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Germ cells are essential for testicular morphogenesis and functional reconstruction in a porcine xenograft model
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  • Published: 23 March 2026

Germ cells are essential for testicular morphogenesis and functional reconstruction in a porcine xenograft model

  • Min-Gi Han1 na1,
  • Yoseop Jeon1 na1,
  • Hyoyoung Maeng1 na1,
  • Donghyeon Kim1 na1,
  • Jeong Tae Do1 na1,
  • Kwonho Hong1 na1,
  • Youngsok Choi1 na1 &
  • …
  • Hyuk Song1 

Scientific Reports , Article number:  (2026) Cite this article

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We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

Subjects

  • Biotechnology
  • Cell biology
  • Developmental biology

Abstract

Reconstruction of functional testis tissue can provide new opportunities for in vitro spermatogenesis and fertility restoration in livestock. Although testis reconstruction has been reported in several species, the germ cell dependent mechanisms underlying de novo testicular morphogenesis remain insufficiently defined in large animals. In this study, we investigated the contribution of porcine spermatogonial stem cells (pSSCs) to testis morphogenesis and spermatogenesis using a xenograft model. Testicular cells containing approximately 10% germ cells combined with a somatic-enriched fraction (SEF) (GF group) or SEF only (FO group) were transplanted subcutaneously into immunodeficient mice and examined after six months. GF tissues developed well-organized seminiferous tubules and showed multiple spermatogenic stages, whereas FO tissues remained fragmented, poorly organized, and lacked stable tubular architecture. Immunofluorescence analyses revealed appropriate localization of germ cells and maturation-associated features of Sertoli cells in GF tissues, whereas FO tissues exhibited aberrant Sertoli cell localization and marker expression patterns. Transcriptomic profiling further showed enrichment of cell cycle– and spermatogenesis-related pathways in GF tissues, while FO tissues exhibited upregulation of coagulation-, inflammation-, and early developmental pathways, consistent with impaired tissue organization. Collectively, these findings demonstrate that pSSCs are indispensable for initiating and stabilizing testicular morphogenesis in xenograft condition and underscore the utility of this model for advancing reproductive biotechnology in livestock.

Data availability

The raw sequencing data generated in this study are available in the NCBI Sequence Read Archive (SRA) under accession number PRJNA1197989.

Abbreviations

ACR:

Acrosin

ACRBP:

Acrosin binding protein

ACTL9:

Actin-like 9

ACVR1:

Activin A receptor, type I

AGTR1:

Angiotensin II receptor type 1

AMH:

Anti-Müllerian hormone

AR:

Androgen receptor

BAG6:

BAG Cochaperone 6

BMP:

Bone morphogenetic protein

C1R:

Complement component 1, r subcomponent

C3:

Complement component 3

CAPN2:

Calpain-2 catalytic subunit

CD109:

Cluster of differentiation 109

CD90:

Cluster of differentiation 90

CFAP:

Cilia and flagella associated protein

CFI:

Complement factor I

DAZAP1:

DAZ-associated protein 1

DMRT1:

Doublesex and mab-3 related transcription factor 1

EGF:

Epidermal growth factor

ESR:

Estrogen receptor, ERα

FGF:

Fibroblast growth factor

FN1:

Fibronectin 1

GDNF:

Glial cell line-derived neurotrophic factor.

GFRα1:

GDNF family receptor alpha 1

GJA5:

Gap junction protein alpha 5

GOBP:

Gene ontology biological process

ITGAV:

Integrin subunit alpha V

JAK:

Janus kinase

KLF7:

Kruppel-like factor 7

LIF:

Leukemia inhibitory factor

MAEL:

Maelstrom spermatogenic transposon silencer

MMP:

Matrix metalloproteinase

NR2F2:

Nuclear receptor subfamily 2, group F, member 2

NR5A1:

Nuclear receptor subfamily 5, group A, member 1

UCHL1:

Ubiquitin carboxyl-Terminal hydrolase L1

PIWIL1:

Piwi-like RNA-mediated gene silencing 1

PRM:

Protamine

RARA:

Retinoic acid receptor alpha

RSPH1:

Radial spoke head component 1

SOX9:

SRY-box transcription factor 9

SPATA6:

Sspermatogenesis associated 6

SRY:

Sex-determining region Y

STAT:

Signal transducer and activator of transcription

STRBP:

Spermatid perinuclear RNA binding protein

TDRD:

Tudor domain containing

TGFBR3:

Transforming growth factor beta receptor 3

TIMP:

Tissue inhibitor of metalloproteinase

TNP:

Transition protein

DDX4 (VASA):

DEAD-box helicase 4; a conserved germ cell-specific RNA helicase

VEGFA:

Vascular endothelial growth factor

WNT:

Wingless-related integration site

WNT5A:

Wingless-type MMTV integration site family, member 5A

WT1:

Wilms’ tumor gene 1

YBX3:

Y-box binding protein 3

ZBTB16:

Zinc finger and BTB domain-containing protein 16

ZCWPW1:

Zinc finger CW-type and PWWP domain containing 1

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Acknowledgements

The authors thank the Sam-Woo livestock for providing porcine testis samples and Editage (www.editage.co.kr) for editing and reviewing this manuscript for English language.

Funding

This study was supported by grants from the National Research Foundation of Korea (NRF)-2022R1A2C1004503.

Author information

Author notes

  1. Min-Gi Han, Yoseop Jeon, Hyoyoung Maeng, Donghyeon Kim, Jeong Tae Do, Kwonho Hong and Youngsok Choi are authors contributed equally to this work.

Authors and Affiliations

  1. Department of Stem Cells and Regenerative Technology, Konkuk University, Seoul, 05029, Republic of Korea

    Min-Gi Han, Yoseop Jeon, Hyoyoung Maeng, Donghyeon Kim, Jeong Tae Do, Kwonho Hong, Youngsok Choi & Hyuk Song

Authors
  1. Min-Gi Han
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Contributions

M.G.H. conducted most of the experiments and analyzed the data. Y.J. and H.M. performed primary cell isolation and xenografts. D.K. contributed to the examination of porcine total cell characteristics. J.T.D., K.H., and Y.S. interpreted the data. M.G.H. and H.S. prepared the manuscript.

Corresponding author

Correspondence to Hyuk Song.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics

This study followed the standard operating guidelines for experimental animals of the Korean Ministry of Food and Drug Safety and Animal and Plant Quarantine Agency. The project, titled “Evaluation of the function of CD14 in the niche homing mechanism of porcine spermatogonia,” was approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University with approval number KU23097. No human cells or samples were involved in this study.

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Han, MG., Jeon, Y., Maeng, H. et al. Germ cells are essential for testicular morphogenesis and functional reconstruction in a porcine xenograft model. Sci Rep (2026). https://doi.org/10.1038/s41598-026-44916-4

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  • Received: 12 August 2025

  • Accepted: 16 March 2026

  • Published: 23 March 2026

  • DOI: https://doi.org/10.1038/s41598-026-44916-4

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Keywords

  • Xenograft
  • Testicular morphogenesis
  • Spermatogonial stem cells
  • Testis reconstruction
  • Porcine testis
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