Characterization of spermatogonial markers in the mature testis of the dogfish (Scyliorhinus canicula L.)

Abstract

In dogfish, spermatogenesis progresses from a restricted germinative zone, which lines the dorsal testicular vessel. Single spermatogonia (A s ), including the spermatogonial stem cells (SSCs), produce successively paired (A p ), undifferentiated (A u4 to A u512 ), and differentiated (A d1 to A d8 ) spermatogonia and preleptotene (PL) spermatocytes through 13 mitoses. Dogfish spermatogonial subpopulations present classical morphological characteristics but cannot be distinguished on the basis of molecular markers. This characterization has been initiated in mammals despite the difficulty to separate each spermatogonial subpopulation. For instance, both glial cell-derived neurotrophic factor family receptor alpha 1 (GFRa1) and promyelocytic leukemia zinc finger protein (PLZF) are markers of undifferentiated spermatogonia, whereas receptor tyrosine kinase C-kit is a marker of differentiated spermatogonia. The aim of this study is to characterize spermatogonial markers and to differentiate several spermatogonial subpopulations. Dogfish cDNA sequences have been identified and validated by phylogenetic analyses for gfra1, plzf, pou2, as well as for high-mobility group box proteins 2 and 3 (hmgb2 and 3) and for mini-chromosome maintenance protein 6 (mcm6). We have used the anatomical advantage of the polarized dogfish testis to analyze the expression of those markers by RT-PCR and in situ hybridization. gfra1, pou2, and plzf have been detected in the testicular germinative zone, suggesting that spermatogonial markers are relatively well conserved among vertebrates but with a less restricted expression for plzf. Moreover, hmgb3 and mcm6 have been identified as new markers of differentiated spermatogonia. Finally, this first molecular characterization of spermatogonial subpopulations in a chondrichthyan model will be useful for further studies on the SSC niche evolution.

Maintenance of Potential Spermatogonial Stem Cells In Vitro by GDNF Treatment in a Chondrichthyan Model (Scyliorhinus canicula L.)1

“…As SSC molecular markers and regulators are relatively well conserved between teleosts and mammals, it was interesting to generate an in vitro model of undifferentiated spermatogonia in a widespread chondrichthyan species to test their conservation through Vertebrates. The germinative area containing potential SSCs was easily identified, as Chondrichthyes harbor a polarized cystic testis, and different spermatogonial subpopulations were distinguished on the basis of morphological criteria [6] and transcripts expressions [29]. In the present study, we first confirm at a protein level that the GDNF receptor GFRα1 was expressed in undifferentiated spermatogonia in the dogfish testis until the first differentiating stages, in accordance with the mRNA expression pattern previously described [29].…”

“…Tissue distributions of proteins observed by Western-blot were consistent with the literature. Thus, GFRα1 protein was expressed in testis and brain in accordance to its mRNA distribution in this species [29]. PLZF protein was also detected in testis and brain, tissues showing a weak mRNA expression [29], in accordance with data in mouse [46].…”

“…An expression pattern in undifferentiated spermatogonia was also reported in many mammalian species such as mouse [7], primates [42], equids [8], domestic cat [43], collared peccary [10] or in fish such as the Nile tilapia [5] and a similar expression pattern extended to spermatocytes, but at a much lower level, was also observed in trout [44]. PLZF expression pattern appeared less restrictive than GFRa1 with an expression extended from undifferentiated spermatogonia to primary spermatocytes, which is consistent with the mRNA localization in germ cells of the dogfish [29]. This extended expression pattern differs from the specific expression observed in undifferentiated spermatogonia of mammals such as mouse [7], primates [45] or equids [8].…”

“…The germinative area containing potential SSCs was easily identified, as Chondrichthyes harbor a polarized cystic testis, and different spermatogonial subpopulations were distinguished on the basis of morphological criteria [6] and transcripts expressions [29]. In the present study, we first confirm at a protein level that the GDNF receptor GFRα1 was expressed in undifferentiated spermatogonia in the dogfish testis until the first differentiating stages, in accordance with the mRNA expression pattern previously described [29]. An expression pattern in undifferentiated spermatogonia was also reported in many mammalian species such as mouse [7], primates [42], equids [8], domestic cat [43], collared peccary [10] or in fish such as the Nile tilapia [5] and a similar expression pattern extended to spermatocytes, but at a much lower level, was also observed in trout [44].…”

“…The molecular characterization of dogfish spermatogonia was recently initiated by our research group. Dogfish differentiated spermatogonia expressed c-Kit receptor, like mammals differentiated spermatogonia [6] and mRNAs of classical undifferentiated spermatogonia markers in mammals like GFRα1, POU2/POU5F1 or PLZF were detected in dogfish undifferentiated spermatogonia in the germinative zone [29]. A culture medium has been previously defined for studying stage-related changes during spermatogenesis such as steroid production by isolated cysts during 12 hours [30] or DNA and protein syntheses by cysts and isolated Sertoli cells during a longer time culture of 15 days in another chondrichthyan, Squalus acanthias [31,32].…”