0610009K11Rik, a testis-specific and germ cell nuclear receptor-interacting protein

Abstract

Using an in silico approach, a putative nuclear receptor-interacting protein 0610009K11Rik was identified in mouse testis. We named this gene testis-specific nuclear receptor-interacting protein-1 (Tnrip-1). Tnrip-1 was predominantly expressed in the testis of adult mouse tissues. Expression of Tnrip-1 in the testis was regulated during postnatal development, with robust expression in 14-day-old or older testes. In situ hybridization analyses showed that Tnrip-1 is highly expressed in pachytene spermatocytes and spermatids. Consistent with its mRNA expression, Tnrip-1 protein was detected in adult mouse testes. Immunohistochemical studies showed that Tnrip-1 is a nuclear protein and mainly expressed in pachytene spermatocytes and round spermatids. Moreover, co-immunoprecipitation analyses showed that endogenous Tnrip-1 protein can interact with germ cell nuclear receptor (GCNF) in adult mouse testes. Our results suggest that Tnrip-1 is a testis-specific and GCNF-interacting protein which may be involved in the modulation of GCNF-mediated gene transcription in spermatogenic cells within the testis.

Figure 1

Restricted expression of Tnrip-1 in adult mouse testes by (A) RT-PCR and (C) Northern blot analyses. Lanes: B, brain; K, kidney; T, testis; U, uterus; Sv, seminal vesicles; E, epididymis; Sp, spleen; Li, liver; O, ovary; Lu, lung; H, heart.

Figure 2

Tnrip-1 expression in mouse testes during postnatal development and its predominant expression in spermatogenic cells of adult mouse testes. (A) QRT-PCR analyses showing the developmentally regulated Tnrip-1 expression in mouse testes. Tnrip-1 mRNA levels from mice at various days after birth were normalized by β-actin mRNA in each samples (mean ± standard errors, n=3). (B) In situ hybridization studies showing the predominant Tnrip-1 expression in pachytene spermatocytes (arrowheads) and round spermatids (arrows) within adult mouse testes.

Figure 3

Tnrip-1 protein expression in adult mouse testes. (A) Western blot analyses showing the restricted expression of Tnrip-1 protein (42 kDa) in the homogenates of adult mouse testes. (B) Immnohistochemical studies showing the expression of Tnrip-1 protein in pachytene spermatocytes (arrowheads) and round spermatids (arrows) in adult mouse testes.

Figure 4

Protein-protein interaction between Tnrip-1 and GCNF in the homogenates of adult mouse testes by Co-IP experiments. Top two panels: equal amounts of testicular proteins were immunoprecipitated (IP) with anti-GCNF antibodies, anti-Tnrip-1 antibodies or their respective preimmune serum IgG (Pre-Sera IgG). Precipitates were then subjected to Western blot analyses (WB) using anti-Tnrip-1 antibodies or GCNF antibodies. Bottom two panels: testicular homogenates for the above Co-IP experiments were probed with anti-Tnrip-1 or anti-GCNF antibodies to show the presence of both Tnrip-1 and GCNF proteins in the samples.