CCR4-associated factor CAF1 is an essential factor for spermatogenesis

Abstract

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.

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.

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.

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, ×200); in the presence of purified polyclonal antibody anti-CAF1 (A2, ×200), or in the presence of CAF1 antibody plus the antigenic peptide (A3, ×200). Immunolocalization of mCAF1 protein in the testis of a mouse fetus at 15.5 days post coitum (B1, ×200); in adult mouse testes (B2 and B3, ×630); in the Müller (M) and Wolfian ducts (W) of mouse testis mesonephros at 15.5 days postcoitum (C1, ×630); and in epididymis from 4-day-old (C2, ×100) and 40-day-old (C3, ×400) mice.

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.

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, ×100; D, ×1,000; G, ×1,000) (left) and mutant testes (B, ×100; C, ×100; E, ×1,000; F, ×1,000; H, ×1,000; I, ×400) (right). Four-month-old wild-type mouse epididymis (J and L, ×1,000) and mutant mouse epididymis (K and M, ×1,000) 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.

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, ×950) were identical to those of wild-type mice (A, ×950). At 19 days, the first vacuoles and disorganization appeared in mutant mice testes (D, ×380) compared to the wild type (C, ×380). At 20 days, the first cluster of abnormal tubules was apparent (E, ×190; F, ×190). At 40 days, an important loss of immature germ cells could be observed in the testis (G, ×380; H, ×380). Then, vacuolization, disorganization, and depletion of germ cells increased during mutant mouse aging (1 year) (I and J, ×950). 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, ×950) and mutant testis sections (L, ×950).

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, ×630.

FIG. 8.

Ultrastructural appearance of 3-month-old normal and mCAF1-deficient mouse testicular cells. (A, ×2,850; D, ×2,375; G, ×9,500; J, ×23,750; M, ×38,000) 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, ×1,900; C, ×19,000; E, ×14,250; F, ×71,250; H, ×9,500; I, ×19,000; K, ×11,400; L, ×19,000; N, ×19,500; O, ×95,000). 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.