A novel regulatory sex-skewing method that inhibits testicular DPY30 expression to increase female rate of dairy goat offspring

Abstract

The demand for goat milk products has increased exponentially with the growth of the global population. The shortage of dairy products will be addressed extraordinarily by manipulating the female rate of goat offspring to expand the goat population and goat milk yield. No studies have reported bioinformatic analyses of X- and Y-bearing sperm of dairy goats, although this will contribute to exploring novel and applied sex-skewing technologies. Regulatory subunit of the histone methyltransferase complex (DPY30) was determined to be the key differentially expressed protein (DEP) among 15 DEPs identified in the present study. The spatiotemporal expression of DPY30 strongly suggested a functional involvement of the protein in spermatogenesis. DPY30 promoted meiosis via upregulating SYCP3, which played a crucial role in mediating sex ratio skewing in goats. Although DPY30 suppressed the self-renewal of spermatogonia stem cells through AKT/PLZF, DPY30 inhibition in the testis did not induce testicular dysgenesis. Based on the biosafety assessment in mice testes, lentivirus-mediated DPY30 knockdown in bucks' testes increased X-bearing sperm proportion and female kids' rate (22.8 percentage points) without affecting sperm quality, pregnancy rate, and kidding rate. This study provides the first evidence of the DEGs in the sexed sperm of dairy goats. DPY30 inhibition in the testes of bucks increased the female kids' rate without influencing reproductive performance. The present study provides evidence for expanding the female dairy goat population to address the concern of dairy product shortage.

Keywords: AKT; DPY30; proteomics; self-renewal; sex control.

Graphical Abstract

Figure 1.

The identification and testicular expression profiles of crucial differentially expressed protein in the X- and Y-bearing of bucks. A. Representative image and statistical analysis of purity analysis of X- and Y-bearing sperm from three bucks. “P” represented the percentage of X/Y purity. B. Differentially expressed proteins were presented with the heatmap. C. The transcriptional level of differentially expressed proteins of X- and Y-bearing sperm in dairy goat testis. D. Western blotting was performed to determine the expression of DPY30 and DYNLL2 in X- and Y-bearing sperm. E. DPY30 mRNA level was evaluated in various tissues of goat. F. Expression levels of DPY30 gene in goat testes of different ages. G. The immunohistochemical of DPY30 was performed in goat testes of different ages (scale bar = 100 μm). A: Spermatogonia; black arrow: spermatocyte; triangle: spermatid. H. The grey scale values (H) were performed. I. The number of DPY30 positive cells (H) was analyzed. At least three biological replicate samples (bucks) for immunohistochemistry were analyzed. (G, H). Three biological replicates were evaluated in (RT-PCR, WB), with three technical replicates per biological replicate (C–F). Data were compared by one-way ANOVA. (a–d) Values with different letters are specific to each group and show significant differences (P < 0.05) (D-I).

Figure 2.

DPY30 ameliorates self-renewal attenuated by retinoic acid via AKT. A. The cells were transfected with Ad-DPY30 for 48 h; the effects of Ad-DPY30 on the pluripotency, self-renewal, differentiation, and meiosis were measured in mGSCs-I-SB. B. The cells were treated with RA for 48 h; the effects of RA on pluripotency, self-renewal, differentiation, and meiosis were measured in mGSCs-I-SB. C. The cells were transfected with Ad-DPY30 for 72 h; the effects of Ad-DPY30 on the signaling proteins were measured in mGSCs-I-SB. D. The cells were treated with RA for 72 h; the effect of RA on the signaling proteins was measured in mGSCs-I-SB. E. The cells were treated with RA for 48 h; the effect of RA on the WDR5, RBBP5, ASH2L, and DPY30 was measured by RT-PCR in mGSCs-I-SB. Three biological replicates were evaluated in (RT-PCR, WB), with three technical replicates per biological replicate (A–E). Data were presented as the mean ± standard deviation (A–E). Data were compared by two-tailed Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001(A–E).

Figure 3

DPY30 promoted self-renewal of goat male germline stem cells via AKT/PLZF. A. The mGSCs-I-SB cells were transfected with Ad-DPY30 for 72 h. Cell lysates were immunoblotted with the indicated antibodies. B. The quantification analyses (A) were shown. C. The mGSCs-I-SB cells were treated with RA for 72 h. Cell lysates were immunoblotted with the indicated antibodies. D. The quantification analyses (C) were shown. E. The mGSCs-I-SB cells were treated with MK2206 for 72 h. Cell lysates were immunoblotted with the indicated antibodies. F. The quantification analyses (E) were shown. G. The mGSCs-I-SB cells were transfected with Ad-DPY30 (24 h), followed by MK2206 (1 μM) treatment for 48 h; PLZF expression was measured by immunofluorescence. H. The quantification analyses (G) were shown. I. The mGSCs-I-SB cells were transfected with Ad-DPY30 (24 h), followed by MK2206 (1 μM) treatment for 48 h; cell lysates were immunoblotted with the indicated antibodies. J. The quantification analyses (G) were shown. K. The mGSCs-I-SB cells were transfected with Ad-DPY30 (24 h), followed by MK2206 (1 μM) treatment for 24 h; RT–qPCR with primers for PLZF was used to analyze the levels of the gene expression. Three biological replicates were evaluated in (RT-PCR, WB, IF), with three technical replicates per biological replicate (A-K). Data were presented as the mean ± standard deviation (A-K). Data were compared by two-tailed Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001 (A-F). Data were compared by one-way ANOVA, ^a–d Values with different letters are specific to each group and show significant differences (P < 0.05) (G-K).

Figure 4

DPY30 inhibition in testis could not induce testicular dysgenesis in mouse. A. The injection of DPY30 interference lentivirus in mice testis. B. Testes were treated with lentivirus for 35 d; the expression of pAKT in testes was analyzed. C. Testes were treated with lentivirus for 35 d; the expression of DPY30 and PLZF was analyzed in the testes. D. The quantitative analysis of DPY30 expression in C. E. The quantitative analysis of PLZF expression in C. F. The DPY30/PLZF positive cell number in C. G. Testes were treated with lentivirus for 35 d; the effect of DPY30 interference lentivirus on mice testicular morphology; H. Testes were treated with lentivirus for 35 d; the effect of DPY30 interference lentivirus on cell counts (spermatogonia, round spermatid cell) in mice testis were calculated. At least three biological replicate samples (mice) for immunohistochemistry and immunofluorescence were analyzed. (B-H) Data were presented as the means ± standard deviation (B-H). Data were compared by two-tailed Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001 (B-H).

Figure 5.

DPY30 inhibition in bucks’ testis increased female kids’ ratio. A. Testes were treated with lentivirus for 43 d; the effect of DPY30 interference lentivirus on goat sperm quality. B and C. The swim-up sperm was stained with Hoechst33342 and measured by flow cytometry to detect the X-bearing sperm proportion. Representative image (B) and statistical analyses (C) of X-bearing sperm proportion in swim-up sperm. D. Testes were treated with lentivirus for 43 d, and the treated semen was used for artificial insemination; the effect of DPY30 interference lentivirus on pregnancy rate of does. E. Testes were treated with lentivirus for 43 d, and the treated semen was used for artificial insemination; the effect of DPY30 interference lentivirus on the kidding rate of does. F. Testes were treated with lentivirus for 43 d, and the treated semen was used for artificial insemination; the effect of DPY30 interference lentivirus on female kids’ rate of offspring. All images are representative of n = 6 (Control) and n = 3 (PLL3.7-shRNA C1) independent experiments (A-F). Data were presented as the means ± standard deviation (A-F). Data were compared by two-tailed Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001(A-F).