Stage-Dependent Expression of Protein Gene Product 9.5 in Donkey Testes

Molecular markers can be used to identify and isolate specific developmental stages of germ cells and Leydig cells. Protein gene product (PGP)9.5 expression in spermatogonia and Leydig cells has been reported in several species. The stages of spermatogonia and Leydig cells expressing PGP9.5 vary depending on the species and reproductive stages. Thus, the objectives of this study were (1) to identify the localization of PGP9.5 in donkey testicular cells, and (2) to compare the expression patterns of PGP9.5 in donkey testicular cells between pre- and post-pubertal stages. Testes samples were collected following the routine field castration of six donkeys. Western blotting was performed to verify the cross-reactivity of the rabbit anti-human PGP9.5 antibody to donkey testes. Immunofluorescence was performed to investigate the expression pattern of PGP9.5 in testicular tissues at different reproductive stages. In Western blotting, the protein band of the PGP9.5 antibody appeared at approximately 27 kDa, whereas the band was not observed in the negative control treated with normal mouse IgG. In the pre-pubertal stage, the expression of deleted in azoospermia-like (DAZL) was found in some spermatogonia in pre-pubertal testicular tissues. However, the immunolabeling of PGP9.5 in testicular tissue was not observed in the seminiferous tubules. In stages 1 and 2, spermatogonia were immunolabeled with either PGP9.5 or DAZL. In contrast, PGP9.5 and DAZL were co-immunolabeled in some of the spermatogonia in stages 3 to 8. Interestingly, some Leydig cells were immunolabeled with PGP9.5 in both pre- and post-pubertal stages. In conclusion, the PGP9.5 antibody can be used as a tool to identify and isolate spermatogonia from seminiferous tubules.

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371 EVALUATION OF ENRICHMENT STRATEGIES FOR PORCINE SPERMATOGONIA BY EXPRESSION OF PROTEIN GENE PRODUCT 9.5, A SPERMATOGONIA-SPECIFIC MARKER IN THE PIG TESTIS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab371 ◽

2006 ◽

Vol 18 (2) ◽

pp. 293

Author(s):

J. Luo ◽

S. Megee ◽

R. Rathi ◽

I. Dobrinski

Keyword(s):

Gene Product ◽

Germ Cells ◽

Protein Gene ◽

Velocity Sedimentation ◽

Seminiferous Tubules ◽

Cell Populations ◽

Specific Marker ◽

Pgp 9.5 ◽

Protein Gene Product ◽

Fraction I

Transplantation of genetically altered male germ cells is under investigation as a novel route to generate transgenic animal models. Identification and isolation of spermatogonial stem cells are a prerequisite for this strategy. The objectives of this study were to validate a marker for identification of undifferentiated porcine spermatogonia, and to use this marker to develop a practical enrichment strategy for spermatogonia from pig testis. We established that expression of protein gene product (PGP) 9.5 is a spermatogonia-specific marker in porcine testis through analysis of its expression pattern in testis cells, by comparison with the expression of the cell-type specific proteins GATA-4 (expressed in Sertoli cells) or PLZF (expressed in undifferentiated mouse spermatogonia) in seminiferous tubules at different ages, and by comparison of expression levels of PGP 9.5 and the germ cell-specific protein VASA in different cell fractions after differential plating. Using expression of PGP 9.5 as a marker, we characterized enrichment of porcine spermatogonia from two-week-old (2wo) and 10-week-old (10wo) pigs by immunofluorescence either after differential plating only or after velocity sedimentation at unit gravity followed by differential plating. After differential plating with overnight culture to deplete testicular somatic cells that firmly attach to culture dishes, spermatogonia (mean � SEM per 1000 cells) were 5-fold enriched (P < 0.05) in cells remaining in suspension (fraction I) (2wo: 54.0 � 9.1; 10wo: 162.7 � 30.5) and in populations slightly attached to the culture plate (fraction II) (2wo: 92.7 � 8; 10wo: 159.5 � 22.5) compared to the initial samples (2wo: 12.3 � 2.7; 10wo: 27.2 � 2.9). Slightly attached spermatogonia appear to be superior for future experiments due to higher viability (>90%) than spermatogonia remaining in suspension (<50%). Cell populations containing up to 70% spermatogonia with good viability (>80%) were achieved by velocity sedimentation isolation followed by differential plating. These results indicate that expression of PGP 9.5 is a useful marker for identification of undifferentiated porcine germ cells. Simple differential adhesion culture of testis cells harvested from pre-pubertal boars can supply cell populations enriched in spermatogonia for subsequent genetic manipulation and transplantation. This work was supported by 1 R01 RR17359-01.

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Immunoexpression of Tyro 3 Family Receptors—Tyro 3, Axl, and Mer—and Their Ligand Gas6 in Postnatal Developing Mouse Testis

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.5a6637.2005 ◽

2005 ◽

Vol 53 (11) ◽

pp. 1355-1364 ◽

Cited By ~ 41

Author(s):

Huizhen Wang ◽

Yongmei Chen ◽

Yehua Ge ◽

Pengpeng Ma ◽

Quanhong Ma ◽

...

Keyword(s):

Sertoli Cells ◽

Leydig Cells ◽

Cytoplasmic Membrane ◽

Expression Patterns ◽

Exact Expression ◽

Seminiferous Tubules ◽

Testicular Cells ◽

Similar Expression Pattern ◽

Weak Staining ◽

Cell Processes

Tyro 3 family receptors contain three members—Tyro 3, Axl, and Mer—that are essential regulators of mammalian spermatogenesis. However, their exact expression patterns in testis are unclear. In this study, we examined the localizations of Tyro 3, Axl, Mer, and their ligand Gas6 in postnatal mouse testes by immunohistochemistry. All three members and their ligand were continuously expressed in different testicular cells during postnatal development. Tyro 3 was expressed only in Sertoli cells with a varied distribution during testis development. At day 3 postnatal, Tyro 3 was distributed in overall cytoplasmic membrane and cytoplasm of Sertoli cells. From day 14 to day 35 postnatal, Tyro 3 appeared on Sertoli cell processes toward the adlumenal compartment of seminiferous tubules. A stage-dependent Tyro 3 immunoexpression in Sertoli cells was shown by adulthood testis at day 56 postnatal with higher expression at stages I-VII and lower level at stages IX-XII. Axl showed a similar expression pattern to Tyro 3, except for some immunopositive Leydig cells detected in mature testis. In contrast, immunostaining of Mer was detected mainly in primitive spermatogonia and Leydig cells, whereas a relative weak signal was found in Sertoli cells. Gas6 was strongly expressed in Leydig cells, and a relative weak staining signal was seen in primitive spermatogonia and Sertoli cells. These immunoexpression patterns of Tyro 3 family receptors and ligand in testis provide a basis to further study their functions and mechanisms in regulating mammalian spermatogenesis.

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