Protein gene product 9.5 is a spermatogonia‐specific marker in the pig testis: Application to enrichment and culture of porcine spermatogonia

Luo, Jinping; Megee, Susan; Rathi, Rahul; Dobrinski, Ina

doi:10.1002/mrd.20529

Cited by 180 publications

(144 citation statements)

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“…The cytoplasmic bridge formed between spermatogonia during colony formation (arrow, b, c) PGP 9.5 positive cells per tubule section) including those were identified as As, Ap and some Aal by morphology [14,25]. Luo et al (2006) established an enrichment method for spermatogonial cells using PGP9.5 marker. Enriched cells containing SSCs were maintained in culture for 2 weeks without losing their PGP9.5 expression [26].…”

Section: Discussionmentioning

confidence: 99%

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Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers

Heidari

Rahmati-Ahmadabadi

Akhondi

et al. 2012

J Assist Reprod Genet

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Introduction Presently the techniques for making transgenic animals are cumbersome, required costly instruments and trained man-power. The ability of spermatogonial stem cells (SSCs) to integrate foreign genes has provided the opportunity for developing alternate methods for generation of transgenic animals. One of the big challenges in this field is development of the methods to identify and purify donor SSCs by antibody mediated cell sorting. Purpose The present study was aimed to identify goat subpopulations of SSCs using polyclonal antibodies against PGP9.5 and c-kit molecular markers as well as the growth characteristics of SSCs during short term culture. Methods One month old goats' testicular samples were subjected for immunohistochemical and immunocytochemical evaluations. The enzymatically isolated SSCs were cultured in DMEM plus FCS supplemented with (treatment) or without (control) growth factors (GDNF, LIF, FGF, and EGF) for 2 weeks. At the end of culture the morphological characteristics of SSCs colonies and immunocytochemical staining were evaluated. Results The number and size of colonies in treatment groups were significantly (P<0.01) higher than corresponding values in controls. The presence of PGP 9.5 and c-kit antigens was confirmed in immunocytochemical evaluation. In immunocytochemical evaluation, the proportion of c-kit and PGP9.5 positive cells were significantly (P<0.001) higher in control and treatment groups, respectively. Conclusions The presence of PGP9.5 and c-kit antigens was confirmed in goat SSCs. Moreover, culture medium supplementation with growth factors could effectively retain the undifferentiation status of SSCs, reflected as a higher population of PGP9.5 positive cells, after short term culture.

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Section: Discussionmentioning

confidence: 99%

“…Luo et al (2006) established an enrichment method for spermatogonial cells using PGP9.5 marker. Enriched cells containing SSCs were maintained in culture for 2 weeks without losing their PGP9.5 expression [26]. However, the successfully isolated SSCs in minipigs could not be maintained in culture for more than 10 days [27].…”

Section: Discussionmentioning

confidence: 99%

Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers

Heidari

Rahmati-Ahmadabadi

Akhondi

et al. 2012

J Assist Reprod Genet

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“…RT-PCR analysis demonstrated that UCHL1, PLZF and NANOG, which characterized undifferentiated spermatogonia in pigs [6,23], were expressed in fresh testicular cells and cultured MGSCs (Fig. 5).…”

Section: Rt-pcr Analysis Of Mgscsmentioning

confidence: 99%

“…Markers for undifferentiated spermatogonia from piglets, UCHL1, PLZF [23] and THY1 [20], were expressed in the colonies. VASA, a marker of differentiating spermatogonia [22], was also expressed in the colonies (Fig.…”

Section: Immunocytochemical Analysis Of Cultured Mgscsmentioning

confidence: 99%

In vitro propagation of male germline stem cells from piglets

Zheng

Tian

Zhang

et al. 2013

J Assist Reprod Genet

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Purpose To study the effects of serum and growth factors on propagation of porcine male germline stem cells (MGSCs) in vitro and develop a culture system for these stem cells. Methods Fresh testicular cells from neonatal piglets were obtained by mechanical dissociation and collagenase-trypsin digestion. After differential plating, non-adhering cells were cultured in media supplemented with different concentrations of serum (0, 1 %, 2 %, 5 %, 10 %). After 10 days of primary culture, the cells were maintained in media supplemented with different concentrations of growth factors (basic fibroblast growth factor and epidermal growth factor at 1, 5, 10 ng/ml). The number of MGSC-derived colonies with different sizes was determined in each treatment to assess the effects of serum concentrations and growth factors. Results The number of MGSC-derived colonies was significantly higher in the presence of 1 % rather than 10 % fetal bovine serum (FBS). Basic fibroblast growth factor (bFGF) at 1, 5 ng/ml and epidermal growth factor (EGF) at 5, 10-ng/ml significantly promoted colony formation. Immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and xenotransplantation assays demonstrated the presence of functional stem cells in cultured cell population.Conclusions In vitro propagation of porcine MGSCs could be maintained in the presence of 1 % FBS and supplementation of growth factors for 1 month.

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“…The resulting cell suspension was immediately submitted to fluorescence-activated cell sorting (FACS) using an in Flux cell sorter (BD Bioscience, San Jose, CA, USA, www.bdbiosciences.com). In some cases, the testicular cell suspension was also submitted to a differential plating method (Luo et al 2006) before FACS. Using this method, somatic cells adhere to the bottom of the plate while germ cells either remain in suspension after 2-3 days of culture or are only loosely associated with the firmly adhering somatic cells (Fig.…”

Section: Expression Of Cyp17a1 and Cyp19a1 Genes In Sorted Testicularmentioning

confidence: 99%

Cyp17a1 and Cyp19a1 in the zebrafish testis are differentially affected by oestradiol

Hinfray¹,

Nóbrega²,

Caulier³

et al. 2013

Journal of Endocrinology

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International audienceOestrogens can affect expression of genes encoding steroidogenic enzymes in fish gonads. However, little information is available on their effects at the protein level. In this context, we first analysed the expression of key steroidogenic enzyme genes and proteins in zebrafish testis, paying attention also to other cell types than Leydig cells. Gene expression was analysed by quantitative PCR on fluorescence-activated cell-sorting fractions coupled or not to differential plating, while protein synthesis was studied by immunohistochemistry using specific antibodies against zebrafish Cyp17a1, Cyp19a1a and Cyp19a1b. Furthermore, we have evaluated the effect of oestrogen treatment (17 beta-oestradiol (E-2), 10 nM) on the localization of these enzymes after 7 and 14 days of in vivo exposure in order to study how oestrogen-mediated modulation of their expression is linked to oestrogen effects on spermatogenesis. The major outcomes of this study are that Leydig cells express Cyp17a1 and Cyp19a1a, while testicular germ cells express Cyp17a1 and both, Cyp19a1a and Cyp19a1b. As regards Cyp17a1, both protein and mRNA seem to be quantitatively dominating in Leydig cells. Moreover, E-2 exposure specifically affects only Leydig cell Cyp17a1 synthesis, preceding the disruption of spermatogenesis. The oestrogen-induced suppression of the androgen production capacity in Leydig cells is a major event in altering spermatogenesis, while germ cell steroidogenesis may have to be fuelled by precursors from Leydig cells. Further studies are needed to elucidate the functionality of steroidogenic enzymes in germ cells and their potential role in testicular physiology

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Protein gene product 9.5 is a spermatogonia‐specific marker in the pig testis: Application to enrichment and culture of porcine spermatogonia

Cited by 180 publications

References 46 publications

Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers

Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers

In vitro propagation of male germline stem cells from piglets

Cyp17a1 and Cyp19a1 in the zebrafish testis are differentially affected by oestradiol

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