Tdrd12 Is Essential for Germ Cell Development and Maintenance in Zebrafish

Abstract

The regularity of Piwi-interacting RNA (piRNA) biogenesis is crucial to germline development. Functioning as Piwi-interacting proteins, Tudor domain-related proteins (Tdrds) have been demonstrated to be involved in spermatogenesis and the piRNA pathway. In this study, zebrafish tdrd12 was identified, and the maternal and germ cell-specific expression patterns of zebrafish tdrd12 were observed. Utilizing TALEN (transcription activator-like effector nuclease) techniques, two independent tdrd12 mutant zebrafish lines were generated. Although no defects were found during the generation of the primordial germ cells (PGCs) in the tdrd12-null fish progenies obtained from the heterozygous tdrd12 mutant parents, all Tdrd12-deficient fish developed into infertile males. The reduced numbers and eventually loss of the germ cells by 35 days post fertilization (dpf) led to masculinization and infertility of the Tdrd12-deficient fish. Meiosis defects of the germ cells in the tdrd12 mutants during the gonad-transitioning period were observed, revealing the indispensable functions of Tdrd12 in gametogenesis. Our studies demonstrated that zebrafish Tdrd12 is essential for germ cell development and maintenance.

Keywords: differentiation; germ cell; knockout; maintenance; tdrd12; zebrafish.

Figure 1

Zebrafish Tdrd12 is conserved across species. (A) The full-length coding region of the putative tdrd12 mRNA was amplified from 120-dpf wild-type zebrafish gonadal cDNA samples. The appearance of the amplified fragment showed a PCR product at 4.1 k base pairs in length; (B) Phylogeny analysis of the putative zebrafish Tdrd12 protein and human TDRD family protein members. The results indicate that the putative zebrafish Tdrd12 is most homologous to human TDRD12. The TDRD proteins of humans were as follows: TDRD1: NP_942090.1; TDRD2/TDRKH (Tudor and KH domain-containing protein): NP_001077432.1; TDRD3a and TDRD3b: NP_001139542.1 and NP_001139543.1; TDRD4/RNF17 (RING finger protein 17): NP_001171922.1; TDRD5: NP_001186014.1; TDRD6: NP_001010870.1; TDRD7: NP_001289813.1; TDRD8c and TDRD8a/STK31 (serine/threonine-protein kinase 31): NP_001247434.1 and NP_113602.2; TDRD9: NP_694591.2; TDRD10/SMNDC1 (survival of motor neuron-related-splicing factor 30): NP_005862.1; TDRD11/SND1 (staphylococcal nuclease domain-containing protein 1): NP_055205.2; TDRD12: XP_011525773.1; (C) Phylogeny analysis of the putative zebrafish Tdrd12 protein and mouse TDRD family protein members. The results indicate that the putative zebrafish Tdrd12 is most homologous to mouse TDRD12. The TDRD proteins of mice were as follows: TDRD1: NP_001002238.1; TDRD2/TDRKH (Tudor and KH domain-containing protein): XP_006502201.1; TDRD3a and TDRD3b: NP_766193.3 and NP_001240684.1; TDRD4/RNF17 (RING finger protein 17): NP_001028215.1; TDRD5: NP_001128213.1; TDRD6: NP_001154838.1; TDRD7: NP_001277404.1; TDRD8/STK31 (serine/threonine-protein kinase 31): NP_084192.2; TDRD9: NP_083332.1; TDRD10/SMNDC1 (survival of motor neuron-related-splicing factor 30): NP_766017.1; TDRD11/SND1 (staphylococcal nuclease domain-containing protein 1): NP_062750.2; TDRD12: XP_017167806.1; and (D) Phylogenetic tree of Tdrd12 proteins in zebrafish, medaka, fly, Bombyx, frog, chicken, mouse, olive baboon, human, dog, and sheep. The proteins are as follows. Drosophila melanogaster (Brother of Yb): NP_649430.1 Drosophila melanogaster (sister of Yb): NP_001245959; Drosophila melanogaster (female sterile 1 Yb): NP_477494.2; Bombyx mori: NP_001037005.1 b; Xenopus tropicalis: XP_012816810.1; Oryzias latipes: XP_011471120.1; Danio rerio: this manuscript; Gallus gallus: XP_015147962.1; Papio anubis: XP_009192354.1; Homo sapiens: XP_011525773.1; Ovis aries: XP_012045510.1; Canis lupus familiaris: XP_013976340.1; Mus musculus: XP_017167806.1.

Figure 2

Maternal and germ cell-related expression patterns of tdrd12. (A) Tissue distribution of endogenous tdrd12 transcripts with RT-PCR indicates its gonad-specific patterns in adult zebrafish; (B) The presence of endogenous tdrd12 transcripts at different developmental stages reveals its maternal expression pattern in unfertilized eggs. The expression of zebrafish tdrd12 is decreased in the early embryonic stage until the 48 hpf stage. The tdrd12 transcripts reappear from 15 dpf to the adult stage in the gonads; (C) Presence of PGCs visualized with injected EGFP-dnd 3’UTR mRNA in control larva (upper panel, injected with control morpholino) and PGC-depleted larva (lower panel, injected with dnd morpholino) at the 24-hpf stage. The arrowheads here show the presence of zebrafish PGCs in larva; and (D) Presence of tdrd12 transcripts detected in adult gonadal tissues of the control fish and PGC-depleted dnd morphants (no ovary). The transcripts of β-actin2 were amplified from the same templates as an internal control to check the quality of the cDNA.

Figure 3

Generation of Tdrd12-deficient zebrafish by TALENs. (A) Binding site of engineered TALENS on the tdrd12 gene exon 2 (exons shown in dark green). The underlined-green fonts indicate the sequences of the two targeting arms of TALENS, and the red fonts show the restriction enzyme Pst1 cutting site. WT, wild-type. Mutant-line1 (M1) and mutant-line2 (M2) are two independent mutant lines in which the restrictive endonuclease site Pst1 is eliminated with 8 and 16 bp deleted, respectively. Mutation confirmation as shown by the sequencing results of the transcripts of the tdrd12 gene from the two mutant lines, M1 and M2; (B) Agarose gel electrophoresis image with the PCR products following Pst1 digestion of the tdrd12 locus of the F2 fish offspring. +/+, WT; +/−, heterozygous; and −/−, homozygous; and (C) Diagram representative of wild-type (WT) and two putative truncated mutant Tdrd12 proteins, M1 and M2, in which the first 24 amino acids (shown in green boxes) are identical to those of the wild-type Tdrd12 protein and the following 19 and 7 amino acids in M1 and M2, respectively (shown in red boxes), are miscoded ones. Both putative mutant forms of Tdrd12 are terminated prematurely.

Figure 4

Tdrd12 deficiency results in masculinization and infertility in zebrafish. (A) Sex ratios of the progenies from the in-cross of tdrd12 heterozygous mutant fish (90 dpf). Among the progenies, all homozygous offspring developed into males. The total number of progenies for this analysis is 109. tdrd12^+/−: heterozygous progenies; tdrd12^+/+: wild-type progenies; tdrd12^−/−: homozygous progenies; (B–D) Appearance of the breeding tubercle (BT) clusters (red arrowheads) in the pectoral fin of all homozygous mutant fish (B) and wild-type males (C), but not in wild-type females (D), scale bar = 250 μm; (E–G) Anatomical views of the gonadal tissues of the Tdrd12-deficient fish and wild-type adults. Only atrophied testes were observed in Tdrd12-deficient adults (E), while normal testes (F) and ovary (G) could be observed in wild-types adults, scale bar = 500 μm; (H–J) Histological analyses of the gonadal tissues indicate no signs of germ cells in the atrophied testes of the Tdrd12-deficient adults (H), while normal spermatogenesis (I) and oogenesis (J) progresses in the wild-type adults, scale bar = 50 μm; (K) The fertilization rates of the mating between wild-type (tdrd12^+/+) males and Tdrd12-deficient fish with wild-type females at the 120-dpf stage were recorded. The average fertilization rates of the 10 fish from three separate experiments were measured. Each group consists of 10 pairs of fish. The data shown here represent the means ± standard error of the mean; and (L,M) Morphological observations of embryonic development at 11 hpf derived from the mating between wild-type females with Tdrd12-deficient adults (L) and wild-type males (M); eggs from wild-type females could be induced by Tdrd12-deficient fish, but no successful fertilization could be found, scale bar = 250 μm.

Figure 5

Existence of gonadal somatic cells, but not germ cells, in Tdrd12-deficient adults. Gene expression levels of several germ cell-specific genes (vasa, dnd, piwil1), a testis Sertoli cell-specific gene (amh), or a Leydig cell-specific gene (cyp11c1) in the filament-like testis of wild-type and Tdrd12-deficient adult testes samples were examined at the 90 dpf stage. ** p < 0.01 vs. wild type. β-actin2 was selected as the most suitable and invariant reference gene for our samples from gapdh, β-actin2, and ef1a testing according to the published reports [33,34].

Figure 6

Masculinization occurred in Tdrd12-deficient zebrafish during the late “juvenile ovary” stage. Histological features with hematoxylin-eosin staining of cryo-sections were assessed from gonadal tissues in Tdrd12-deficient zebrafish and their wild-type siblings at various stages. (A–D) At the 18-dpf stage, typical stage Ia in the “juvenile ovary” was observed in all wild-type fish gonadal tissue (A, 7/7), and most Tdrd12-deficient fish gonadal tissue (C, 6/7). However, typical pyknotic cells (pc) in early testes could not been found in wild-type gonadal samples (B, 0/7) and only some of Tdrd12-deficient fish gonadal tissue samples (D, 1/7); (E–H) At the 25-dpf stage, typical pyknotic cells (pc) and spermatogonia (sg) in early testes were observed in some of the wild-type fish gonadal tissue (E, 2/7) and all Tdrd12-deficient fish gonadal tissues were examined (G, 6/6). However, the typical epo stage ((“early” perinucleolar oocytes): early stage IB) in early ovaries could be found in some of the wild-type gonadal tissue samples (F, 5/7). No ovary-like gonads could be found in Tdrd12-deficient gonadal samples (H); (I–L) At 35 dpf, typical spermatogonium (I) or oogonium (J) could be seen in the gonadal samples of the wild-type males (I, 3/7) and females (J, 4/7). However, only the filament-like testes with some Sertoli-like and Leydig-like cells, but no spermatogonium-like cells, were seen in the gonadal samples of the Tdrd12-deficient fish (K, 6/6). No ovary-like tissue was found in mutant fish (L, 0/6); (M–P). At 70 dpf, various spermatogonium (I) or oogonium (J) stages could be seen in the gonadal samples of the wild-type males (M, 3/5) and females (N, 2/5). However, only the filament-like testes without any sign of the presence of germ cells could be seen in all the gonadal samples of Tdrd12-deficient fish (O, 5/5). No ovary-like tissue has been found in mutant fish (L, 0/5). The gonadal tissues in (A–H) are circled with a black dotted line, and the white arrowheads indicate typical cell types in each pictures. (A–H,I,K): 1000×; (J,M,O): 400×; and (N): 100×. Scale bar for all pictures represents 50 μm. Pyknotic cells (pc), spermatogonia (sg), sperm (sp), spermatocytes (sc); stages of oogenesis: stage I, stage I is divided into stage Ia and Ib. (epo (“early” perinucleolar oocytes): early stage of Ib); lpo (“late” perinucleolar oocytes): late stage of Ib), II, III, and IV.

Figure 6

Masculinization occurred in Tdrd12-deficient zebrafish during the late “juvenile ovary” stage. Histological features with hematoxylin-eosin staining of cryo-sections were assessed from gonadal tissues in Tdrd12-deficient zebrafish and their wild-type siblings at various stages. (A–D) At the 18-dpf stage, typical stage Ia in the “juvenile ovary” was observed in all wild-type fish gonadal tissue (A, 7/7), and most Tdrd12-deficient fish gonadal tissue (C, 6/7). However, typical pyknotic cells (pc) in early testes could not been found in wild-type gonadal samples (B, 0/7) and only some of Tdrd12-deficient fish gonadal tissue samples (D, 1/7); (E–H) At the 25-dpf stage, typical pyknotic cells (pc) and spermatogonia (sg) in early testes were observed in some of the wild-type fish gonadal tissue (E, 2/7) and all Tdrd12-deficient fish gonadal tissues were examined (G, 6/6). However, the typical epo stage ((“early” perinucleolar oocytes): early stage IB) in early ovaries could be found in some of the wild-type gonadal tissue samples (F, 5/7). No ovary-like gonads could be found in Tdrd12-deficient gonadal samples (H); (I–L) At 35 dpf, typical spermatogonium (I) or oogonium (J) could be seen in the gonadal samples of the wild-type males (I, 3/7) and females (J, 4/7). However, only the filament-like testes with some Sertoli-like and Leydig-like cells, but no spermatogonium-like cells, were seen in the gonadal samples of the Tdrd12-deficient fish (K, 6/6). No ovary-like tissue was found in mutant fish (L, 0/6); (M–P). At 70 dpf, various spermatogonium (I) or oogonium (J) stages could be seen in the gonadal samples of the wild-type males (M, 3/5) and females (N, 2/5). However, only the filament-like testes without any sign of the presence of germ cells could be seen in all the gonadal samples of Tdrd12-deficient fish (O, 5/5). No ovary-like tissue has been found in mutant fish (L, 0/5). The gonadal tissues in (A–H) are circled with a black dotted line, and the white arrowheads indicate typical cell types in each pictures. (A–H,I,K): 1000×; (J,M,O): 400×; and (N): 100×. Scale bar for all pictures represents 50 μm. Pyknotic cells (pc), spermatogonia (sg), sperm (sp), spermatocytes (sc); stages of oogenesis: stage I, stage I is divided into stage Ia and Ib. (epo (“early” perinucleolar oocytes): early stage of Ib); lpo (“late” perinucleolar oocytes): late stage of Ib), II, III, and IV.

Figure 7

Meiotic defects and failure of germ cell maintenance in Tdrd12-deficient zebrafish. (A) Normalized expression of the meiotic recombination-specific gene sycp3 (synaptonemal complex protein 3) in the developing gonads were examined in gonadal tissue samples in Tdrd12-deficient fish and their wild-type siblings at 13, 18, 25, and 35 dpf; (B) In situ cell death in the immature testis of tdrd12^+/+ and tdrd12^−/− at 35 dpf, with abundant signals (brown) in the Tdrd12-deficient testis. Normalized expression of the germ cell-specific genes dnd (C), vasa (D), and piwil1 (E) in the developing gonads were examined in gonadal tissue samples in Tdrd12-deficient fish and their wild-type siblings at 13, 18, 25, and 35 dpf. The tails of the larvae were collected for genotyping, while the rest of the body was used for total RNA isolation. The numbers of the examined fish for the assays at the 13, 18, 25, and 35 dpf stages for each genotype were 25, 20, 12, and 10, respectively. The experiments were performed with three biological repeats. * indicates the difference at p < 0.05 vs. wild type; ** indicates the significant difference at p < 0.01 vs. wild type. β-actin2 was selected as the most suitable and invariant reference gene for our samples from gapdh, β-actin2, and ef1a testing according to the published reports [33,34].