Nxf3 is expressed in Sertoli cells, but is dispensable for spermatogenesis

Abstract

In eukaryotes, mRNA is actively exported to the cytoplasm by a family of nuclear RNA export factors (NXF). Four Nxf genes have been identified in the mouse: Nxf1, Nxf2, Nxf3, and Nxf7. Inactivation of Nxf2, a germ cell-specific gene, causes defects in spermatogenesis. Here we report that Nxf3 is expressed exclusively in Sertoli cells of the postnatal testis, in a developmentally regulated manner. Expression of Nxf3 coincides with the cessation of Sertoli cell proliferation and the beginning of their differentiation. Continued expression of Nxf3 in mature Sertoli cells of the adult is spermatogenesis stage-independent. Nxf3 is not essential for spermatogenesis, however, suggesting functional redundancy among Nxf family members. With its unique expression pattern in the testis, the promoter of Nxf3 can be used to drive postnatal Sertoli cell-specific expression of other proteins such as Cre recombinase.

Figure 1

Comparison of the NXF family members. (A) Functional domains of NXF. Unlike NXF1/NXF2, NXF3 lacks the C-terminal NUP (nucleoporin)-binding NES (nuclear export signal), but contains Crm1-binding NES. NLS, nuclear localization signal; LRR, leucine-rich repeat. (B) Testis-specific expression of NXF3. Equal amounts (30 μg) of protein extracts from each mouse tissue were used. Blots were probed with anti-NXF3, anti-NXF2, and anti-β-actin antibodies respectively. Note that heart contains little β-actin (a non-muscle cytoskeletal actin).

Figure 2

Nxf3 is expressed in Sertoli cells in the testis. RNA in situ hybridization (ISH) of Nxf3 was performed on adult mouse testis cryo-sections using a digoxigenin (DIG)-labeled cRNA probe that contained 1056 bases of Nxf3 sequence (nucleotides 763-1818). (A) ISH analysis of Nxf3 in adult wild type testes. ISH with Nxf3 anti-sense probe (upper panel) showed that Nxf3 transcripts were detected only in Sertoli cells (indicated by arrowheads) located at the periphery of seminiferous tubules. Nxf3 sense control probe produced no signal in testis sections (lower panel). (B) ISH analysis of Nxf3 in adult XX^Y* testes. Scale bars, 50 μm.

Figure 3

Immunofluorescent analysis of the NXF3 protein in testes. Sections of wild type adult testes were double immunostained with anti-NXF3 (A) and anti-GATA1 (B) antibodies. While Sertoli cells in the right tubule (indicated by asterisks) expressed both NXF3 and GATA1, Sertoli cells in the left tubule (indicated by arrows) expressed NXF3 but not GATA1. Scale bar, 50 μm.

Figure 4

Temporal expression of NXF3 in developing testes. (A) Western blotting analyses on post-natal wild type testes. The number of days after birth is indicated. XX^Y*, germ cell-deficient adult testis (Hunt and Eicher, 1991). NXF2, germ cell-specific control (Wang et al., 2001). β-actin, loading control. Note the two closely migrating NXF3 bands, most prominently in XX^Y* testis. (B) Identification of two alternatively spliced Nxf3 transcripts. Exons are depicted as rectangles and are designated by numbers shown above. Coding regions are shown in black. Non-coding regions (5′ and 3′ UTRs) are shown in open rectangles. Exon 2′ is an alternative exon that is only present in the Nxf3-2 transcript. The asterisk (*) indicates the location of start codon.

Figure 5

Inactivation of the Nxf3 gene in mouse. (A) The Nxf3 targeting construct and various Nxf3 alleles. In the Nxf3^fl allele, one loxP site is inserted in intron 1 and one in intron 8. Exons 2–8 encode amino acids 2-271. (B) Western blot analysis of adult wild type and Nxf3^−/Y testes. The anti-NXF3 antibody UP2048 (raised against aa 203-303) was used. (C) Immunofluorescence analysis of NXF3 protein in adult wild type and Nxf3^−/Y testes. The anti-NXF3 antibody UP1992 (raised against aa 355-553) was used for immunostaining. Scale bar, 50 μm.