Figla Favors Ovarian Differentiation by Antagonizing Spermatogenesis in a Teleosts, Nile Tilapia (Oreochromis niloticus)

Abstract

Figla (factor in the germ line, alpha), a female germ cell-specific transcription factor, had been shown to activate genetic hierarchies in oocytes. The ectopic expression of Figla was known to repress spermatogenesis-associated genes in male mice. However, the potential role of Figla in other vertebrates remains elusive. The present work was aimed to identify and characterize the functional relevance of Figla in the ovarian development of Nile tilapia (Oreochromis niloticus). Tissue distribution and ontogeny analysis revealed that tilapia Figla gene was dominantly expressed in the ovary from 30 days after hatching. Immunohistochemistry analysis also demonstrated that Figla was expressed in the cytoplasm of early primary oocytes. Intriguingly, over-expression of Figla in XY fish resulted in the disruption of spermatogenesis along with the depletion of meiotic spermatocytes and spermatids in testis. Dramatic decline of sycp3 (synaptonemal complex protein 3) and prm (protamine) expression indicates that meiotic spermatocytes and mature sperm production are impaired. Even though Sertoli cell (dmrt1) and Leydig cell (star and cyp17a1) marker genes remained unaffected, hsd3b1 expression and 11-KT production were enhanced in Figla-transgene testis. Taken together, our data suggest that fish Figla might play an essential role in the ovarian development by antagonizing spermatogenesis.

Fig 1. Phylogenetic and synteny analysis of Figla gene in vertebrates.

(A) Phylogenetic tree representing the evolutionary relationship among vertebrate homologues of Figla, with tilapia Max (Myc-associated factor X) as an out-group. Values on the tree represent bootstrap scores from 1000 trials, indicating the credibility of each branch. Branch lengths are proportional to the number of amino acid changes on the branch. (B) Synteny maps showing the Figla locus organization in vertebrates. Gene symbols are described according to Ensembl database. The bar lengths are not proportional to the distances between genes. The direction of the arrow indicates the gene orientation.

Fig 2. Expression profile of Figla gene in tilapia.

(A) Expression of Figla in adult tilapia tissues by real-time PCR. HK, head kidney; M, muscle; K, kidney; O, ovary; B, brain; T, testis; G, gill; H, heart; L, liver; I, intestine; S, spleen. (B) Ontogenic expression of Figla gene in tilapia gonads analyzed by real-time PCR. Data were represented as means ± S.E.M. of three independent samples. The expression level was normalized using the geometric mean of the levels of three internal control genes (gapdh, ef1a, and actb). Different lower-case letters indicate a significant difference in Figla mRNA levels (P<0.05).

Fig 3. Cellular localization of Figla expression in tilapia gonads by IHC.

Figla was abundantly observed in early primary oocytes at 90 (A) and 150 dah (B) of XX fish. No signal was detected in the control testis (C and D). Arrows indicate positive immunostaining.

Fig 4. Screening, analysis and histological investigations of Fig1a-transgene fish.

(A) Confirmation of transgene insertion by genomic PCR and sequencing. Lane 1 and 6, DNA marker; Lane 2, XY fish carrying Figla-transgene; Lane 3 and 4, using plasmid and water as template, respectively; Lane 5, XY control fish. (B) Comparison of GSI (Gonadal Somatic Index) of control and Fig1a-transgene XY fish. Data were expressed as means ± S.E.M. of control (n = 4) and Fig1a-transgene XY (n = 13) fish. Different letters indicate statistical differences at P<0.05. (C) Expression analysis of Figla gene in the testis of the Fig1a-transgene and control XY fish by real-time PCR. (D) Expression profiles of Figla in the gonad of control XX, Figla-transgene XY fish and control XY fish at 90 dah analyzed by Western blotting. β-Actin was used as reference protein to validate equal loading for Western blot analysis. O, Ovary; T, Testis; #1–4, four individuals of Figla-transgene XY. (E) Spermatogonia and all phases of spermatogenic cells could be detected in the testis of the control XY fish. However, only spermatogonia cells, but not later phase spermatogenic cells of spermatocytes and spermatids, were detected in Fig1a-transgene XY testes (F). Abundant expression of hrGFP (H) and Figla (J) were detected in the Fig1a-transgene XY testis, while no positive immunostaining was observed for hrGFP (G) and Figla (I) in the control testis. Arrows indicate positive immunostaining of respective genes. Inset represents the low magnified view of the respective gonads.

Fig 5. Effects of Fig1a over-expression on subcellular re-organization of somatic and germ cell markers.

No significant difference of Dmrt1, Cyp17a1 and StAR were detected between Fig1a-transgene (A, C, E) and control group (B, D, F). Abundant expression of Sycp3 was detected in the testis of the control fish (H), while no positive immunostaining for Sycp3 was detected in the testis for the Figla-transgene fish (G). Arrows indicate positive immunostaining for each gene.

Fig 6. Comparative transcriptional profiling of Figla-transgene and control XY fish.

Expression of dmrt1, prm, cyp17a1, hsd3b1 and star in Figla-transgene and control fish at 90 dah were measured by real-time PCR. Relative mRNA levels were represented as means ± S.E.M. of 3 independent samples for the control fish and 5 samples for the Figla-transgene fish. *, indicates the significant difference (P<0.05) between the control and Figla-transgene fish.

Fig 7. Comparison of serum 11-KT levels between Figla-transgene and control XY fish.

Results were presented as means ± S.E.M. of 6 independent samples, respectively.