Elsevier

Theriogenology

Volume 113, June 2018, Pages 221-228
Theriogenology

Expression patterns and role of SDF-1/CXCR4 axis in boar spermatogonial stem cells

https://doi.org/10.1016/j.theriogenology.2018.03.008Get rights and content

Highlights

  • SDF-1 expression was observed in PGP 9.5-positve spermatogonia in all developing stages of boar testis.

  • CXCR4 expression was detected in spermatogonia from 5-day-old boar testis.

  • SDF-1 and CXCR4 expression was observed in cultured pSSCs from 5-day-old boar testes.

  • Inhibition of the CXCR4 receptor signaling pathway significantly decreased the colony formation of pSSCs.

Abstract

The signaling of chemokine stromal cell-derived factor (SDF)-1 and its receptor C-X-C motif chemokine receptor 4 (CXCR4) is involved in the cellular proliferation, survival, and migration of various cell types. Although SDF-1/CXCR4 has been implicated in the maintenance of the spermatogonial population during mouse testis development, their expression patterns and functions in boar testis remain unclear. In the present study, the expression pattern of SDF-1 and CXCR4 was determined during pre-pubertal and post-pubertal stage boar testes and in vitro cultured porcine spermatogonial stem cells (pSSCs). The role of these proteins in colony formation in cultured pSSCs was also investigated. Interestingly, SDF-1 expression was observed in PGP 9.5-positve spermatogonia in all developing stages of boar testis; however, CXCR4 expression was only detected in spermatogonia from 5-day-old boar testis. In addition, SDF-1 and CXCR4 expression was observed in cultured pSSCs from 5-day-old boar testes, and inhibition of the CXCR4 receptor signaling pathway by AMD3100 significantly decreased the colony formation of pSSCs. These results suggest that SDF-1 and CXCR4 are useful markers for detecting stage-specific spermatogonia in boar testis. Our results reveal the role of the SDF-1/CXCR4 axis in pSSC in vitro culture.

Introduction

The mammalian testis is formed in a specialized microenvironment niche that regulates the spermatogenesis and homeostasis of testicular cells. The niche for spermatogonial stem cells (SSCs) is the basal compartment of the seminiferous tubules that includes SSCs, Sertoli cells, and peritubular myoid cells [1]. SSCs are stem cells in the testis that generate haploid functional spermatozoa during a male's life after puberty [2]. Sertoli cells are epithelial cells that support SSCs and differentiating germ cells by providing nutrients and mediating external signals to support spermatogenesis [3]. Peritubular myoid cells provide structural integrity to seminiferous tubules and are involved in regulating spermatogenesis and testicular function [4].

Biomarkers of testicular cells in the boar testes have been reported in several studies. Markers for boar SSCs and undifferentiated spermatogonia include protein gene product 9.5 (PGP9.5) [5], lectin Dolichos biflorus agglutinin [6], promyelocytic leukemia zinc finger and Nanog [7], glial cell line-derived neurotrophic factor receptor-alpha 1 [8], stage-specific embryonic antigen-1 [9], undifferentiated embryonic cell transcription factor-1 [10], and sex-determining region Y-box 2 [11]. In additions, our previous study described matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 1 (MMP1), glutathione peroxidase 1 (GPX1), chemokine receptor 1 (CCR1), insulin-like growth factor binding protein 3 (IGFBP3), CD14, CD209, and Kruppel-like factor 9 (KLF9) were detected in boar SSCs [12]. Synaptonemal complex protein 3 [10], Sal-like protein 4, and c-Kit [13] were reported as markers for primary spermatocytes, and GATA4 is expressed in Sertoli cells in pre-pubertal boar testis [14]. However, membrane-specific and stage-specific genes expressed following germ cell development require further examination.

Chemokines, which are small pro-inflammatory chemoattractant cytokines that bind to specific G-protein-coupled seven-span transmembrane receptors present on the plasma membranes of target cells, are major regulators of cell trafficking. Stromal cell-derived factor 1 (SDF-1 or CXCL12) is a chemotactic chemokine that binds to C-X-C chemokine receptor type 4 (CXCR4) to regulate the trafficking of various cell types including immune cells, stem cells, and tumor cells [15]. In mouse testis, signaling of SDF-1 and its receptor CXCR4 are involved in maintaining the spermatogonial population during postnatal development [16]. In addition, this signaling plays a critical role in the maintenance of cultured SSCs in vitro [17]. However, the role of the SDF-1/CXCR4 axis in non-murine testes has not been examined. Therefore, the present study aimed to identify the expression pattern of SDF-1/CXCR4 axis in boar testes, specifically cells in seminiferous tubules, and determine its possible role in cultured boar SSCs in vitro.

Section snippets

Material and methods

All animals were handled according to the guidelines approved by the Institutional Animal Care and Use Committee at the NIAS (approval no. NIAS2015-120) and Sam-Woo Breeding Farm in Korea.

Localization and expression of SDF-1 and PGP9.5 in boar testis tissue

The expression and localization of SDF-1 and PGP9.5, which is known as a spermatogonia marker for pig [10] over a broad developmental period from postnatal days 5–150, were investigated using direct immunofluorescence techniques. Most PGP9.5-positive spermatogonia expressed SDF-1 in the testes collected from the pre-pubertal stage of pig at 5, 30, and 60 days of age (Fig. 1A–C), and both SDF-1- and PGP9.5-positive cells were observed in the inner space of the seminiferous cord contained

Discussion

In mice, the transcription levels of SDF-1 are relatively low in the neonatal testis, but is stable throughout the developmental stages of testes and adult testes [17,19]. Yang et al. observed the expression of SDF-1 in an undifferentiated spermatogonial population of mouse testes from both pubertal and adult animals [17] and found that CXCR4 was expressed in a fraction of SDF-1-positive cells that possessed stem cell capacity [17]. In addition, CXCR4 expression was identified in

Declarations of interest

None.

Funding

This work was supported by the Science Research Center [grant number 2015R1A5A1009701] from the National Research Foundation of Korea, Republic of Korea.

Acknowledgements

The authors are grateful to the Sam-Woo Breeding Farm (Yang Pyung, Korea) for providing boar testes.

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