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. 2023 Jan 3:101:skac393.
doi: 10.1093/jas/skac393.

The regulatory role of BMP4 in testicular Sertoli cells of Tibetan sheep

Keyan Ma  1   2 , Nana Chen  1   2 , Huihui Wang  1   2 , Qiao Li  1   2 , Huibin Shi  1   2 , Manchun Su  1   2 , Yong Zhang  3 , Youji Ma  1   2 , Taotao Li  1   2
Affiliations

The regulatory role of BMP4 in testicular Sertoli cells of Tibetan sheep

Keyan Ma et al. J Anim Sci. .

Abstract

This study aimed to determine the regulatory mechanism of bone morphogenetic protein 4 (BMP4) gene in the testes of Tibetan sheep and its role in the blood-testis barrier (BTB). First, we cloned BMP4 gene for bioinformatics analysis, and detected the mRNA and protein expression levels of BMP4 in the testes of Tibetan sheep pre-puberty (3-mo-old), during sexual maturity (1-yr-old), and in adulthood (3-yr-old) by qRT-PCR and Western blot. In addition, the subcellular localization of BMP4 was analyzed by immunohistochemical staining. Next, BMP4 overexpression and silencing vectors were constructed and transfected into primary Sertoli cells (SCs) to promote and inhibit the proliferation of BMP4, respectively. Then, CCK-8 was used to detect the proliferation effect of SCs. The expression of BMP4 and downstream genes, pathway receptors, tight junction-related proteins, and cell proliferation and apoptosis-related genes in SCs were studied using qRT-PCR and Western blot. The results revealed that the relative expression of BMP4 mRNA and protein in testicular tissues of 1Y group and 3Y group was dramatically higher than that of 3M group (P < 0.01), and BMP4 protein is mainly located in SCs and Leydig cells at different development stages. The CDS region of the Tibetan sheep BMP4 gene was 1,229 bp. CCK-8 results demonstrated that the proliferation rate of BMP4 was significantly increased in the overexpression group (pc-DNA-3.1(+)-BMP4; P < 0.05). In addition, the mRNA and protein expressions of SMAD5, BMPR1A, and BMPR1B and tight junction-related proteins Claudin11, Occludin, and ZO1 were significantly increased (P < 0.05). The mRNA expression of cell proliferation-related gene Bcl2 was significantly enhanced (P < 0.05), and the expression of GDNF was enhanced (P > 0.05). The mRNA expression of apoptosis-related genes Caspase3 and Bax decreased significantly (P < 0.05), while the mRNA expression of cell cycle-related genes CyclinA2 and CDK2 increased significantly (P < 0.05). It is worth noting that the opposite results were observed after transfection with si-BMP4. In summary, what should be clear from the results reported here is that BMP4 affects testicular development by regulating the Sertoli cells and BTB, thereby modulating the spermatogenesis of Tibetan sheep.

Keywords: Sertoli cells; Tibetan sheep; bone morphogenetic protein 4; testis.

Plain language summary

The fertility of male Tibetan sheep is mainly affected by testicular development and spermatogenesis. In these processes, Sertoli cells (SCs) play a central role and are regulated by a variety of genes and factors. BMP4 is mainly distributed in Sertoli cells, and its expression level increases with age. Overexpression of the BMP4 gene in Tibetan sheep testis SCs revealed elevated expression of BMP4 protein and its downstream genes SMAD5, pathway receptor proteins BMPR1A and BMPR1B; followed by elevated expression levels of cell proliferation-related genes and decreased expression levels of apoptosis-related genes. Meanwhile, the expression of tight junction proteins was also elevated. These results indicate that BMP4 affects testicular development by regulating the Sertoli cells and blood–testis barrier, thereby affecting the spermatogenesis of Tibetan sheep.

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Conflict of interest statement

The authors declare no real or perceived conflicts of interest.

Figures

Figure 1.
Figure 1.
Cloning results of Tibetan sheep BMP4 gene. (A) PCR amplification products of the Tibetan sheep BMP4 coding sequence (CDS).M, DL 2000 marker; 1–3, PCR product. (B) Alignment of NCBI amino acid sequence and cloned amino acid sequence of Tibetan sheep BMP4. (C) Secondary structure of the BMP4 protein. (D) Tertiary structure of the BMP4 protein. (E) Phylogenetic tree of BMP4 gene. (F) Alignment of the deduced amino acid sequences of Tibetan sheep BMP4 with that of Bioson_bison_bison, Bos_taurus, Bubalus_bubalis, Goat, Physeter_catodon.
Figure 2.
Figure 2.
The relative expression levels of BMP4 in sheep testis at different ages. (A) Expression of BMP4 mRNA in sheep testis at different ages. **P < 0.01; 3 M, 3-mo-old; 1Y, 1-yr-old; 3Y: 3-yr-old, the same below. (B) Expression of BMP4 protein in sheep testis at different ages.
Figure 3.
Figure 3.
Localization of immunopositive signal for BMP4 protein in Tibetan sheep testis and (20×). (A1–A3) Negative controls; (B1–B3) Testis from 3-mo-old, 1-yr-old, and 3-yr-old, respectively. SC, Sertoli cell; LC, Leydig cell.
Figure 4.
Figure 4.
Evaluation of recombinant plasmid and detection of transfection efficiency. (A–E) BMP4 gene silencing or overexpression efficiency; (F) Evaluation of recombination plasmids.
Figure 5.
Figure 5.
The expression of BMP4 protein in SCs after overexpression or silence.
Figure 6.
Figure 6.
Effect of BMP4 gene silencing and overexpression on the proliferation, apoptosis, and cycle of SCs. (A, B) CCK-8 detects the cell proliferation rate after overexpression and silencing of BMP4; (C–F) the effect of overexpression or silencing of BMP4 gene on the expression of proliferation, apoptosis, and cycle-related genes.
Figure 7.
Figure 7.
Changes in expression of pathway receptors BMPR1A and BMPR1B after overexpression or silencing of BMP4 gene. (A, B) Relative expression level of BMPR1A and BMPR1B mRNA; (C, D) relative expression level of BMPR1A and BMPR1B protein; (E) Western blot analysis for BMPR1A and BMPR1B protein.
Figure 8.
Figure 8.
Changes in expression of SMAD5 after overexpression or silencing of BMP4 gene. (A, B) Relative expression level of SMAD5 mRNA; (C, D) relative expression level of SMAD5 protein; (E) Western blot analysis for SMAD5 protein.
Figure 9.
Figure 9.
Changes in expression of Claudin11, Occludin, and ZO1 after overexpression or silencing of BMP4 gene. (A, B) Relative expression level of Claudin11, Occludin, and ZO1 mRNA; (C, D) relative expression level of Claudin11, Occludin, and ZO1 protein; (E) Western blot analysis for Claudin11, Occludin, and ZO1 protein.
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