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Molecular and Cellular Biology, July 2004, p. 5808-5820, Vol. 24, No. 13
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.13.5808-5820.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

CCR4-Associated Factor CAF1 Is an Essential Factor for Spermatogenesis

Cyril Berthet,1,{dagger} Anne-Marie Morera,2 Marie-Jeanne Asensio,2 Marie-Agnes Chauvin,2 Anne-Pierre Morel,1 Frederique Dijoud,3 Jean-Pierre Magaud,1 Philippe Durand,2 and Jean-Pierre Rouault1,2*

UMR INSERM 418-INRA 1245-Université Claude Bernard Lyon I,2 Service d'Anatomopathologie, Hopital Debrousse, F 69322 Lyon Cedex 05,3 INSERM U453, CLB, F 69373 Lyon Cedex 08, France1

Received 8 September 2003/ Returned for modification 16 December 2003/ Accepted 9 April 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The CCR4-associated protein CAF1 has been demonstrated to play several roles in the control of transcription and of mRNA decay. To gain further insight into its physiological function, we generated CAF1-deficient mice. They are viable, healthy, and normal in appearance; however, mCAF1–/– male mice are sterile. The crossing of mCAF1+/– mice gave a Mendelian ratio of mCAF1+/+, mCAF1+/–, and mCAF1–/– pups, indicating that haploid mCAF1-deficient germ cells differentiate normally. The onset of the defect occurs during the first wave of spermatogenesis at 19 to 20 days after birth, during progression of pachytene spermatocytes to haploid spermatids and spermatozoa. Early disruption of spermatogenesis was evidenced by Sertoli cell vacuolization and tubular disorganization. The most mature germ cells were the most severely depleted, but progressively all germ cells were affected, giving Sertoli cell-only tubes, large interstitial spaces, and small testes. This phenotype could be linked to a defect(s) in germ cells and/or to inadequate Sertoli cell function, leading to seminiferous tubule disorganization and finally to a total disappearance of germ cells. The mCAF1-deficient mouse provides a new model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The yeast protein yeast CAF1yPOP2 (CCR4-associated factor) belongs to the CCR4-NOT complex (24). These proteins were initially characterized through genetic analyses in Saccharomyces cerevisiae. This complex regulates a number of genes via several pathways, including control of transcription and mRNA decay (20). This complex has been implicated in the transcriptional control of a variety of genes involved in nonfermentative growth and cell wall integrity. It affects the expression of genes both positively and negatively, apparently through contacts made with the TFIID, the SAGA complex, and SRB9-11 proteins (2, 6, 12, 15, 16, 19, 23, 42-45, 59, 70).

The role of yeast CAF1yPOP2 within this complex, although essential, is not clearly established yet. It may act as a bridge between CCR4 and the NOT proteins (3, 10, 21, 24, 44, 48). Furthermore, yeast CAF1yPOP2 and CCR4 have been shown to be major actors of mRNA decay control. Yeast CAF1yPOP2 associates with DHH1p, a component of a decapping complex, and consequently could be linked to mRNA degradation (34, 47). Moreover, yeast CAF1yPOP2 and yeast CCR4 have been directly involved in RNA metabolism. Yeast CCR4 and the cofactor, yeast CAF1pop2, have been shown to be the major cytoplasmic deadenylases in S. cerevisiae (9, 74-76). Another study has further suggested that yeast CAF1yPOP2 could itself degrade mRNA (17).

It has also been reported that these proteins are implicated in cell cycle control. Yeast CCR4 and yeast CAF1yPOP2 associate with DBF2, a cell cycle kinase implicated in late telophase-G1 phase transition (46). Furthermore, yeast CAF1yPOP2 is phosphorylated on Thr97 by YAK1, a DYRK family kinase, in response to a glucose deprivation signal. When Thr97 is mutated, the resulting yeast strain shows a defect in G1 phase arrest upon glucose depletion (54). These results drew our attention to the fact that the cell cycle, at least in S. cerevisiae, could be regulated by the control of mRNA stability through the CCR4-NOT complex activity.

The proteins implicated in this complex are structurally conserved through evolution, and a homologous multisubunit CCR4-NOT complex has been characterized in mammalian cells (25, 53). Additionally, two homologs of yeast CAF1yPOP2, human CAF1 and human CALIF, were identified in humans (1, 27). Furthermore, we have also demonstrated that mCAF1 associates with all the BTGAPRO/TOB proteins (32, 35, 66, 79). This family of proteins, whose molecular role is poorly understood, is involved in negative control of the cell cycle, since their ectopic overexpression or conversely their elimination provokes cell cycle arrest abnormalities (50, 65, 67, 80). Moreover, BTG2 expression is upregulated by the tumor suppressor proteins P53 and ARF, and BTG2 also transcriptionally represses cyclin D1 (31, 41, 65). From these results, it appears that, in metazoans, CAF1 could contribute to cell cycle arrest mediated by the P53-BTG2 pathway through the CCR4-NOT complex.

CAF1 may be part of an original negative cell cycle control (for instance, from P53 to control of mRNA stability), since the functions elicited by this complex in S. cerevisiae are conserved. Ectopic overexpression of CAF1 is also antiproliferative in mammalian cells (8). mCAF1, like BTG1 and -2, has been implicated as a cofactor in estrogen receptor {alpha}-dependent transcription (62). Although experiments showing that metazoan CCR4 and CAF1 proteins are involved in RNA stability have not been reported, analysis of their sequence indicates that they harbor a putative exonuclease domain (55, 74). The CCR4-NOT-CAF1 complex is likely to achieve a fundamental function in cellular homeostasis. CAF1, a key subunit of this complex, enables its formation. Therefore, through its association with BTGAPRO/TOB proteins, CAF1 could integrate multiple pathways leading to cell cycle arrest. To gain insight into CAF1 function, we generated mutant mice that lack murine CAF1 (mCAF1). In this work, we report that mCAF1-deficient mice are viable but males are sterile.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of mCAF1 targeting vector. Two overlapping mouse genomic clones were isolated from a 129/Sv mouse library of phage {lambda} by standard techniques, spanning 25 kb of DNA and containing the complete mCAF1 sequence (7 exons). A 1.5-kb NheI/XbaI fragment, containing a portion of the 5' untranslated region sequence and beginning of exon 2, was subcloned into pBluescript II-KS+/–. The herpes simplex virus thymidine kinase gene cassette was inserted adjacent to the 5' end of this genomic fragment for the negative selection. Similarly, a 6.8-kb PstI fragment, encompassing exons 5 to 7, was subcloned. Both fragments were transferred into pGNAß. In the final construct, the 3' portion of exon 2 (including the start codon) to exon 4 (amino acids 1 to 206 were deleted according to mCAF1 GenBank accession number U21855) was deleted and replaced with the ß-galactosidase gene and a phosphoglycerate kinase-neomycin resistance cassette.

Generation of targeted ES cells and mCAF1-deficient mice. ENS embryonic stem (ES) cells were electroporated with the linearized targeting vector and selected with G418 (250 µg/ml) and ganciclovir (0.5 µg/ml) as described previously (65). DNA from resistant ES cell clones was digested with BglII or BamHI and analyzed by Southern blotting with a 1.4-kb StuI-SacII or a PstI-HindIII genomic fragment, respectively, as the 5' and 3' probes, located outside of the region used for the targeting vector. We injected two of these clones individually into 3.5-day C57BL/6 blastocysts, and the resulting male chimeras were crossed with wild-type C57BL/6 females to generate mCAF1+/– offspring. The genotype of agouti pups was determined by Southern blotting of tail DNA as described above. Subsequent genotyping was done by PCR with two wild-type mCAF1-specific primers (5'-TTTCTGTTTGGGCAGGGACCGTT-3' and 5'-TCTTGCAACAACGCCTGGAAACTC-3') and two primers specific for the targeted mCAF1 allele (5'-GAGTGAATTGAACTCGGAGCAAATCT-3' and 5'-CCTCTTCGCTATTACGCCAGCTGG-3').

Histology. mCAF1-deficient and wild-type mice were killed by CO2 asphyxiation. Several tissues, including endocrine organs, were frozen in liquid nitrogen and stored at –80°C or fixed in Bouin solution for 24 h or in AFA fixative for 4 h at room temperature, then dehydrated and embedded in paraffin; 5-µm sections were transferred to microscope slides and stained with hematoxylin, eosin, and saffron or the periodic acid-Schiff reagent to better identify germ cell nuclei and counterstained with Harris hematoxylin.; 10-µm sections of unfixed frozen testes were cut with a cryostat microtome (HM560 Microm) and collected on silane-coated slides. Lipids were stained with the lipid-soluble dye oil red O (Sigma), osmium, or Soudan III in isopropanol-H2O (60%) for 10 min at room temperature. After nuclear counterstaining, the sections were rinsed in tap water and mounted in Permafluor.

Immunohistochemistry. Paraffin sections (5 µm) of 4% paraformaldehyde-fixed mouse tissues were mounted on silane-coated objective slides. Immunocytochemical localization of mCAF1 was performed with a polyclonal CAF1 antiserum. The antibodies directed against human CAF1 were raised in a rabbit by immunization with a specific synthetic peptide (amino acids 242 to 254, REMFFEDHIDDAK) and immunopurified by affinity chromatography against the CAF1 protein. Slides were dewaxed, and endogenous peroxidase activity in tissue sections was blocked with 3% hydrogen peroxide (vol/vol) for 10 min at room temperature; then the slides were treated with 0.1% Triton for 10 min. After washing, tissues were incubated with CAF1 antibody diluted at 1:100 in antibody diluent with background reducing components (Dako) for 1 h at room temperature. After several washes with optimal wash buffer (Biogenex), antibody binding to mCAF1 was detected with the horseradish peroxidase-conjugated anti-rabbit immunoglobulin secondary antibody Envision (Dako) for 30 min at room temperature. The immunoreaction was revealed in a mixture containing 0.02% 3,3'-diaminobenzidine (Vector Laboratory) and 0.002% H2O2 in imidazole-HCl buffer, pH 7.5. The slides were then washed in water for 10 min to stop the reaction, counterstained with Harris hematoxylin, dehydrated, and mounted in Clearium (Surgipath). Controls were performed by suppressing the first or second antibody or by saturating CAF1 antibody with the antigenic peptide. Immunolocalization of vimentin was done with monoclonal vimentin antibody 13.2 LN6 from Sigma; 5-µm slides of AFA (acetic acid, formol, ethanol, water, 5:12:75:18)-fixed mouse testes were incubated with vimentin antibody diluted at 1:50 in antibody diluent (Dako) for 1 h at room temperature, and anti-mouse immunoglobulin secondary antibody Envision (Dako) was added for 30 min at room temperature. The immunoreaction was revealed with 3-amino-9 ethylcarbazole from Dako.

Electronic microscopy. Adult testes were fixed with 2% glutaraldehyde for 15 min at room temperature and in 4% glutaraldehyde plus 0.2 M cacodylate buffer (vol/vol) for 15 min. After washing in the same buffer, the tissues were cut into small pieces, immersed in the same fixative for 2 h at 4°C, rinsed with 0.2 M cacodylate plus 0.4 M (vol/vol) saccharose at 4°C, and then fixed in OsO4. Thereafter, the samples were dehydrated through a graded ethanol series and then embedded in Epon. Ultrathin sections were cut on an ultramicrotome (model MTX) and stained with uranyl acetate and lead citrate. Slides were visualized with a JEM-1200 EX transmission electron microscope.

Leydig, Sertoli, and germ cell preparations. The interstitial cells removed by collagenase-dispase (0.05%) digestion of 20-day-old mouse testes are referred to as the Leydig-enriched fraction. This fraction was subjected to Percoll gradient separation to increase the purity of the Leydig cell preparation. Sertoli cells were prepared from 20-day-old wild-type mice by successive collagenase treatments. Enrichment was close to 90% (contamination essentially by peritubular myoid cells). Adult germ cells were isolated from 3-month-old mCAF1+/– mouse testes by trypsinization (52). The resulting crude germ cell population (containing germ cells from all developmental steps) was subjected to centrifugal elutriation with a Beckman JE-6 rotor (Beckman, Palo Alto, Calif.) as described before (51). Two fractions were harvested, the pachytene spermatocyte fraction (enrichment of 95%) and the early spermatid fraction (enrichment of 80%). After collection, the different cell populations were processed for RNA extraction.

Western blot analysis. At the time of sacrifice, testes were immediately frozen at –180°C, homogenized at 4°C in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.25% sodium dodecyl sulfate) plus antiprotease complete (1 tablet/10 ml of RIPA) centrifuged at 10,000 xg at 4°C for 30 min. Proteins in the supernatant were measured by the BCA protein assay (Interchim) and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12% acrylamide gel) and electrophoretically transferred to nitrocellulose membranes. After treatment with a blocking solution (5% nonfat milk in Tris-buffered saline) for 3 h, mCAF1 was detected by incubation of the membrane overnight at 4°C with a polyclonal rabbit anti-human CAF1. The membrane was washed and incubated with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody (Envision; Dako) (1:25 in Tris-buffered saline). Immunoreactive proteins were visualized by the enhanced chemiluminescent detection system (Amersham Pharmacia Biotech).

TUNEL. For terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), testis was immersion fixed in 4% paraformaldehyde-phosphate buffer at room temperature. The fixed tissues were embedded in paraffin and processed for detection of apoptotic cells (5); 5-µm sections were treated with 20 µg of proteinase K per ml in water for 15 min at room temperature, and then DNA free ends were labeled with the addition of fluorescein dUTP at strand breaks by terminal deoxyribonucleotidyltransferase. Slides were analyzed by fluorescence microscopy (Zeiss).

RT-PCR. Total cellular RNA was isolated from fresh and frozen tissues with the guanidium thiocyanate method (13). First-strand cDNA synthesis was carried out in 30 µl of reaction mixture containing 1 µg of total RNA, 6 µl of random hexamers (50 µM), 6 µl of 100 mM dithiothreitol (DTT), and 600 U of Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen). The reverse transcriptase-negative control was performed without the addition of reverse transcriptase. For PCR, 2 µl of the reverse-transcribed cDNA template (2 µg) was added to a final volume of 20 µl of reaction buffer containing 1.5 mM MgCl2, 50 mM KCl, 0.2 mM deoxynucleoside triphosphates, 15 pM each primer, and 0.5 U of Taq polymerase in 3 µl of Master Taq polymerase enhancer (Eppendorf). The primers for mCAF1 were mCAF1A, 5'-ATGCCAGCAGCAACCGTA-3', and mCAF1B, 5'-AAGAAGACTATTTCCTGTCATG-3'. After an initial denaturation at 95°C for 4 min, the reaction was subjected to 30 cycles at 95°C for 30 s, 50°C for 30 s, and 72°C for 60 s. The hypoxanthine phosphoribosyltransferase gene was used as a control, 5'-CCTGCTGGATTACATTAAAGCACTG-3' and 5'-TCCACCTTCTCCAACTTCACGG-3'. The sizes of the amplicon produced were 900 and 340 bp for mCAF1 and hypoxanthine phosphoribosyltransferase, respectively.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Targeted disruption of the mouse CAF1 gene. The mouse CAF1 gene, which encompasses seven exons, was mutated by replacing coding exons 3 and 4 and part of exon 2 with a lacZ-neo cassette (Fig. 1A). About two-thirds of the mCAF1 protein, from the initiation codon to the {alpha}-helix-9 region according to the crystal structure and including the main RNase catalytic site, was thus deleted (74). ES cells (ENS26) were transfected with the resulting construction, and two independent properly recombined clones were chosen for blastocyst (C57BL/6) microinjection. These clones were checked for the absence of visible chromosomal abnormalities and randomly integrated exogenous vector DNA. Germ line transmission was observed, and offspring from matings between chimeric mice (C57BL/6) were intercrossed in order to generate mice of different mCAF1 genotypes (+/+, +/–, and –/–) (Fig. 1B and C).



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  		FIG. 1. mCAF1 targeting in ES cells and generation of mCAF1–/– mice. (A) Genomic organization and disruption strategy for mCAF1, showing the gene, the targeting construct, and the recombined mCAF1 allele. (B) Southern blot analysis of the resulting DNAs. (C) Immunoblot analysis of mCAF1 protein in cellular extracts from different mCAF1 genotypes.

 
mCAF1–/– male phenotype. Both male and female mCAF1–/– mice were viable and apparently healthy. The offspring of mCAF1+/– intercrosses were born at the normal Mendelian frequency (22.9% mCAF1+/+, 55.6% mCAF1+/–, and 23.1% mCAF1–/–), and 50.8% of mCAF1–/– offspring were males (total, 627). The growth of mCAF1–/– pups from birth to adulthood was indistinguishable from that of their wild-type littermates (data not shown). The mCAF1–/– mice exhibited no significant abnormalities in external appearance, and histological examination of young mutant mice (3 months) showed that the majority of tissues were normal (lung, liver, kidney, heart, pituitary gland, and ovary) (data not shown) except for the male gonad, which displayed pathological features. The mCAF1–/– males were completely infertile despite apparently normal sexual behavior: vaginal copulatory plugs were seen when they were crossed with females. mCAF1 was apparently dispensable for normal development and the function of most organs but essential for male fertility. During fetal, postnatal, and prepubertal life, the testes developed normally, and external and testicular descent appeared normal. The testis weights of normal and mutant mice were similar in prepubertal animals. By contrast, the testis weights of adult (8 months) mCAF1–/– mice were lower (38.5 ± 16 mg, mean ± standard deviation) than those of controls (96.3 ± 20 mg). However, there was no significant difference in seminal vesicle development (442 ± 23 mg versus 423 ± 27 mg) or in the epididymis (37.5 ± 9.6 mg versus 40 ± 6 mg) (Fig. 2). In contrast to mCAF1–/– male mice, heterozygote males exhibited normal fertility.



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  		FIG. 2. Mutant reproductive tract phenotype. Anatomy of the reproductive tract of a 4-month-old wild-type male mouse (right) and homozygous mutant CAF1 mouse (left). T, testis; SV, seminal vesicle; B, bladder; E, epididymis. Testes from mutant (left) and wild-type (right) mice are shown in the inset.

 
mCAF1 expression in testis. To facilitate the interpretation of the mCAF1–/– phenotype, we sought to determine mCAF1 expression in the testis by RT-PCR and immunochemistry. Immunohistological detection of mCAF1 was performed with a polyclonal antibody (Fig. 3). Although staining of mCAF1 was not very intense, the testes of mice at 15.5 days post coitum (Fig. 3B1) were distinctly stained, indicating that they expressed mCAF1 very early in the differentiated male gonad. Moreover, the immunological expression of mCAF1 persisted in the testis of young postnatal, prepubertal (data not shown), and adult male mice (Fig. 3A2, B2, and B3). All the cells of the adult testis were immunoreactive for mCAF1 (Fig. 3A2 and B3). The staining appeared cytoplasmic (Fig. 3B2 and B3) and was greatly decreased when the anti-CAF1 antibody was preincubated with the antigenic peptide (Fig. 3A3). Immunostaining with CAF1 antibody indicated the presence of mCAF1 in epididymis epithelium (Fig. 3C2 and C3). Besides, staining of the Wolfian duct at 15.5 days post coitum could be observed in the wild-type mouse fetus (Fig. 3C1).



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  		FIG. 3. Immunohistochemical localization of mCAF1. Localization of mCAF1 was studied in the testes of young adult wild-type mice (A). Testis sections were incubated in the absence of CAF1 antibody (A1, control, x200); in the presence of purified polyclonal antibody anti-CAF1 (A2, x200), or in the presence of CAF1 antibody plus the antigenic peptide (A3, x200). Immunolocalization of mCAF1 protein in the testis of a mouse fetus at 15.5 days post coitum (B1, x200); in adult mouse testes (B2 and B3, x630); in the Müller (M) and Wolfian ducts (W) of mouse testis mesonephros at 15.5 days postcoitum (C1, x630); and in epididymis from 4-day-old (C2, x100) and 40-day-old (C3, x400) mice.

 
The pattern of expression of mCAF1 RNA determined by RT-PCR (Fig. 4A) indicated that it was expressed mainly in the testis and the epididymis and at a relatively high level in the brain, pituitary gland, and kidney. In other tissues such as the liver, heart, lung, and thymus, expression was lower. Moreover, the purification of adult Leydig cells, Sertoli cells, and total germ cells and the separation of distinct subpopulations of germ cells (pachytene spermatocytes and round spermatids) by elutriation showed that all these different testicular cells expressed mCAF1 at similar levels (Fig. 4B). Furthermore, mCAF1 was expressed in highly purified Sertoli and Leydig cells at 19 days. Testes obtained from mice at different ages (10, 15, 20, 30, 45, 60, 90, and 120 days) showed the presence of mCAF1 mRNA at every age tested (Fig. 4C). Western blot analysis confirmed these results (data not shown). Our results indicate that mCAF1 is highly expressed in the testis. In normal mice, mCAF1 is expressed very early during testis development, and its level remained similar between 10 and 120 days post partum.



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  		FIG. 4. Expression of mCAF1 mRNA in wild-type mice. (A) Adult wild-type tissues. (B) Testes of adult wild-type mice (left) and prepubertal wild-type mice (right). L, Leydig cells; G, total germ cells; P, pachytene spermatocytes; RS, round spermatids; S, Sertoli cells. (C) Total testis during aging. HPRT, hypoxanthine phosphoribosyltransferase.

 
Histological examination of mCAF1–/– testis. Histological examination of aged mutant adult male mouse testes (12 months) revealed the presence of severely damaged and small seminiferous tubules (Fig. 5B), round vacuoles (Fig. 5F) located mainly at the periphery of a great number of seminiferous tubules (Fig. 5B), and signs of apical germ cell sloughing and shedding (Fig. 6H). The total number of elongating spermatids was decreased, and mature sperm was rare or even absent (Fig. 5B and I). Cells in the seminiferous tubules were disorganized, lacking the characteristic basal to luminal maturation of germ cells (Fig. 5B, E, and H). Some seminiferous tubules of mutant mice were found with no germ cells and progressive disappearance of Sertoli cells could even be observed in some tubules (Fig. 5I and next paragraph). The interstitial tissue seemed enlarged with a number of Leydig cells (Fig. 5A and C).



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  		FIG. 5. Histological comparison of testis (A to I) and epididymis (J to M) of a wild-type mouse (+/+) and mCAF1-deficient mouse (–/–). One-year-old wild-type mouse testes are presented in the upper part of the figure (A, x100; D, x1,000; G, x1,000) (left) and mutant testes (B, x100; C, x100; E, x1,000; F, x1,000; H, x1,000; I, x400) (right). Four-month-old wild-type mouse epididymis (J and L, x1,000) and mutant mouse epididymis (K and M, x1,000) are shown in the lower part. PM, peritubular myoid; S, Sertoli cells; B, basal membrane; RSP, round spermatid; ESP, elongated spermatid; V, vacuole; L, Leydig cells; Sy, symplast; Spz, spermatozoa; ST, seminiferous tubule.

 


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  		FIG. 6. Ontogeny of spermatogenic defect in mCAF1–/– mice. Sections of wild-type (+/+) and mCAF1–/– (–/–) testes at different stages of spermatogenic development were stained with hematoxylin, eosin, and saffron. At 8 days post partum, testes from mCAF1–/– (B, x950) were identical to those of wild-type mice (A, x950). At 19 days, the first vacuoles and disorganization appeared in mutant mice testes (D, x380) compared to the wild type (C, x380). At 20 days, the first cluster of abnormal tubules was apparent (E, x190; F, x190). At 40 days, an important loss of immature germ cells could be observed in the testis (G, x380; H, x380). Then, vacuolization, disorganization, and depletion of germ cells increased during mutant mouse aging (1 year) (I and J, x950). Mutant Sertoli cells were characterized by the presence of vimentin (I, control for immunohistochemistry; J, immunostaining with antivimentin). The basal membrane was localized by Masson's trichrome coloration of collagen (blue) in wild-type testis sections (K, x950) and mutant testis sections (L, x950).

 
In contrast to the young adult wild-type epididymis (Fig. 5J and L), those of mCAF1–/– mutants (Fig. 5K and M) contained only sparse elongated or shaped nuclear profiles characteristic of differentiated spermatozoa along with immature and malformed cells and unidentified cellular debris (Fig. 5K). Viscous luminal fluid was alcian blue positive, suggesting the presence of a high level of mucous polysaccharide in mCAF1-deficient mouse epididymal fluid (data not shown). The few spermatozoa present in the deferent duct of young adult mutant mice remained immobile when suspended in phosphate-buffered saline (even if diluted to lower the viscosity of the epididymal fluid), whereas under the same conditions, almost all spermatozoa removed from the wild-type caudal epididymis became highly mobile.

Furthermore, examination at higher magnification of epididymis epithelium from the mCAF1 mutant (Fig. 5M) also revealed the presence of double-nucleated cells (Fig. 5L).

Chronology of testicular defects in mCAF1–/– mouse testis. The severe pathological observations in mCAF1–/– adult testes prompted examination of earlier stages of postnatal development. In the mouse, spermatogenesis begins neonatally, and during the first wave of germ cell differentiation, this process is rather synchronized (68). In sexually immature mice at 8 days of age, seminiferous tubules of mCAF1–/– mice were comparable in both size and morphology to those of their wild-type littermates (Fig. 6A and B). Similarly, examination of the testes at days 15 and 17 did not reveal abnormal histology in mCAF1–/– testes (data not shown). Histological abnormalities were first detected at day 19 in mCAF1–/– testes (Fig. 6C and D) and consisted in a few tubules with vacuoles. Therefore, the first abnormalities were seen at the time of the meiotic divisions. By 20, 21, and 22 days of age when germ cells in wild-type mouse testes progressed to the haploid stage, the seminiferous tubules of mCAF1–/– mice showed reduced numbers of germ cells (Fig. 6F).

The tubular pathology worsened progressively, and at 40 days, a massive loss of immature germ cells could be observed in the seminiferous tubules of mutant mice (Fig. 6H). Pachytene spermatocytes persisted during the first months of life in a great number of tubules despite a progressive loss of round spermatids (Fig. 6F and H). These observations localized the onset of the spermatogenetic defect preferentially at the stage when pachytene spermatocytes progress to spermatids. Indeed, these mCAF1 mutant adult mice presented an increased number of TUNEL-positive spermatids (Fig. 7). However, the classical figures of nuclear fragmentation were not seen in these cells. Testis abnormalities increased with the age of the mouse. At 1 year, we observed a great number of Sertoli cell-only tubules (Fig. 5I and 6J), showing that spermatocytes and spermatogonia were also progressively affected and deleted. One year-old mutant mice testes presented (i) a great number of symplasts of degenerating elongated spermatids (Fig. 5H) and (ii) tubules containing only Sertoli cells as confirmed by vimentin immunostaining (Fig. 5I and 6J). In 18-month-old animals, the number of Sertoli cells was even more drastically affected (2.6-fold less in the mutant compared to the wild type). The tubular membranes gradually become thickened, with an increased number of peritubular myoid cells as evidenced by alpha-smooth muscle actin immunostaining (data not shown). Basal membrane components infiltrated between Sertoli cells in the tubules as distinctly shown by trichrome coloration (Fig. 6L).



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  		FIG. 7. TUNEL assay. Two-month-old mouse testis was fixed in 4% paraformaldehyde and processed for detection of apoptotic cells by the TUNEL method. DNA free ends were labeled with fluorescein dUTP (A, mCAF1+/+; B, mCAF1–/–). Magnification, x630.

 
Electron microscopy of adult testis. To better understand the spermatogenic defect in mutant testicular cells, electron micrographs of 3-month-old mCAF1–/– testes were done (Fig. 8). Several unusual features were observed. Numerous necrotic figures were present in all testicular cell types. The cytoplasm of Sertoli cells appeared impaired, with large vacuolization surrounded by endoplasmic reticulum membrane, and numerous figures of phagocytosis could be observed (Fig. 8H, I, K, and L). In testes from mCAF1–/– mice, intercellular spaces were observed, suggesting a disruption of the junction between testicular cells (Fig. 8E and F). Moreover, pachytene spermatocytes presented some defects in their nuclear membrane creating a dissociation between external and internal membranes and intra nuclear vacuoles (Fig. 8B). Numerous spermatozoa were phagocytized by Sertoli cells (Fig. 8H, I, and L). Figure 8L shows defects in chromatin condensation. Abnormal localization of outer dense fibers was also observed (Fig. 8O). Cross sections of axonema of wild-type mouse spermatozoa showed the typical organization constituted of nine microtubular doublets surrounding two central microtubules (Fig. 8M). This organization was partially destroyed in mutant mice axonema (Fig. 8N and O), and the sheath surrounding the axonema was impaired, with numerous destructions of the external membrane.



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  		FIG. 8. Ultrastructural appearance of 3-month-old normal and mCAF1-deficient mouse testicular cells. (A, x2,850; D, x2,375; G, x9,500; J, x23,750; M, x38,000) represent sections of wild-type mouse testis. A, normal interstitial compartment; D, normal pachytenes; G, normal elongated spermatids; J, longitudinal section of normal spermatozoa flagella; M, transverse section of normal flagella. The other panels represent sections of young adult mutant mouse testis (B, x1,900; C, x19,000; E, x14,250; F, x71,250; H, x9,500; I, x19,000; K, x11,400; L, x19,000; N, x19,500; O, x95,000). B, abnormal interstitial compartment and vacuole in pachytene nucleus; C, excess of membrane in peritubular myoid; E and F, dysjunction in plasma membrane; H and I, phagocytosis of degenerated elongated spermatids and spermatozoa; K, excess of endoplasmic reticulum; L, degenerate elongated spermatids; N and O, transverse section of spermatozoal flagella with abnormal membrane.

 
Furthermore, the absence of classical nuclear fragmentation observed by TUNEL detection (Fig. 7) was confirmed by transmission electronic microscopy. The degenerate testicular cells lacked characteristic features of apoptosis. This is consistent with a phagocytic activity of the defectuous germ cells and numerous figures of necrosis and membrane autolysis.

Finally, in young adult male mutant mice, a great number of Leydig cells presented a cytoplasm containing numerous large mitochondria with tubular cristae, abundant smooth endoplasmic reticulum, and numerous lipid droplets characteristic of functional secreting Leydig cells, although several Leydig cells presented signs of necrosis (Fig. 8B). Testosterone concentrations measured by radioimmunoassay in mutant adult mouse blood were not significantly different from that of normal mice (data not shown). The peritubular cells seemed slightly affected, since the membrane appeared thickened with an excess of endoplasmic reticulum (Fig. 8C). Thus, electronic microscopy revealed that the abnormalities induced by mCAF1 suppression were associated with a defect in membrane formation and/or turnover.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
By gene targeting, we have demonstrated an essential role for mCAF1 during spermatogenesis. Testosterone levels and copulatory function were not perturbed in mCAF1–/– mice, whereas these homozygote mutant males were sterile; this should indicate a primary spermatogenic defect.

The observation that mCAF1+/– males were fertile and gave a Mendelian ratio of mCAF1–/–, mCAF1+/–, and mCAF1+/+ offspring when crossed with mCAF1+/– females indicates that haploid mCAF1 spermatids can differentiate normally and give normal spermatozoa. This demonstrates that the absence of mCAF1 in haploid cells does not prevent the final step of spermatogenesis, leading to fertile spermatozoa, as long as Sertoli cells heterozygous for mCAF1 support these cells and/or these haploid germ cells are issued from diploid heterozygous precursors. This result might suggest that the primary defect is located in Sertoli cells, but a primary defect of diploid germ cells cannot be excluded. Indeed, the localization and rather constant expression levels of mCAF1 in both Sertoli and germ cells during development are indicative of either a direct (in germ cells) or an indirect (via Sertoli cells) role of mCAF1 in the normal formation of spermatozoa.

In wild-type mice, the first wave of spermatogenesis begins shortly after birth; the first meiotic divisions occur at about postnatal day 20, and at day 35 the first spermatozoa are produced (51). In mutant mice the first abnormalities (vacuolization in seminiferous tubules) were observed at day 19 postpartum, and in young adult knockout mice the defects reached the elongating spermatids. Early steps of spermatogenesis were present in prepubertal mutant mice, whereas loss of immature germ cells was observed later on in mCAF1–/– adult male mice. In these mice, Sertoli cells could be affected either directly by the absence of mCAF1 or indirectly by the degenerating spermatids, since it has been shown that defective spermatids can release protamines, which are known to exert deleterious effects on epithelial cells (61). In this case, the disruption of Sertoli cell function would appear to be a secondary consequence.

The TUNEL data indicated that mCAF1 deficiency resulted in germ cell apoptosis mainly at the spermatid stage in young adult mice. However, the classical figures of nuclear fragmentation were not seen in these cells, as observed in Bclw-deficient mice, which have an impairment of spermatogenesis with several similarities to that of mCAF1–/– mice (69). However, Bclw expression was not modified in our case (data not shown). Autophagy could represent an alternative pathway for the degradation of cellular components. In this process, portions of the cytoplasm are engulfed within double-membraned vesicles. Autophagy involves dynamic rearrangement of cellular membranes (40), as observed by electron microscopy in mCAF1–/– testicular cells. Finally, enlargement of the interstitial compartment and basal membrane thickening were observed in mutant testes, but they represent a general feature of seminiferous tubule degeneration (68).

We compared our findings with those reported for mice having spermatogenic defects elicited by disruption of different genes. mCAF1–/– mice did not exhibit a stage-specific arrest of spermatogenesis as generally observed in mutant mice in which male sterility is due to a germinal cell primary defect (7, 14, 22, 39, 49, 57, 58, 60, 64, 72, 77). The absence of a stage-specific defect in mCAF1–/– pathology would thus suggest a defect in seminiferous tubule homeostasis. Indeed, nonspecific damage to germ cells is generally observed when Sertoli cell functions are altered (38, 78).

The fact that vacuolization of Sertoli cells preceded degeneration of germ cells could suggest that dysfunction of the Sertoli cells is the primary defect in mCAF1–/– mice. In the same manner, Sertoli cell toxicants typically induce an irreversible testicular atrophy (4), with vacuolization of the Sertoli cell cytoplasm, apical germ cell sloughing or shedding, and death, followed by the disappearance of all categories of germ cells. It seems possible that it is the progressive loss of Sertoli cell function, in aging mutant mCAF1–/– mice, which is the cause of immature germ cell depletion.

Spermatogenesis proceeds in an appropriate environment provided by Sertoli cells. However, germ cells also regulate the biological activities and gene expressions of Sertoli cells (36, 71). Recent data suggest that the cycle and wave of the seminiferous epithelium originate early during fetal development, and their maintenance in the early postnatal cords depends essentially on the somatic cell lineages. After this period, it is likely that precise orchestration between all the cells in the testis is needed to ensure optimal spermatogenesis (73). Moreover, some proteins involved in germ cell DNA repair have a role in maintaining genome stability in somatic cells (30).

Numerous testicular genes are regulated by interactions involving germ cells and Sertoli cells (73). Differentiation of germ cells and their translocation from the basal to the adluminal compartment of the seminiferous tubules requires a complex and well-regulated network of cellular interactions. Junctional proteins could be considered an example of cell-cell interaction between somatic cells or somatic and germ cells. Hence, disruption of testicular junctions can induce male sterility (29, 37). It has been proposed that junctional proteins may be internalized by endocytosis and recycled to the new site of junction during germ cell translocation (26). More recently, another mechanism involving a dynamic system of protease and antiprotease actions has been proposed for germ cell translocation (28, 56). Electron microscopy studies showed that mCAF1–/– mouse testicular cells present abnormalities of the cellular membrane which could be the cause of some defects in cellular junctions and induce the disorganization of the seminiferous tubule architecture, with concomitant premature release and degeneration of developing germ cells. In addition, we observed that mCAF1 mutant mice present an aberrant localization of gap and tight junction proteins connexin 43 and claudin 11, respectively (unpublished data).

Hence, modification of the intercellular communications between Sertoli cells and/or Sertoli and germ cells might be linked with the spermatogenic impairment, but these results do not tell us what the primary defect is.

Since CAF1 is associated with all the BTGAPRO proteins, it is worth noting that FOG-3, the orthologous BTGAPRO gene in Caenorhabditis elegans is also implicated in the control of spermatogenesis. FOG-3 is required for germ cells to initiate spermatogenesis (11). BTG1, which is also expressed in the Sertoli cells and the germinal cells of the rat (63), has been implicated in the control of differentiation (50). Therefore, CAF1 could play a major role through the functions mediated by BTG1 (50), and this pathway could be important in the Sertoli cell and germ cell differentiation processes. The control mechanism in both models remains to be established, but CAF1–/– testicular cells could constitute an interesting tool with which to study this pathway.

Human male infertility is estimated to affect 5% of the male population, and approximately 40% of cases are idiopathic (33). Gene-knockout studies in mice have identified autosomal genes involved in spermatogenesis, and some of them have been implicated in human pathologies. However, these genes account for only a small proportion of inherited spermatogenic defects. The majority of infertile human males have no other identifiable phenotypic abnormalities in somatic organs. Thus, it appears that the study of genes that have essential functions confined to the testis (i.e., CAF1) is of potential interest for human pathology. So is this mCAF1 mutant mouse, since mCAF1 is highly homologous to its human counterpart. Consequently, mCAF1 mutant mice can be used as a model of genetically determined azoospermia and may be useful for a better understanding of some mechanisms of the spermatogenesis process. The eventual contribution of CAF1 deficiency to human male sterility is under investigation.


   ACKNOWLEDGMENTS
 
We thank Martine Roussel, Tadashi Yamamoto, and Marc Billaud for valuable discussions. We thank Bertrand Pain for continuous support and for the gift of the ENS26 cells. We thank Scott Moorefield and Deborah Hodge for critical reading of the manuscript. We thank Hervé Le Mouellic for the gift of the pGNAß vector and Elisabeth Delolme for technical assistance.

These works were supported in part by grants from the Ligue Nationale Contre le Cancer, comités départementaux du Rhône, de la Saône et Loire et de la Savoie, from ARC (9556). J. P. Rouault is an investigator of the CNRS.


   FOOTNOTES
 
* Corresponding author. Mailing address: UMR INSERM 418- INRA 1245-UCBL, Hop. Debrousse, 29 rue Soeur Bouvier, F 69322 Lyon cedex 05, France. Phone: (33) (0)4.72.38.55.11. Fax: (33) (0)4.78.25.61.68. E-mail: rouault{at}lyon.inserm.fr. Back

{dagger} Present address: National Cancer Institute, Frederick, MD 21702-1201. Back


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Molecular and Cellular Biology, July 2004, p. 5808-5820, Vol. 24, No. 13
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.13.5808-5820.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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