Inactivation of Nxf2 causes defects in male meiosis and age-dependent depletion of spermatogonia

Abstract

In eukaryotes, mRNA is actively transported from nucleus to cytoplasm by a family of nuclear RNA export factors (NXF). While yeast harbors only one such factor (Mex67p), higher eukaryotes encode multiple NXFs. In mouse, four Nxf genes have been identified: Nxf1, Nxf2, Nxf3, and Nxf7. To date, the function of mouse Nxf genes has not been studied by targeted gene deletion in vivo. Here we report the generation of Nxf2 null mutant mice by homologous recombination in embryonic stem cells. Nxf2-deficient male mice exhibit fertility defects that differ between mouse strains. One third of Nxf2-deficient males on a mixed (C57BL/6x129) genetic background exhibit meiotic arrest and thus are sterile, whereas the remaining males are fertile. Disruption of Nxf2 in inbred (C57BL/6J) males impairs spermatogenesis, resulting in male subfertility, but causes no meiotic arrest. Testis weight and sperm output in C57BL/6J Nxf2(-/Y) mice are sharply reduced. Mutant epididymal sperm exhibit diminished motility. Importantly, proliferation of spermatogonia in Nxf2(-/Y) mice is significantly decreased. As a result, inactivation of Nxf2 causes depletion of germ cells in a substantial fraction of seminiferous tubules in aged mice. These studies demonstrate that Nxf2 plays a dual function in spermatogenesis: regulation of meiosis and maintenance of spermatogonial stem cells.

Fig. 1

Inactivation of Nxf2 causes male meiotic arrest in mice of mixed (C57BL/6 × 129) backgrounds. (a) The Nxf2 targeting construct and various Nxf2 alleles. The mouse Nxf2 gene consists of 23 exons and spans a 22-kb genomic region on the X chromosome. In the Nxf2^flox allele, one loxP site is inserted in intron 2 and one in intron 11. Exons 3-11 encode amino acids 44-345. (b) Western blot analysis of adult wild type and Nxf2^-/Y testes. Equal amounts (30 μg) of testis protein extracts were loaded. Three blots were probed with anti-NXF2, anti-NXF1, and anti-β-actin antibodies respectively. The abundance of NXF1 did not differ between Nxf2^-/Y and wild type testes. (c) Testis weight of adult Nxf2^-/Y mice. Adult Nxf2^-/Y males have either small or large testes. The average testis weight ± standard deviation is shown for each group (22 mice each). (d, f) Histological analysis of testes from adult wild type mice. (e, g) Histological analysis of small testes from 4-month-old Nxf2^-/Y mice reveals meiotic arrest. Arrows indicate normal chromosome segregation in wild type anaphase I spermatocytes (f) and chaotic chromosome segregation in Nxf2^-/Y anaphase I spermatocytes (g). Scale bars, 50 μm.

Fig. 2

Analyses of crossovers, bivalent formation, and apoptosis in Nxf2-deficient spermatocytes from mice of mixed (C57BL/6 × 129) backgrounds. Adult Nxf2-deficient mice with small testes (∼60 mg) were analyzed. (a, b) The number of MLH1 foci is comparable between wild type and Nxf2-deficient pachytene spermatocytes. (c) 20 bivalents in wild type metaphase I spermatocytes. (d) Prevalence of univalent chromosomes in Nxf2-deficient metaphase I spermatocytes. Of 30 chromatin masses, 10 are bivalent and 20 univalent. (e) TUNEL analysis of wild type tubules. Apoptotic cells are absent in stage XII tubules. (f) Massive apoptosis of germ cells (presumably anaphase I spermatocytes) in Nxf2^-/Y Stage XII tubules.

Fig. 3

Age-dependent loss of spermatogonia in C57BL/6J Nxf2^-/Y mice. (a, b) Histological analysis of testes from 2.5-month-old mice. The diameter of seminiferous tubules is smaller in Nxf2^-/Y mice (b) than in wild type (a). (c, d) Histological analysis of testes from 9-month-old mice at medium magnification (200×). Sertoli cell-only (SCO) tubules (indicated by asterisks) are prevalent in Nxf2^-/Y mice but not in wild type. Groups of Sertoli cells indicated by arrows are sloughed off the epithelium. (e, f) Histological analysis of testes from 9-month-old mice at high magnification (400×). Note the absence of germ cells in the Nxf2-deficient SCO tubule. Scale bars: 50 μm.