Study of the potential spermatogonial stem cell compartment in dogfish testis, Scyliorhinus canicula L.

Abstract

In the lesser-spotted dogfish (Scyliorhinus canicula), spermatogenesis takes place within spermatocysts made up of Sertoli cells associated with stage-synchronized germ cells. As shown in testicular cross sections, cysts radiate in maturational order from the germinative area, where they are formed, to the opposite margin of the testis, where spermiation occurs. In the germinative zone, which is located in a specific area between the tunica albuginea of the testis and the dorsal testicular vessel, individual large spermatogonia are surrounded by elongated somatic cells. The aim of this study has been to define whether these spermatogonia share characteristics with spermatogonial stem cells described in vertebrate and non-vertebrate species. We have studied their ultrastructure and their mitotic activity by 5'-bromo-2'-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) immunodetection. Additionally, immunodetection of c-Kit receptor, a marker of differentiating spermatogonia in rodents, and of alpha- and beta-spectrins, as constituents of the spectrosome and the fusome, has been performed. Ultrastructurally, nuclei of stage I spermatogonia present the same mottled aspect in dogfish as undifferentiated spermatogonia nuclei in rodents. Moreover, intercellular bridges are not observed in dogfish spermatogonia, although they are present in stage II spermatogonia. BrdU and PCNA immunodetection underlines their low mitotic activity. The presence of a spectrosome-like structure, a cytological marker of the germline stem cells in Drosophila, has been observed. Our results constitute the first step in the study of spermatogonial stem cells and their niche in the dogfish.

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

“…Moreover, plzf transcripts were detected in dogfish Sertoli cells from stage I to stage III. This may be explained by the fact that in dogfish, those cells actively proliferate until complete cyst formation, which occurs at stage IIIa (Stanley 1966, Holstein 1969, Loppion et al 2008. These data suggest that PLZF may have a main conserved function in stem cell fate regulation in vertebrates.…”

“…In this model, the number of spermatogonial divisions from stage I to stage VI was estimated to be 13 by Holstein (1969) on the basis of the number of spermatoblasts per cyst. Our previous observations (Loppion et al 2008) have suggested that the germinative zone located between the testicular capsule and the main testicular blood vessel contains single spermatogonia, paired spermatogonia, and (at least) clusters of four interconnected spermatogonia. Therefore, undifferentiated spermatogonia were found to be distributed from stage I (germinative zone) to stage II, which could correspond to the successive types of A s , A p , and A al described for rodents.…”

“…In vertebrates and invertebrates, conserved SSC characteristics such as low mitotic activity, mottled chromatin, and contact with the basal membrane have been described for mammals (De Rooij & Russell 2000, Chiarini-Garcia et al 2003, zebrafish (Leal et al 2009), dogfish (Loppion et al 2008), and Drosophila (Joti et al 2011, Sheng & Matunis 2011. In mammals, the molecular characterization of the different spermatogonial subpopulations is in progress and a few markers have been characterized.…”

“…Cysts are composed of spermatoblasts (480-500 per cyst) in which one Sertoli cell is associated with one spermatogonium initially and with 64 spermatids in the post-meiotic stages. Cysts are formed after a proliferating phase for both spermatogonia and Sertoli precursors upon leaving the germinative zone constituting the potential stem cell compartment (Loppion et al 2008), and cysts then proceed in maturational order toward the opposite margin of the gonad. Zonation thus allows to observe all spermatogenesis stages on a cross section of the testis and to dissect the pre-meiotic state (zone A, stages I-VI), meiotic state (zone B, stages VII-X), early spermiogenesis (zone C, stages XI and XII), and late spermiogenesis (zone D, stages XIII-VVII) regions (Loir & Sourdaine 1994, Loir et al 1995.…”

“…Zone A can be further dissected to separate the area A0, which corresponds to the germinative zone (stage I) and the remaining area, A-. This area corresponds to the progenitor proliferating area (stage II), the cyst formation (stage IIIa) coinciding with the end of Sertoli cell divisions (Stanley 1966, Holstein 1969, Loppion et al 2008, and to the further four mitosis of differentiated spermatogonia leading to preleptotene (PL) spermatocytes (stages IIIb, IV, V, and VI). In this model, the number of spermatogonial divisions from stage I to stage VI was estimated to be 13 by Holstein (1969) on the basis of the number of spermatoblasts per cyst.…”