thereafter the difference in testosterone production between the experimental and the control remained constant.
|
Kitl Mediates Testosterone Production Through the PI 3-Kinase Pathway
In KitY719F/KitY719F mice, Kit-mediated PI 3-kinase activation is impaired. Therefore, we investigated whether Kitl-induced testosterone production in Leydig cells in vitro is dependent on Kit-mediated PI 3-kinase activation. Purified C57Bl/6J Leydig cells were stimulated with 1) Kitl alone, 2) Kitl together with the PI 3-kinase inhibitor wortmannin, 3) LH alone, or 4) LH together with wortmannin (Fig. 4B). Although Kitl alone doubled testosterone production over basal levels (Fig. 4B, dotted line), administration of Kitl together with wortmannin (10-6 M) reduced testosterone production to basal levels, suggesting that Kitl mediates its effects via activation of PI 3-kinase or activation of a PI 3-kinase-like enzyme such as mTOR (rapamycin). In contrast, LH-mediated testosterone production was not affected by wortmannin. LH alone produced a large increase in testosterone production over basal levels, but coadministration with wortmannin (10-6 M) had no effect, indicating that LH-mediated steroidogenesis is not dependent on PI 3-kinase activation.
Leydig Cells Isolated from KitY719F/KitY719F Mice Are Steroidogenically More Competent than Normal Leydig Cells
To investigate the role of Kit-mediated PI 3-kinase signaling in Leydig cells in vivo, we analyzed the stimulatory effects of Kitl on Leydig cells isolated from KitY719F/KitY719F mice (Fig. 5). Although basal levels of testosterone production in mutant and littermate control Leydig cells were comparable, Leydig cells from KitY719F/KitY719F animals were steroidogenically more competent (by approximately 2-fold) than those from wild-type controls when stimulated with either LH or 25-hydroxy-cholesterol (a diffusible substrate of testosterone biosynthesis). Equal stimulatory capacity was observed only after treatment of mutant and control Leydig cells with Kitl. Therefore, in contrast to stimulation by LH and 25-hydroxy-cholesterol Kitl-induced testosterone production was not enhanced in mutant Leydig cells, consistent with a partial impairment of Kit function due to lack of Kit-mediated PI 3-kinase activity.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Previously, a role for Kitl as a survival factor in Leydig cells had been inferred from experiments examining the recovery of Leydig cells from ethylene dimethane sulfonate-induced ablation [29]. However, there was no evidence for a role of Kitl in normal development of Leydig cells. We now propose a role for Kit in Leydig cell steroidogenesis.
Testosterone production by Leydig cells is regulated by LH, and serum LH concentrations are controlled by the pituitary through a feedback mechanism that registers the concentrations of testosterone in circulation. KitY719F mutant mice had serum testosterone concentrations that appeared to be slightly lower than those of littermate controls (without differing significantly), and the intratesticular testosterone concentrations per animal were indistinguishable from those of controls. The increase in LH concentrations among mutant animals may be explainable as a response of the androgen negative feedback system controlling the pituitary. If the number of Leydig cells in mutant mice had been reduced, the consequent lowering of serum testosterone concentrations would have triggered the pituitary to secrete higher concentrations of LH. However, Leydig cell numbers were normal in the mutant animals, whereas LH concentrations were increased 5-fold. Mutant Leydig cells were morphologically indistinguishable from those of normal adult mice, and a lowering of serum testosterone concentrations in the KitY719F/KitY719F mutant could thus not have been caused by an immature Leydig cell phenotype. We infer that the steroidogenic capacity of mutant Leydig cells was deficient initially as a result of abrogated Kit signaling but was subsequently increased in response to chronically elevated LH concentrations.
Kit expression has been identified in the pars intermedia of the pituitary [30]. Therefore, it was possible that the increased LH concentrations were due to deficient Kit signaling either at the pituitary or at the gonadal level. The castration experiment, in which concentrations of LH in mutant and wild-type mice rose by the same amount subsequent to castration, demonstrated that the hyporesponsiveness of testosterone production by mutant animals to the elevated concentrations of LH circulating in the blood is due to a gonad-specific effect and not to a change in pituitary secretion.
In support of our hypothesized role of Kit signaling in Leydig cell steroidogenesis, we observed that Kitl stimulates testosterone production by Leydig cells in vitro, and Kit-mediated PI 3-kinase signaling appears to be critical for this effect. There have been no prior studies of the effects of Kitl on the testosterone biosynthetic pathway, and the cellular targets of the upregulation are not known. We examined four enzymes involved in testosterone biosynthesis in C57BL/6J wild-type mice: P450 side chain cleavage (P450scc), 3ß-HSD, P450c17, and 17ß-HSD. Two of these enzymes, P450sccand P450c17, are upregulated (P < 0.05, signed-rank test) upon Kitl stimulation (R. Ge, unpublished results), which indicates that P450 enzymes are more subject to regulation by Kit-mediated signaling than are enzymes such as 3ß-HSD, which are members of the aldo-keto reductase superfamily [31].
In keeping with the elevated LH concentrations, Leydig cells isolated from KitY719F/KitY719F mice were more steroidogenically competent in short-term culture assays than were littermate Leydig cells. The mutant Leydig cells produced testosterone at rates that were 2-fold higher than those of controls. However, with Kitl as agonist, the levels of stimulation were not increased, suggesting that the Kit response was impaired by the KitY719F mutation.
Leydig cells have a distinct morphology that changes as they mature. In contrast to the spindle-shaped precursor cell from which they are derived, immature mouse Leydig cells are round, possess numerous lipid droplets in which cholesterol is stored, and contain abundant SER [32]. The cells develop a capacity for steroidogenesis concurrent with the expansion of SER. Immature Leydig cells synthesize high levels of the androgen metabolites, both precursors of testosterone such as androstenedione and 5-reduced steroids such as 5-androstane-3,17ß-diol. The 5-reduced androgens are secreted at higher rates relative to adult Leydig cells because androgen-metabolizing enzymes are expressed at higher levels in immature animals. Leydig cells continue to increase in size beyond the immature stage, primarily resulting from further expansion in the volume of the SER. In adult Leydig cells, the activities of androgen-metabolizing enzymes decline and testosterone becomes the dominant steroid end product [33]. Membranous whorls of SER appear in the cytoplasm with the transition from immature to adult Leydig cell [31]. Electron microscopic characterization of mutant testes indicated that Leydig cells are fully differentiated, based on ultrastructural characteristics.
Use of mice with the KitY719F mutation allowed us to examine whether the formation of seminiferous tubules and Sertoli cells is sufficient for postnatal Leydig cell development and differentiation. Kit loss-of-function mutations affect several aspects of gametogenesis, including the primordial germ cell stages, oogenesis, and spermatogenesis [11, 34]. In general, mutant Kit alleles affect the primordial germ cell stages, and therefore the gonads in mutant animals are either depleted of germ cells or numbers are reduced [1, 4, 35]. The KitY719F allele does not affect primordial germ cells; germ cell numbers at the time of birth are normal. However, germ cell development in the testis is arrested at the spermatogonial stages, and consequently seminiferous tubules are depleted at a very young age (2–3 wk). Development of mature Leydig cells occurs postnatally. In KitY719F mutant mice, normal numbers of mature differentiated Leydig cells are found in 8- to 9-wk-old testes. Therefore, germ cell maturation is not required for this process. These results are in agreement with earlier studies indicating that the formation of seminiferous cords is not a prerequisite for the formation of differentiated Leydig cells [36]. KitlSl17H is a kit ligand mutation that has a weak effect on primordial germ cell numbers [35]. In KitlSl17H mutant male mice, spermatogenesis is also affected, leading to sterility. However, in contrast to KitY719F mice, in KitlSl17H mutant mice the first synchronous wave of germ cell maturation proceeds normally but subsequent cycles are impaired and seminiferous tubules are depleted. The jsd (juvenile spermatogonial depletion) mutant mouse is similar to the KitlSl17H mutant mouse in that spermatogenesis is not affected in the first maturation cycle; however, in the jsd mouse tubules subsequently become depleted, leading to sterility [37–39]. In part, elevated intratesticular testosterone concentrations could contribute to tubule depletion in KitlSl17H mutant mice. In KitlSl17H mice, it is possible that impairment of Kit-mediated steroidogenesis in Leydig cells and consequent negative feedback by LH could lead to increased intratesticular testosterone in partially depleted tubules, which would contribute to maintenance of azoospermia in adult mice.
Adult Leydig cells appear to be dependant on Sertoli cells for proper growth, survival, and function [9, 39, 40]. Based on examination of Kitl expression and expression of other markers of Sertoli cells and the observation of normal FSH levels in mutant mice, the KitY719F mutation does not appear to affect Sertoli cell development and function. Thus, it is unlikely that the effect of the KitY719F mutation on Leydig cells is secondary to a Sertoli cell abnormality. In the testis, both spermatogonia and Leydig cells require Kitl for Kit receptor-mediated cellular responses. Sertoli cells, however, are the sole intratesticular source of Kitl. Kitl probably is produced both on the adluminal side and the basolateral side of Sertoli cells to provide for juxtacrine Kit receptor signaling in spermatogonia and release of soluble Kitl into the interstitial space for mediating Kit responses in Leydig cells. Serum may be an additional source of Kitl for Leydig cells, although the concentrations of soluble Kitl in the serum are most likely too low to produce Kit-mediated cellular responses. Therefore, both targeted expression of Kitl on the basolateral surface of Sertoli cells and proteolytic cleavage of membrane Kitl to produce soluble Kitl are critical steps for Kit receptor signaling in Leydig cells.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Matthew P. Hardy, Center for Biomedical Research, Population Council, 1230 York Ave., New York, NY 10021. FAX: 212 327 7678; hardy{at}popcbr.rockefeller.edu
3 Correspondence: Peter Besmer, Memorial-Sloan Kettering Cancer Center, 1275 York Ave., New York, NY 10021. FAX: 212 717 3623; p-besmer{at}ski.mskcc.org
Received: 12 December 2002.
First decision: 2 January 2003.
Accepted: 8 May 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  IN MEMORIAM Matthew P. Hardy, Ph.D. 1957-2007 Biol Reprod, March 1, 2008; 78(3): 563 - 564. [Full Text] [PDF]
|
|
|
|
 |
|
 F. Raucci and M. M. Di Fiore The c-kit receptor protein in the testis of green frog Rana esculenta: seasonal changes in relationship to testosterone titres and spermatogonial proliferation Reproduction, January 1, 2007; 133(1): 51 - 60. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Prabhu, M. L Meistrich, E. A McLaughlin, S. D Roman, S. Warne, S. Mendis, C. Itman, and K. L. Loveland Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis. Reproduction, March 1, 2006; 131(3): 489 - 499. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. P. Lehmann, S. Phillips, M. Sar, P. M. D. Foster, and K. W. Gaido Dose-Dependent Alterations in Gene Expression and Testosterone Synthesis in the Fetal Testes of Male Rats Exposed to Di (n-butyl) phthalate Toxicol. Sci., September 1, 2004; 81(1): 60 - 68. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. S. Sarkar, S. Paul, J. Han, and S. Reddy Six5 is required for spermatogenic cell survival and spermiogenesis Hum. Mol. Genet., July 15, 2004; 13(14): 1421 - 1431. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Wang and M. P. Hardy Development of Leydig Cells in the Insulin-Like Growth Factor-I (IGF-I) Knockout Mouse: Effects of IGF-I Replacement and Gonadotropic Stimulation Biol Reprod, March 1, 2004; 70(3): 632 - 639. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
