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Biology of Reproduction 67, 70-79 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Apoptosis, Onset and Maintenance of Spermatogenesis: Evidence for the Involvement of Kit in Kit-Haplodeficient Mice1

Fabrice Guerifa, Veronique Cadoreta, Valerie Rahal-Perolaa, Jacques Lansaca, Florence Bernexb, Jean Jacques Panthierb, Marie Thérèse Hochereau-de Reviersa, and Dominique Royere2,,a

a Physiopathologie de la Reproduction, Unité Mixte de Recherche 6073 "Physiologie de la Reproduction et des Comportements," Institut National de Recherche Agronomique, Centre National de la Recherche Scientifique, Université de Tours, Nouzilly, France b UMR 955 Institut National de Recherche Agronomique, Ecole Nationale Veterinaire d'Alfort, 94704 Maisons-Alfort, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Kit/stem cell factor (SCF ) has been reported to be involved in survival and proliferation of male differentiating spermatogonial cells. This kinetics study was designed to assess the role of Kit/SCF during spermatogenesis in mice, and the extent of male programmed germ cell death was measured between 8 and 150 days of age. For this purpose, 129/Sv inbred mice in which one Kit allele was inactivated by an nlslacZ sequence insertion (KitW-lacZ/+) were compared with 129/Sv inbred mice with wild-type alleles at the Kit locus. Four different approaches were used: 1) morphometric study to assess spermatogenesis, 2) flow cytometry to study testicular cell ploidy, 3) in situ end labeling to detect apoptosis, and 4) follow-up of reporter gene expression. Spermatogenesis was lower in KitW-lacZ/+ heterozygous mice both at the onset of spermatogenesis and during adulthood. Indeed, greater apoptosis occurred at the onset of spermatogenesis. This was followed in the adult by a smaller seminiferous tubule diameter and a lower ratio between type B spermatogonia and type A stem spermatogonia in KitW-lacZ/+ mice compared with Kit+/+ mice. These differences are probably related to the Kit haplodeficiency, which was the only difference between the two genotypes. Germ cell counts and testicular cell ploidy revealed delayed meiosis in KitW-lacZ/+ mice. Reporter gene expression confirmed expression of the Kit gene at the spermatogonial stage and also revealed Kit expression during the late pachytene/diplotene transition. These results suggest involvement of Kit/SCF at different stages of spermatogenesis.

apoptosis, developmental biology, meiosis, polypeptide receptors, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is a dynamic and complex process of germ cell proliferation and differentiation. Germ cell degeneration has been fully established as a factor that limits spermatogenesis efficiency [1, 2]; apoptosis has been reported in 32-day-old rats with DNA fragmentation in primary spermatocytes, and a study using GnRH antagonists suggests that the process is controlled by hormones [3]. The wave of apoptosis has recently been described in normal mice around 3 wk of age, with clear involvement of the Bcl2 gene family. Prevention of this apoptotic wave was followed by drastic changes in spermatogenesis and fertility in adults [4]. The physiological significance and molecular mechanisms of male germ cell death remain largely unknown. It has been suggested that adjusting the number of proliferating germ cells to match the number of supporting Sertoli cells might ensure the quality of the gametes produced [4, 5].

Among the different candidates that may influence the apoptotic process during spermatogenesis, the Kit/stem cell factor (SCF) system has received specific attention. The system has well-documented effects on hematopoiesis, melanogenesis, and gametogenesis [6, 7]. Numerous mutations at the white-spotting locus (W) or the Steel locus (Sl) have been described in mice [8–13]. They result in similar phenotypes (defects in pigmentation, anemia, and sterility) due to a lack of progenitor cells belonging to the melanocytic, hematopoietic, and germ cell lineages [6, 14, 15]. The proto-oncogene Kit maps to W [16, 17] on chromosome 5, SCF maps to Sl [10] on chromosome 10.

The main product of the Kit gene is a transmembrane tyrosine-kinase receptor that belongs to the same family as {alpha} and ß platelet-derived growth factors and colony stimulating factor-1 receptors [18, 19]. The structural organization of this glycoprotein includes an immunoglobulin-like extracellular domain, connected by a single transmembrane domain to an intracellular tyrosine-kinase domain. Reports have demonstrated that the Kit gene is expressed in differentiating type A spermatogonia [20–22], in type B spermatogonia [23], in premeiotic [23] and meiotic spermatocytes [24], and in a truncated form in haploid cells [25–27]. The SCF ligand has been detected both as a soluble protein and as a membrane-bound protein in Sertoli cells [28, 29]. It has been found in Sertoli cells during all development stages, with some ontogenetic variation, depending on the length of the transcripts that encode membrane-associated or soluble forms of the protein [30, 31].

The protective action of the Kit/SCF system on primordial germ cells and type A spermatogonia survival and its effect on their proliferation have been documented both in vivo [20, 32] and in vitro [33–39]. The PI3 kinase domain from the intracellular part of the membrane-associated receptor is reported to be involved at premeiotic stages in the signal-transduction pathways of kit gene action [40–42].

The extent to which Kit/SCF is involved in early spermatogenesis and adulthood is not fully understood. We therefore used 129/Sv mice with Kit haplodeficiency and in which one allele of Kit was inactivated by an nlslacZ sequence insertion (KitW-lacZ/+), and compared them with 129/Sv coisogenic mice (Kit+/+), which were used as controls. We investigated 1) whether spermatogenesis was impaired in this model, 2) whether germ cell apoptosis was affected, and 3) the types of germ cells that express Kit. A 50% reduction in the level of the Kit gene product led to reduced spermatogenesis and demonstrates that Kit is haploinsufficient. We also show that apoptosis is increased at the onset of spermatogenesis and during adulthood. Finally, we show that Kit is expressed at two distinct stages of spermatogenesis (i.e., in differentiated spermatogonia and in late pachytene/diplotene spermatocytes).


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

We used an inbred strain of 129/Sv mice in which Kit was inactivated by an nlslacZ sequence insertion in the first exon of the Kit gene [43]. Homozygous Kit-defective animals could not be used because they died soon after birth due to megaloblastic anemia. We analyzed heterozygous mice (KitW-lacZ/+) that presented no apparent defect in embryo development or survival, and we used wild type mice (Kit+/+) as controls. As required by European rules for animal maintenance, mice were maintained at 22 ± 2°C in 12L:12D cycles, and they had ad libitum access to food pellets (UAR, Villemoisson, Epinay sur Orge, France) and water.

At least 50 animals per genotype were killed at different ages by cervical dislocation under anesthesia, taking into account the progression and maintenance of spermatogenesis (from 8 days until 150 days of age, at least 3 animals per age). At 8 days, KitW-lacZ/+ animals were easily identified by their unpigmented tail tip. Testes were surgically removed and weighed. Epididymides were carefully dissected and minced in equilibrated potassium simplex optimized medium [44] for 20 min at 37°C in order to allow sperm to swim out into the medium. After suspension recovery, the final volume was adjusted to 1 ml before sperm counts were evaluated. Unless specifically noted, all reagents were purchased from Sigma Chemical Company (St. Quentin Fallavier, France).

Testicular Endocrine Status

In order to evaluate androgen secretion, seminal vesicles were removed and weighed, and serum testosterone concentrations were assayed after pooling blood samples from mice of the same age and genotype. Trunk blood was allowed to clot for 3 h at room temperature; serum was then separated by centrifugation (200 x g at room temperature for 10 min) and stored frozen at -20°C until assayed. Testosterone concentrations were determined by radioimmunoassay following solvent extraction [45]. The sensitivity of the assay was 15 pg per sample.

Histomorphological Evaluation of Testes

Testes were immersion-fixed in Bouin-Hollande solution for 24 h, then histological study was performed with 3- to 5-µm paraffin sections. Relative volumes of seminiferous tubules and interstitial tissue were determined using a 25-point integrator on 20 fields per testis, as already described [46]. Testis cross-diameters were measured on the largest histological sections for each animal to determine approximate testis volumes. Total volumes of seminiferous tubules and interstitial tissue were then calculated by multiplying the relative volume by the total testis volume. Mean seminiferous tubule diameter (20 tubules per animal) and mean number of Sertoli cells (10 tubules per animal) were measured. Type A stem spermatogonia were distinguished from type B spermatogonia at 8 days of age. Staging was based on the steps of spermiogenesis and also on the full association of germ cells. Then, type A stem spermatogonia (stages V–VIII), type B spermatogonia (stages V–VI), leptotene and zygotene spermatocytes (stages VIII–XI), pachytene spermatocytes (stages VII–VIII), diplotene spermatocytes (stages X–XI), round spermatids (stages VII–VIII), and elongated spermatids (stages V–VII) were counted on the basis of an evaluation of 10 cross-sections of seminiferous tubules per stage per animal. In order to avoid bias in evaluating germ cell populations in both genotypes, we expressed numbers of each type of germ cell as a ratio of the numbers of Sertoli cells. The yields of spermatogonial divisions (B:A stem spermatogonia and leptotene-zygotene spermatocytes:B spermatogonia), meiosis (round spermatids:leptotene-zygotene spermatocytes ratio), and spermiogenesis (elongated spermatids:round spermatids) were then calculated.

In Situ End Labeling

Testes were immersion-fixed in 4% paraformaldehyde for 12 h. For in situ end labeling (ISEL) we used direct labeling by fluorescein nucleotide triphosphate transfer to the 3' ends of DNA by terminal desoxynucleotidyl transferase (Apoptag Fluorescein Direct Kit, S7160; Appligen-Oncor, Illkirch, France) with slight modifications. Briefly, instead of protease treatment, we heated slides in a 10 mM citrate buffer pH 6 in 3-min cycles using a microwave oven [47, 48]. We used Evans Blue instead of DAPI as a counterstain mainly because Evans Blue provides sufficient contrast when using a long-pass filter for fluorescein isothiocyanate (excitation 490 nm, emission 520 nm). We used an apoptotic index (number of stained cells/number of round tubular sections) with minimal counting (100 round tubular sections). In order to incorporate the variations in seminiferous tubule section area with age, these results were weighted by dividing the result by the mean square diameter of tubules at each age and for each genotype. We used also ISEL with peroxidase staining and hematoxylin counterstaining (S7101; Apoptag Plus Peroxidase, Appligen-Oncor) in order to characterize which types of germ cells were affected by apoptosis.

Flow Cytometry Analysis of Ploidy of Testicular Cells

The tunica albuginea was removed after surgical excision of the testes, then testicular tissue was minced with scissors, and seminiferous tubules were grossly separated from most of the interstitial tissue by two gravity sedimentation steps in Leibovitz medium (L15; Gibco BRL Life Technologies, Cergy Pontoise, France). Seminiferous tubules were then mechanically dissociated to disperse cells before filtering through nylon mesh (80-µm pore size) to remove large clumps of cells. Cell suspensions were adjusted to 2 x 106 cells per tube, then centrifuged at 200 x g for 10 min. The pellet was resuspended in 1 ml PBS. Cell suspensions were filtered through nylon mesh (60-µm pore size), then incubated with 10 µg/ml propidium iodide (Molecular Probes Europe BV, Leiden, The Netherlands) and 0.1% v/v Nonidet P40 at room temperature for 20 min in the dark. Samples were immediately analyzed using a flow cytometer (Becton Dickinson FACStarplus, San Jose, CA). Red fluorescence (610LP filter) emitted from individual cells was recorded from 10 000 cells per sample after excitation with a 488 nm argon-ion laser (80 mW). Data were analyzed using the Lysys-II processing program.

Biochemical Assay of ß-Galactosidase Activity

Testes were rapidly excised and chilled before weighing. The tunica albuginea was then rapidly stripped from the testicular parenchyma and the tissue was homogenized mechanically in ice-cold 15 mM Tris buffer, pH 7.9, containing 60 mM KCl, 15 mM NaCl, 2 mM EDTA, and 0.4 mM PMSF. The homogenate was centrifuged at 2000 x g for 10 min and the pellet was discarded. The supernatant was used as the source of ß-galactosidase. ß-Galactosidase activity in these extracts was measured fluorimetrically (450 nm) using 4-methylumbelliferyl ß-D-galactopyranoside as a substrate [49] and was expressed per gram of testis.

Histochemical Detection of ß-Galactosidase

Whole testes from mice of both genotypes were fixed in 0.1 M PBS, 0.1% saponin, and 4% paraformaldehyde for 2 h at 4°C, then half-sectioned and fixed again in the same conditions. ß-Galactosidase staining was performed after 3 rinses at room temperature in 0.1 M PBS by overnight immersion at 32°C in the following solution: 0.4 mg/ml 4-chloro-5-bromo-3-indolyl-D-galactopyranoside, 4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM MgCl2, 0.1% saponin, and 0.01% Tween-20 in 0.1 M PBS, pH 7.4. Testes were briefly rinsed with 0.1 M PBS, dehydrated in 50%–100% ethanol, embedded in paraffin, and 5-µm sections were cut. Paraffin-embedded sections were dewaxed in xylene, rehydrated through a graded alcohol series to distilled water, counterstained with hematoxylin, dehydrated, and mounted in Depex.

Preliminary Fertility Assessment

In order to evaluate the consequences of kit deficiency on in vivo fertility, we performed matings using four females (9–15 wk old) per male (3 mo old) with two types of allocations. Matings occurred between KitW-lacZ/+ males (n = 166) and Kit+/+ females, and between Kit+/+ males (n = 10) and Kit+/+ females. Fewer matings of only Kit+/+ mice were allowed in order to preserve the heterozygous genetic background. Results were expressed as the percentage of pregnant females and mean litter size.

Statistical Analysis

Quantitative data resulting from histomorphological studies, ISEL, and enzymatic studies were expressed as mean ± SEM (at least 3 animals per age per genotype). Results were analyzed on a Power Macintosh computer using Statview 4.1 (Abacus Concepts, Berkeley, CA). ANOVA was used to determine statistical significance, and analysis of covariance was used to study the effects of age and genotype. Post hoc comparisons between genotypes were performed using the Bonferroni/Dunn test. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
General Findings on Sexual Organs

Genital development and status of Kit-haplodeficient mice (KitW-lacZ/+) were assessed to define the phenotype. A significant increase in testis weight occurred with age in mice of both genotypes (Fig. 1A). However, the increase was lower in KitW-lacZ/+ mice compared with Kit+/+ mice (from 18 days to 5 mo; 49 ± 10 mg vs. 116 ± 5 mg; P < 0.001). By contrast, there was no difference in seminal vesicle weight between mice of either genotype at any age (Fig. 1B) or in total body weight (data not shown). Relative interstitial tissue volume (Fig. 1D) was higher in KitW-lacZ/+ mice than in Kit+/+ mice at various ages (at 18 days, then from 45 days onward). However, there was no difference between mice of either genotype in calculated total volume of interstitial tissue (3.6 ± 0.2 mm3 vs. 3.9 ± 0.0 mm3 at 45 days, 2.6 ± 0.1 mm3 vs. 2.9 ± 0.6 mm3 at 5 mo for KitW-lacZ/+ and Kit+/+ mice, respectively. There also was no difference in Leydig cell density between mice of either genotype (data not shown).



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  		FIG. 1. Testis weights (A), seminal vesicle weights (B), seminiferous tubule diameters (C), and relative interstitial tissue volumes (D) were compared between kit+/+ (black) and kitW-lacZ/+ (gray) mice. Values are expressed as mean ± SEM (n = 3 animals per age and genotype). Letters above bars denote a statistically significant difference between genotypes (a, P < 0.05; b, P < 0.01; c, P < 0.001)

Serum testosterone assessment revealed no difference between mice of either genotype at any age (mean for whole population studied, 0.57 ± 0.31 ng/ml vs. 0.64 ± 0.49 ng/ml for KitW-lacZ/+ and Kit+/+ mice, respectively). Finally, total sperm counts in epididymides were significantly lower in KitW-lacZ/+ mice than in Kit+/+ mice (4.0 ± 1.0 x 106 vs. 12.3 ± 1.0 x 106 at 45 days for KitW-lacZ/+ mice and Kit+/+ mice, respectively; P < 0.001). Gonadal development was considerably affected by Kit haplodeficiency.

Histomorphological Analysis of Seminiferous Tubules

Seminiferous tubule diameter increased with age, but the increase was less in KitW-lacZ/+ mice than in Kit+/+ mice (Figs. 1C and 2). The number of Sertoli cells per tubule section was similar in mice of both genotypes (Fig. 3A; 16.2 ± 0.6 and 16.3 ± 0.3 per section for Kit+/+ and KitW-lacZ/+ mice at 45 days, respectively). The type A stem spermatogonia:Sertoli cell ratio did not differ between genotypes (Fig. 3B). However, the number of type B spermatogonia per tubule section (data not shown) and the type B spermatogonia:Sertoli cell ratio (Fig. 3C) were both considerably lower in KitW-lacZ/+ mice than in Kit+/+ mice. The ratio increased with puberty in mice of both genotypes, but in KitW-lacZ/+ mice it remained lower than in Kit+/+ mice, and a further decrease occurred at a later age (Fig. 3C).



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  		FIG. 2. Testis sections from kit+/+ (A and C) and kitW-lacZ/+ (B and D) mice at 45 days and 150 days of age. Bars = 100 µm



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  		FIG. 3. Histomorphological quantitative analysis of germ cell populations in testes of kit+/+ (black) and kitW-lacZ/+ (gray) mice from 8 days until 150 days. Numbers of Sertoli cells per seminiferous tubule cross-sections (A), type A stem spermatogonia (B), type B spermatogonia (C), leptotene-zygotene primary spermatocytes (D), pachytene primary spermatocytes (E), diplotene primary spermatocytes (F), round spermatids (G), and elongated spermatids (H) are expressed as a ratio of the Sertoli cell number. Values are expressed as mean ± SEM (n = 3 animals per age and genotype). Letters above bars denote a statistically significant difference between mice of both genotypes (a, P < 0.05; b, P < 0.01; c, P < 0.001)

Similarly, fewer more-advanced germ cell populations were found in KitW-lacZ/+ mice with all methods of evaluation (total cell number per section, data not shown) and as a ratio of the Sertoli cell number (Fig. 3D, leptotene-zygotene:Sertoli cells; Fig. 3E, pachytene:Sertoli cells; Fig. 3F, diplotene spermatocytes:Sertoli cells; Fig. 3G, round spermatids:Sertoli cells; and Fig. 3H, elongated spermatids:Sertoli cells). Recovery at different stages during spermatogenesis showed contrasting results. Indeed, the ratio of type B to type A stem spermatogonia was lower in KitW-lacZ/+ mice, with lower levels at several ages (30 and 45 days) during pubertal development and a strong decrease at later ages in adult mice (Fig. 4A). However, no change was observed in mice of either genotype in the ratios between leptotene-zygotene spermatocytes and type B spermatogonia (Fig. 4B), round spermatids and leptotene-zygotene spermatocytes (Fig. 4C), or elongated and round spermatids (Fig. 4D). The occurrence of agametic tubules, which were devoid of haploid germ cells, became more frequent from maturity until later ages in KitW-lacZ/+ mice than in Kit+/+ mice (Fig. 2, B and D). Indeed, the percentage of agametic tubules remained low in both genotypes until 45 days of age (0.2% in both genotypes). However, a greater increase in this percentage was observed later in KitW-lacZ/+ mice compared with Kit+/+ mice (19.9% vs. 0.3% for KitW-lacZ/+ and Kit+/+ mice, respectively; P < 0.05).



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  		FIG. 4. Histomorphological quantitative analysis of germ cell populations in testes of kit+/+ (black) and kitW-lacZ/+ (gray) mice from 8 days until 150 days. Various ratios between germ cell populations were compared between kit+/+ and kitW-lacZ/+ mice. Type B spermatogonia:type A stem spermatogonia (A), leptotene-zygotene primary spermatocytes:type B spermatogonia (B), round spermatids:leptotene-zygotene primary spermatocytes (C), and elongated spermatids:round spermatids (D). Values are expressed as mean ± SEM (n = 3 animals per age and genotype). Letters above bars denote a statistically significant difference between mice of both genotypes (a, P < 0.05; b, P < 0.01; c, P < 0.001)

Evolution of Ploidy of Testicular Cells During the First Cycle of Spermatogenesis

Flow cytometry allowed us to monitor the ploidy distribution of testicular cells from 4C to 1C condensed cells according to genotype and age (Fig. 5). Four different major peaks of fluorescence were observed in the testicular cell suspensions from animals with fully completed spermatogenesis, involving 2C diploid cells (mainly spermatogonia with secondary spermatocytes and few somatic cells), 4C tetraploid cells (G2 spermatogonia and primary spermatocytes), and two types of 1C haploid cells (from elongating spermatids with condensed chromatin to spermatozoa [Fig. 5a, 1C condensed cells] or round spermatids [Fig. 5b, 1C cells], respectively). At 8 days, the ploidy distribution was the same for KitW-lacZ/+ mice and Kit+/+ mice (4C population, 6.6% ± 0.2% and 7.1% ± 0.5% in KitW-lacZ/+ and Kit+/+ mice, respectively). Cell ploidy in testes of both genotypes differed at 13 days onward. First, a smaller 4C population was observed in KitW-lacZ/+ mice than in Kit+/+ mice (45% less at 13 days, P < 0.001).



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  		FIG. 5. Cumulative representative DNA flow cytograms of propidium-iodine-stained testicular cells of mice as a function of age and genotype (kit+/+ mice are shown in blue; kitW-lacZ/+ are shown in red; n = 3 to 6 animals per age and per genotype, depending on the cell density). Four different major peaks of fluorescence may be observed: 2C diploid cells (mainly spermatogonia with secondary spermatocytes and a few somatic cells), 4C tetraploid cells (G2 spermatogonia and primary spermatocytes), and two types of 1C haploid cells (from elongated spermatids with condensed chromatin to spermatozoa (a) or round spermatids (b), respectively). Statistically significant differences between mice of both genotypes for each peak are shown (*P < 0.01; **P < 0.001)

The 1C cell population was smaller in KitW-lacZ/+ mice than in Kit+/+ mice (69% less at 24 days, P < 0.001) and a smaller proportion of 1C condensed cells in KitW-lacZ/+ mice was found at 36 days, although it was not statistically significant (39% less compared with Kit+/+ mice). Cell ploidy distribution was similar in mice of both genotypes at 45 days. Later, in 5-mo-old adults, lower 1C and higher 2C levels in KitW-lacZ/+ mice than in Kit+/+ mice (P < 0.01) were the only differences observed between mice in both genotypes. KitW-lacZ/+ mice were thus characterized by a lower proportion of the more-advanced germ cell population compared with Kit+/+ mice, both during the first cycle of spermatogenesis and later in adulthood.

Increase in Germ Cell Apoptosis in KitW-lacZ/+ Mice

ISEL studies were performed to check for apoptotic cells (Fig. 6, A and B). The cellular targets of apoptosis were spermatogonia and spermatocytes, some of which were in metaphase. A wave of apoptosis was observed between 8 and 45 days of age in mice of both genotypes, with a peak at 13 days of age (Fig. 6C). However, the apoptotic index in KitW-lacZ/+ mice was about twice as great as that observed in Kit+/+ mice from 13 until 37 days of age. In adult mice, the apoptotic index remained higher in KitW-lacZ/+ mice than in Kit+/+ mice. The increase in germ cell apoptosis thus occurred in both young and adult male Kit-deficient mice.



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  		FIG. 6. Apoptotic cells were observed and counted using a fluorescent label in kit+/+ (A) and kitW-lacZ/+ mice (B) from 8 until 150 days of age. The apoptotic index was calculated as the ratio between the number of apoptotic cells per 100 tubule sections that was weighted by the square mean diameter of the tubule section (C) (kit+/+ mice, black; and kitW-lacZ/+ mice, gray). At least 100 sections were counted for each animal. Values are expressed as mean ± SEM (n = 3 animals per age and genotype). Letters above bars denote a statistically significant difference between mice of both genotypes (a, P < 0.05; b, P < 0.01; c, P < 0.001). Bars = 60 µm

LacZ Reporter Gene Expression in KitW-lacZ/+ Mice

Insertion of the nlslacZ sequence in the first exon of the Kit gene allowed us to evaluate the ontogenic expression of Kit in KitW-lacZ/+ mice. As expected, very low levels of ß-galactosidase in whole testes were measured in Kit+/+ mice at all ages (lower than 0.033 IU/g). Contrasting with this faint activity, ß-galactosidase activity was high in KitW-lacZ/+ mice. It was, however, higher at two different periods in KitW-lacZ/+ mice. From 12 days until 21 days of age, ß-galactosidase activity was slightly higher than it was at 8 days, but this was not statistically significant (0.13 to 0.18 IU/g). Then, a second large increase (more than 60 times the previous level) was observed in 24- and 27-day-old mice (1.34 ± 0.46 IU/g and 2.0 ± 0.29 IU/g, respectively), with a slight decrease afterward.

To complete this quantitative approach, in situ detection of ß-galactosidase allowed us to investigate its testicular expression according to age and genotype. Contrasting with the absence of ß-galactosidase in germ cells from Kit+/+mice, KitW-lacZ/+ mice were characterized by two distinct chronological stages of ß-galactosidase expression. First, nuclei of type A differentiated spermatogonia were clearly stained at 10 days of age (data not shown). Then at 17 and 24 days of age, type A differentiated spermatogonia remained stained, although neither leptotene nor zygotene spermatocytes expressed ß-galactosidase. Similarly, no expression was observed in either young or mid-pachytene spermatocytes. By contrast, intense staining was observed in late pachytene and diplotene spermatocytes (Fig. 7, A and B). The same features of ß-galactosidase expression were observed in adult mice (5 mo), with staining of type A differentiated spermatogonia, late pachytene and diplotene spermatocytes, and secondary spermatocytes and round spermatids, mainly for steps 1 to 4 in spermiogenesis. At later steps, spermatids were characterized by faint cytoplasmic staining. Cytoplasmic staining disappeared when sperm were released from seminiferous epithelium, whereas residual bodies were stained (Fig. 7, C and D). Leydig cells in adult mice were also examined for ß-galactosidase expression (Fig.7, C and D). Precursor Leydig cells showed weak staining at earlier ages (Fig. 7B). Thus, ß-galactosidase expression in germ cells occurred at two distinct stages during spermatogenesis: in differentiated spermatogonia and in late pachytene/diplotene primary spermatocytes.



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  		FIG. 7. In situ detection of ß-galactosidase at three different ages in kitW-lacZ/+ mice during the first ongoing cycle of spermatogenesis (A, 17 days; B, 24 days) then in adult mice (C and D at 5 mo, at different stages). Spg, Spermatogonia; L/ZSpy, leptotene-zygotene primary spermatocytes; YgSpy, young pachytene primary spermatocytes; Mspy, mid-pachytene primary spermatocytes; PSpy, late pachytene primary spermatocytes; DSpy, diplotene primary spermatocytes; rSpd, round spermatids; eSpd, elongated spermatids; Rb, residual bodies. Bars = 50 µm

Preliminary Fertility Assessment

Considerably fewer females became pregnant through matings with KitW-lacZ/+ males than through matings with Kit+/+ males (6.0% vs. 15% for KitW-lacZ/+ males and Kit+/+ males, respectively; P < 0.05) without any impairment in sexual behavior (similar frequency of copulatory vaginal plugs). In addition, mean litter size was lower in matings with KitW-lacZ/+ males than in matings with Kit+/+ males (6.3 ± 1.2 vs. 7.4 ± 0.9 for KitW-lacZ/+ males and Kit+/+ males, respectively; P < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Kit-haplodeficient mice were used to evaluate the role of Kit during onset and maintenance of spermatogenesis. Mice with the same genetic background were used as controls to allow quantitative comparison [6]. Study of heterozygous mice was based on the hypothesis of a single "dose-effect" for the Kit gene in such mice, which is fairly infrequent in natural mutations of the Kit when fertility occurs [17]. This hypothesis was confirmed when we observed severe impairment of sperm production and a drastic increase in germ cell apoptosis in KitW-lacZ/+ mice compared with Kit+/+mice.

We observed a smaller increase in testis weight in KitW-lacZ/+ mice compared with Kit+/+mice. By contrast, there was no difference in total body weight, seminal vesicle weight, or testosterone plasma level between mice of either genotype. Therefore, the KitW-lacZ/+ phenotype was not related to a general somatic effect or changes in androgen secretion by the testes. Interstitial tissue volume and Leydig cell density did not differ between genotypes. Although total testis volume was smaller in KitW-lacZ/+ mice, interstitial tissue volume was kept constant by a decrease in the volume occupied by the seminiferous tubules. This does not exclude the possibility that Leydig cell function may be dependent on the Kit gene, because Kit involvement in Leydig cell proliferation and survival has been reported [50]. Indeed, we observed reporter gene expression (ß-galactosidase) in Leydig cells both during and after the onset of spermatogenesis.

An interesting finding was the decrease in type B spermatogonia in KitW-lacZ/+ mice compared with Kit+/+mice. However, there was no difference in numbers of Sertoli cells nor in type A stem spermatogonia between genotypes. This agrees with previous reports on Kit involvement for maintenance and proliferation of type A differentiated spermatogonia [20–22]. The role of the Kit gene has recently been described in differentiated type A spermatogonia maintenance but not in undifferentiated spermatogonia proliferation or survival [51]. Moreover, the similar numbers of type A stem spermatogonia in mice of both genotypes suggests Kit haplosufficiency in the migration and proliferation of primordial germ cells. KitW-lacZ/+ mice had fewer germ cells of all types, from type B spermatogonia to spermatozoa, compared with Kit+/+mice, suggesting both a decrease in number and a delay in appearance. One striking observation was provided by the investigation of the 3 main stages of spermatogenesis, in which neither meiosis nor spermiogenesis appeared to be affected, compared with spermatogonia differentiation, which was clearly smaller in KitW-lacZ/+ mice than in Kit+/+mice. To complete this histomorphological approach, we decided to investigate cell ploidy in testicular cells using propidium iodide as a marker. The successive increase in 4C, then 1C, and thereafter 1C condensed cell populations, may be interpreted as the respective appearance of primary spermatocytes, then round spermatids, followed by elongated spermatids. The smaller proportion of the more-advanced cell population during the first cycle of spermatogenesis in KitW-lacZ/+ mice compared with Kit+/+ mice could be interpreted as a delay in the beginning of spermatogenesis or as a lower rate of spermatogenesis. Our comparative histomorphological data did not show any disruption in spermatogenesis staging that might be expected with an alteration in the rate of spermatogenesis. Thus a delay in initiation of spermatogenesis cannot be excluded. Whether this delay might be the consequence of germ cell depletion in the progression of spermatogenesis or whether it might partly involve the Kit gene remains unknown. Despite the similar ploidy distribution that was observed between mice of both genotypes at 45 days, these are proportions of testicular cells and not absolute numbers. In fact, both genotypes clearly differed in their spermatogenesis efficiency at this age as confirmed by epididymal sperm counts. Moreover, the 1C population at 5 mo was smaller in KitW-lacZ/+ mice than in Kit+/+mice, which also argues for an ongoing involvement of Kit deficiency in later ages.

Our observation of a wave of apoptosis between 8 and 45 days of age corroborates previous reports of an increase in apoptosis during the first cycle of spermatogenesis [3, 4]. However, our model provides 2 additional results. The first is the greater increase in apoptosis observed in KitW-lacZ/+ mice compared with Kit+/+mice during the first cycle of spermatogenesis. The greater increase in apoptotic index was observed at 13 days of age, meaning that meiosis had begun, because leptotene, zygotene, and young pachytene spermatocytes were present. The cellular targets of the apoptotic process were both spermatogonia and meiotic spermatocytes. The second result is the significantly higher apoptosis index at later ages in adult KitW-lacZ/+ mice. This might be linked to the histomorphological findings showing increasing differences in spermatogenesis between adult mice of both genotypes with time. This was confirmed for testis weight, seminiferous tubule diameter, and type B/type A stem spermatogonia ratio. Moreover, this ongoing apoptotic process may at least in part explain the presence of some agametic tubules deprived in haploid cells with increasing age in KitW-lacZ/+ mice compared with Kit+/+ mice. The presence of agametic tubules has been reported in homozygous Kit mutant mice (KitWf/Wf mice) and was linked to a deficiency in primordial germ cells [52]. However, in our study, seminiferous tubule involution might be interpreted as a progressive depletion in meiotic cells, because agametic tubules were deprived in haploid germ cells. The precise mechanism, which might associate germ cell apoptosis with Kit gene expression, is not fully understood. Various reports have focused on the role of the kit gene in preventing apoptosis of melanocytes [53, 54] and hematopoietic progenitor cells [55]. Recent results have been reported on the p53 dependency of germ cell apoptosis in Kit deficient mice using double-deficient KitW-v/W-v:Trp53-/- mice [56].

Our model gave us the opportunity to follow the expression of the Kit gene, using lacZ reporter gene expression. Use of the lacZ reporter gene has been fully established for gene expression studies, without phenotypic consequences [57, 58]. Moreover, it has already been reported that gonadal expression of the lacZ reporter gene does not impair spermatogenesis in mice [59]. Indeed, the lacZ gene is under the control of the Kit gene promoter [43]. The greater increase in ß-galactosidase activity in whole testis was observed from 24 days onward, when meiosis occurs. Moreover, the in situ approach to ß-galactosidase expression gave additional information, because in addition to expression in spermatogonia, a clear and distinct expression was observed in late pachytene/diplotene spermatocytes, whereas no staining was observed in earlier spermatocyte stages. Such results only partly agree with previous observations reporting Kit mRNA expression using in situ hybridization from type A2 to preleptotene spermatocytes [23]. The latter study focused on the transcriptional level of Kit gene expression, whereas our report involved the translational level using a reporter gene with increased sensitivity of detection. It could be hypothesized that insertion of the reporter lacZ gene in the Kit gene might modify some elements of translational control and RNA stability, thus altering protein expression. However, the expression of ß-galactosidase at two distinct stages of spermatogenesis (type A differentiated spermatogonia and pachytene/diplotene spermatocytes) without any expression at the beginning of meiosis does not support such a hypothesis. Because this reporter gene remained under the control of the major promoter of the gene, we may assume that late pachytene spermatocytes were able to express the longer transcript of the Kit gene rather than the shorter one, which is under the control of a cryptic promoter [60]. Moreover, our results are in agreement with the in vitro observation of Kit expression in pachytene spermatocytes [24]. Although in vitro observations may suggest the possible involvement of KIT receptor in an ongoing meiotic process, the precise level and the signal transduction pathway remain to be demonstrated.

Finally, the severe impairment of spermatogenesis in KitW-lacZ/+ mice compared with Kit+/+ mice demonstrated in this study suggests that a single copy of the Kit gene is not sufficient for maintaining genuine spermatogenesis in mice, as haplodeficiency in the Kit gene is the sole genotypic difference between KitW-lacZ/+ and Kit+/+ 129/Sv mice. In our model, germ cell apoptosis was increased during both the first cycle of spermatogenesis and during adult spermatogenesis. This should have real consequences on the fertility of KitW-lacZ/+ mice compared with Kit+/+ mice. Given the drastic reduction in sperm numbers, preliminary results seem to confirm that the fertility of KitW-lacZ/+ male mice is compromised, without any impairment in sexual behavior. Whether the decrease in litter size may be related to sperm deficiency in KitW-lacZ/+ or Kit defective expression in some embryos is unclear. In vivo and in vitro studies are now in progress to characterize the fertility impairment in KitW-lacZ/+ mice. The decrease in type B/type A spermatogonia is related to the well-established role of Kit at one crucial stage of spermatogonia differentiation. The expression of Kit at one stage of meiosis raises the question of its biological role independently of its known action on cell survival.


   ACKNOWLEDGMENTS
 
We acknowledge Claude Cahier and Michel Vigneau for their help in caring for 129/Sv mice and facilities, Christine Perreau and Genevieve Chauveau for their technical assistance in histological studies, Dominique Kerboeuf and Yves Le Vern for their help in the management and analysis of cytofluorimetric studies, and the staff of the assay laboratory for their technical help with testosterone assays. Doreen Raine is kindly acknowledged for assisting with the English text.


   FOOTNOTES
 
First decision: 24 October 2001.

1 This work was supported by the French Ministry of Education and Scientific Research, the National Institute for Agricultural Research, and the National Centre for Scientific Research as part of UMR 6073. V.C. held a grant from Organon Research Foundation (FARO, France). Back

2 Correspondence: D. Royere, Biologie de la Reproduction, Centre Hospitalier Universitaire Bretonneau, 37044 Tours Cedex, France. FAX: 33 02 47 47 84 99; royere{at}med.univ-tours.fr Back

Accepted: January 15, 2002.

Received: September 26, 2001.


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