J Vet Sci.  2018 Sep;19(5):592-599. 10.4142/jvs.2018.19.5.592.

In vitro differentiation of spermatogonial stem cells using testicular cells from Guangxi Bama mini-pig

• Zhao H
• Nie J
• Zhu X
• Lu Y
• Liang X
• Xu H
• Yang X
• Zhang Y
• Lu K
• Lu S

• KMID: 2420927
• DOI: http://doi.org/10.4142/jvs.2018.19.5.592

Abstract

In this study, we attempted to establish a culture system for in vitro spermatogenesis from spermatogonial stem cells (SSCs) of Bama mini-pig. Dissociated testicular cells from 1-month-old pigs were co-cultured to mimic in vivo spermatogenesis. The testicular cells were seeded in minimum essential medium alpha (α-MEM) supplemented with Knockout serum replacement (KSR). Three-dimensional colonies formed after 10 days of culture. The colonies showed positive staining for SSC-associated markers such as UCHL1, PLZF, THY1, OCT4, Dolichos biflorus agglutinin, and alkaline phosphatase. Induction of SSCs was performed in α-MEM + KSR supplemented with retinoic acid, bone morphogenetic protein 4, activin A, follicle-stimulating hormone, or testosterone. The results showed that STRA8, DMC1, PRM1, and TNP1 were upregulated significantly in the colonies after induction compared to that in testis from 1-month-old pigs, while expression levels of those genes were significantly low compared to those in 2-month-old testis. However, upregulation of ACROSIN was not significant. Replacement of α-MEM and KSR with Iscove's modified Dulbecco's medium and fetal bovine serum did not upregulate expression of these genes significantly. These results indicate that SSCs of Bama mini-pig could undergo differentiation and develop to a post-meiotic stage in α-MEM supplemented with KSR and induction factors.

Keyword

mini pig; spermatogenesis; stem cell; testicular

MeSH Terms

Acrosin
Activins
Alkaline Phosphatase
Bone Morphogenetic Protein 4
Dolichos
Follicle Stimulating Hormone
Humans
In Vitro Techniques*
Infant
Infant, Newborn
Spermatogenesis
Stem Cells*
Swine
Testis
Testosterone
Tretinoin
Up-Regulation
Acrosin
Activins
Alkaline Phosphatase
Bone Morphogenetic Protein 4
Follicle Stimulating Hormone
Testosterone
Tretinoin

Figure

• 

  Fig. 1 Histology of testis tissues. (A) Image of a 1-month-old testis. Spermatogonial stem cells were observed (asterisk), but no spermatids or sperm were observed. (B) Image of a 2-month testis. Round spermatids (arrowhead) and elongated sperm (arrow) were observed. Scale bars = 50 µm (A and B).

• 

  Fig. 2 Cultivation of testicular cells in vitro. Three-dimensional colonies were formed when testicular cells were cultured in minimum essential medium alpha (α-MEM) and Knockout serum replacement (KSR), flat colonies were formed when testicular cells were cultured in Dulbecco's modified Eagle medium nutrient mixture F-12 (DMEM/F12) and KSR. IMDM, Iscove's modified Dulbecco's medium; FBS, fetal bovine serum. Scale bars = 100 µm.

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  Fig. 3 Colony formation process. After 3 days of culture (A), after 4 to 5 days of culture (B), after 6 days of culture (C), and morphology of colonies observed as finally formed (D). Scale bars = 100 µm (A–D).

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  Fig. 4 Characterization of spermatogonial stem cells (SSCs) in 1-month-old testis tissue. SSCs were stained with UCHL1 (A) and double stained with Dolichos biflorus agglutinin (B). (C) Counterstaining was carried out with Hoechst 33342. (D) Panels A–C in Fig. 4 merged. (E) Alkaline phosphatase staining result. Scale bars = 50 µm (A–E).

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  Fig. 5 Identification of the cultured colonies. The colonies expressing UCHL1, PLZF, THY1, and OCT4 showed DBA- and alkaline phosphatase (AP)-positive staining results. Scale bars = 100 µm.

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  Fig. 6 Immunocytochemical staining of the induced cells. (A) Stra8 expression was observed in the cytoplasm of cells cultured in Medium 1 or Medium 2. (B) Scp3 expression was observed in cell nuclei. Scale bars = 10 µm (A and B).

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  Fig. 7 Relative expressions of the marker genes involved in spermatogenesis. Relative expression levels of Stra8, DMC1, PRM1, TNP1, and ACROSIN were quantified by quantitative reverse transcription polymerase chain reaction in differentiated cells that were cultured in Medium 1 or Medium 2 compared to those in 1-month testis (Testis 1), 2-month testis (Testis 2), and the control cultures. The average values were from three experiments. The results are shown as mean ± SEM. The data were analyzed by one-way analysis of variance by using PASW Statistics software (ver.18.0; IBM). Double asterisks indicate p < 0.01.

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  Fig. 8 DNA content as determined by using flow cytometry.

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