A novel role of Krüppel-like factor 4 in Zhikong scallop Chlamys farreri during spermatogenesis

Abstract

Krüppel-like factor 4 (KLF4) is a kind of zinc finger transcription factor, which is involved in terminal differentiation of epithelial cells and reprogramming of somatic cells to induced pluripotent stem (iPS) cells in mammals. In the present study, we identified a full-length cDNA of Klf4 in Zhikong scallop Chamys farreri (Cf-Klf4) and found that Cf-Klf4 presented a sexual dimorphic expression characteristic in C. farreri gonads. Cf-Klf4 expression was significantly higher in testes than in ovaries from growing stage to mature stage detected by quantitative real-time PCR, and was located in male gametes, except for spermatozoa during spermatogenesis through in situ hybridization and immunohistochemistry, while no positive signal was visible in female gametes during oogenesis. Furthermore, the knockdown of Cf-Klf4 in testes by means of in vivo RNA interference led to an obviously developmental retardance, lower gonadosomatic index, less male gametes and more apoptotic spermatocytes. Interestingly, we found that two out of eight scallops showed a hermaphroditic phenotype characteristic of male-to-female sex reversal when the Klf4 mRNA and protein levels were knocked down in males. These results verified that Klf4 plays an important role in testis functional maintenance and is necessary in spermatogenesis of C. farreri.

Fig 1

Multiple sequence alignment (a) and phylogenetic analysis (b) among the KLF4 sequences from different species. Identical residues are highlighted in black. The transcriptional activation domain, transcriptional repression domain and nuclear localization signal are in box I to III. The three Cys₂His₂ type zinc fingers are in box IV, VI and VIII, its linkers are in box V and VII.

Fig 2. Expression of Cf-Klf4 mRNA in C. farreri gonads at different stages detected by qRT-PCR.

Data are indicated as the mean ± SEM from five independent samples with triplicate. The expression level in the testes at resting stage is set as 1.00 to calibrate the relative levels in gonads at other stages. Different letters indicate significant differences (P < 0.05).

Fig 3. Localization of Cf-Klf4 mRNA in gonads of C. farreri demonstrated by in situ hybridization.

Positive signals with an antisense probe are indicated in blue; controls with sense probe are indicated in mature ovary (e) and testis (l). (a)-(e), ovary; (a), resting stage; (b), proliferative stage; (c), growing stage; (d), mature stage. (f)-(l), testis; (f), resting stage; (g), proliferative stage; (h), growing stage; (j), mature stage; (i) and (k), magnified image of (h) and (j), respectively. Og, oogonium; Oc, oocyte; MO, mature oocyte; Sg, spermatogonium; Sc, spermatocyte; St, spermatid; Sz, spermatozoon. Bar, 40 μm for a-e; 20 μm for (h), (j), (l); 10 μm for others.

Fig 4. Localization of Cf-KLF4 protein in gonads of C. farreri demonstrated by immunohistochemistry.

Positive signals with anti-KLF4 are indicated in brown; controls with negative serum are indicated in mature testis (h) and ovary (i). (a), specific test of KLF4 polyclonal antibody shows that only a specific band appears, meaning KLF4 polyclonal antibody is specific. (b)-(h), testis; (b), resting stage; (c), proliferative stage; (d), growing stage; (e), mature stage; (f), (g), magnified image of (e). (i)-(m), ovary; (j), resting stage; (k), proliferative stage; (l), growing stage; (m), mature stage; Og, oogonium; Oc, oocyte; MO, mature oocyte; Sg, spermatogonium; Sc, spermatocyte; St, spermatid; Sz, spermatozoon. Bar, 10 μm for (b), (f); 20 μm for others.

Fig 5

Level of the Cf-Klf4 mRNA and Cf-KLF4 protein in C. farreri testes for 72 h post-injection detected by qRT-PCR (a) and Western blot (b). (a) Level of the Cf-Klf4 mRNA detected by qRT-PCR. Data are indicated as the mean ± SEM from five independent samples with triplicate. The expression level in the testes of blank group is set as 1.00 to calibrate the relative levels in testes of other groups. Different letters indicate significant differences (P < 0.05). (b) Level of the Cf-KLF4 protein for 72 h post the second injection detected by Western blot. 1, blank group; 2, PBS group; 3, dsKLF4 group.

Fig 6. Characteristics of C. farreri testes when Cf-Klf4 knockdown for 24 d.

(a), phenotype; (b), GSI (n = 8); (c), Gamete number in testis follicles of a 2500 μm² area (n = 5, five sights each individual, respectively); (d), percentage of various types of germ cells in a total number of 200 gametes (n = 5, five sights each individual, respectively). Data are means ± SEM. Different letters indicate significant differences (P < 0.05).

Fig 7. Histological characteristics of C. farreri testes in RNAi experiment.

(a), testes at the start of experiment; (b), testes at the 10^th day of blank group; (c), testes at the 10^th day of dsKLF4 group; (d)-(i), testes after RNAi for 24 days. (d), blank group; (e), PBS group; (f), dsKLF4 group; (h), the hermaphroditic gonads in the dsKLF4 group after RNAi for 24 days; (g) and (i), magnified images of (f) and (h), respectively. Sg, spermatogonium; Sc, spermatocyte; St, spermatid; Sz, spermatozoon; Og, oogonium; Oc, oocyte. Bar, 10 μm for (g) and (i); 20 μm for others.

Fig 8. Cell apoptosis in the testes demonstrated by TUNEL after RNAi for 24 days.

Positive signals are indicated in brown. (a)-(c), blank group; (d)-(f), dsKLF4 group; (g)-(i), the hermaphroditic gonad in the dsKLF4 group; (b), (e), (h), magnified images of the gonads; (c), (f), (i), negative control without terminal deoxynucleotidyl transferase of TUNEL experiment. Sg, spermatogonium; Sc, spermatocyte; St, spermatid; Sz, spermatozoon; Og, oogonium; Oc, oocyte. Bar, 10 μm for (b), (e), (h); 20 μm for others.

Fig 9. Location of a female-specific gene Cf-foxl2 demonstrated by in situ hybridization in the hermaphroditic gonads of dsKLF4 group.

Positive signals with an antisense probe are indicated in blue. (b), the magnified image of (a). Sz, spermatozoon; Og, oogonium; Oc, oocyte. Bar, 20 μm.