Spermatogonial stem cell organization in felid testis as revealed by Dolichos biflorus lectin

Abstract

Spermatogonial stem cells are being exploited in many species as a tool to recover fertility, but may also be used to manipulate the genetic pool. Whatever the purpose, these cells must be fully characterized and easily identifiable, and our goal was to improve this procedure in the domestic cat, used as an animal model for endangered felid species and for some human diseases/physiological processes. We have therefore screened several markers that might be used to distinguish and study the undifferentiated spermatogonia population in situ and in vitro via immunohistochemistry applied to tissue sections and whole mounts of the domestic cat seminiferous tubules. Our results show that, although they label the cytoplasm and nucleus of gonocytes and spermatogonia in pre-pubertal animals, PGP9.5 and FoxO1 cannot be considered markers of undifferentiated spermatogonia in adult animals, as almost all spermatogonia, namely type A and B, express these proteins. Nonetheless, the Dolichos biflorus agglutinin (DBA lectin) was able to label the cell surface and cytoplasm of a small type A spermatogonial population in the adult animals. Analysis of the number and distribution of the DBA-labeled cells showed they were present in low number, which did not vary with epithelium seminiferous stage. Morphometric analysis revealed that DBA-labeled cells present tropism to a peculiar area of the seminiferous tubules, namely the area in direct contact with Leydig cells. Whole mounts of DBA-stained seminiferous tubules revealed the arrangement of DBA-stained cells in small clones up to eight cells. Noteworthy, the clonal cells presented variable staining intensity suggesting the existence of asymmetric distribution of O-glycosylated proteins within each clone. Our results strongly suggest that the DBA lectin is a marker of undifferentiated spermatogonia in domestic cat, and illustrate the peculiar characteristics of spermatogonial stem cell development and organization in this species.

Spermatogonial stem cells identified by molecular expression of PLZF, integrin β1 and reactivity to Dolichos biflorus agglutinin in alpaca adult testes

“…The loss of GalNAc epitopes is one of the earliest cell surface change that can be assigned to differentiating cells (Nash et al, ). Our results are supported by previous reports, where the cell and cytoplasm of a spermatogonia population, especially undifferentiated spermatogonia, in felid testis were labelled by DBA in situ and in vitro by immunohistochemistry (Escada‐Rebelo et al, ).…”

“…Likewise, we identify alpaca SSCs in adult individuals through flow cytometry with the fluorescent DBA probe, which recognises type A spermatogonia in different species such as cattle, buffalos and cats (Ahmad et al, ; Aponte et al, ; Escada‐Rebelo et al, ; Fujihara, Kim, Minami, Yamada, & Imai, ; Izadyar, Matthijs‐Rijsenbilt, et al, ; Izadyar, Spierenberg, et al, ). Unlike what occurs in pigs, SSC recognition with DBA reactivity identifies gonocytes and primitive spermatogonia in the neonatal stage.…”

Can we induce spermatogenesis in the domestic cat using an in vitro tissue culture approach?

“…PGP9.5 (Protein gene product 9.5), also known as UCHL-1 (Ubiquitin carboxy-terminal hydrolase 1), which is expressed in spermatogonia in a variety of species, including mouse [ 30 ], monkey [ 31 ], human [ 32 ], bovine [ 33 ] and pig [ 34 ]. In the domestic cat, PGP9.5 labels gonocytes and spermatogonial stem cells in immature animals and all spermatogonia and early spermatocytes (pre-leptotene) in pubertal and adult animals [ 35 ]. In our cat organ culture the anti-PGP9.5 antibody labeled all the germ cells present indicating that no germ cell had crossed the meiotic barrier.…”

Molecular phenotyping of domestic cat (Felis catus) testicular cells across postnatal development – A model for wild felids

“…Unlike in mice or humans, characterisation of feline testicular cell types, and in particular, subtypes of spermatogonia, is still challenging since there is a paucity of antibodies raised against feline antigens. Indeed, the few studies where immunostaining of feline testicular cells has been performed relied on antibodies validated for cross-reactivity in various species, but not in felines [ 17 – 21 ]. Additionally, disparate tissue fixation, embedding, antigen retrieval and immunostaining protocols across research groups are hurdles that produce conflicting results that are difficult to reconcile.…”