Gtsf1l and Gtsf2 Are Specifically Expressed in Gonocytes and Spermatids but Are Not Essential for Spermatogenesis

Abstract

The unknown protein family 0224 (UPF0224) includes three members that are expressed in germ-line cells in mice: Gtsf1, Gtsf1l, and BC048502 (Gtsf2). These genes produce proteins with two repeats of the CHHC Zn-finger domain, a predicted RNA-binding motif, in the N terminus. We previously reported that Gtsf1 is essential for spermatogenesis and retrotransposon suppression. In this study, we investigated the expression patterns and functions of Gtsf1l and Gtsf2. Interestingly, Gtsf1l and Gtsf2 were found to be sequentially but not simultaneously expressed in gonocytes and spermatids. Pull-down experiments showed that both GTSF1L and GTSF2 can interact with PIWI-protein complexes. Nevertheless, knocking out Gtsf1, Gtsf2, or both did not cause defects in spermatogenesis or retrotransposon suppression in mice.

Fig 1. GTSF1L and GTSF2 have two N-terminal CHHC-type Zn-finger domains.

(A) Domain architecture of mouse UPF0224-family proteins. The N-terminal region has two CHHC Zn-finger domains. The black lines indicate the regions used to raise rabbit antibodies against GTSF1L and GTSF2. (B) Alignment of the amino-acid residues of the mouse UPF0224-family proteins using CLUSTAL-X. The first and second CHHC Zn-finger domains are indicated in blue and red, respectively. Asterisks indicate amino-acid residues that are conserved among GTSF1, GTSF1L, and GTSF2. Pluses indicate amino-acid residues that are conserved between GTSF1L and GTSF2.

Fig 2. Gtsf1l and Gtsf2 expression in male germ-cell development.

(A) RT-PCR of Gtsf1l, Gtsf2, and Gapdh mRNAs using total RNA from the whole embryo at E7.5 and from the testes at various time points from E13.5 to 8 weeks. P: postnatal day. (B-K) Frozen sections of adult testis were immunostained with anti-GTSF1L and anti-GTSF2 antibodies (green), revealing GTSF1L (B-F) and GTSF2 (G-K) protein expression in the seminiferous tubules at developmental stages I-III (B, G), IV-VI (C, H), VII-VIII (D, I), IX-X (E, J), and XI-XII (F, K). Nuclei were stained with DAPI (blue). Scale bar: 100 μm.

Fig 3. GTSF1L and GTSF2 interact with PIWI and Tudor complexes.

(A) Diagram showing the GST-fused GTSF1L or GTSF2 protein with various deletions. FL: the full-length protein; dC: the protein with a C-terminal deletion; ZnF: the N-terminal region of the protein, which contains the Zn-finger domains; dZnF: the protein with an N-terminal deletion; CR: the central region. (B) Pull-down experiments using of GST-fused GTSF1L or GTSF2 or their deletion mutants. Pulled-down proteins were analyzed by western blotting with antibodies against MILI, MIWI, TDRD1, or TDRD6. Testis lysates were untreated (-) or pretreated (+) with RNase A. Part of each pull-down fraction was separated by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB).

Fig 4. Normal spermatogenesis in mice lacking Gtsf1l and/or Gtsf2.

(A) Representative images of testes from 8-week-old Gtsf1l^+/-/Gtsf2^+/-, Gtsf1l^-/-/Gtsf2^+/-, Gtsf1l^+/-/Gtsf2^-/-, and Gtsf1l^-/-/Gtsf2^-/- mice. Scale bar: 1 mm. (B-I) Hematoxylin/eosin-stained sections of the testis (B, D, F, and H) and cauda epididymis (C, E, G, and I) from 8-week-old Gtsf1l^+/-/Gtsf2^+/- (B, C), Gtsf1l^-/-/Gtsf2^+/- (D, E), Gtsf1l^+/-/Gtsf2^-/- (F, G), and Gtsf1l^-/-/Gtsf2^-/- (H, I) mice. Scale bar: 200 μm. (J, K) Mean weight of the testis (J) and cauda epididymis (K) in 8-week-old mice. Error bars represent standard deviation. (L) Total sperm counts per mg cauda epididymis in 8-week-old mice. Error bars represent standard deviation.