Professional phagocytic granulocytes of the bony fish gilthead seabream display functional adaptation to testicular microenvironment

Abstract

It has been shown previously that professional phagocytic granulocytes are present in the testis of the gilthead seabream, a seasonal breeding teleost that offers an excellent model for studying the testicular regression process that occurs in seasonal testicular involution and sex change. It is unexpected that testicular granulocytes produce interleukin-1␤, a regulator for spermatogonia proliferation in mammals, but are not involved in the elimination of degenerative germ cells. Here, we show that phagocytosis and reactive oxygen intermediate (ROI) production were suppressed dramatically in testicular phagocytic granulocytes, compared with their level of activity in the head-kidney, the main hematopoietic organ in fish. Furthermore, testicular-conditioned media modulated migration, phagocytosis, and ROI production of head-kidney phagocytic granulocytes, and the addition of testicular cells impaired their ROI production capacity. Until now, monocytes/ macrophages were believed to be the only innate immune cells able to develop into functional subsets, whereas neutrophils only infiltrate the tissues upon infection or inflammation. Our findings demonstrate, however, that fish professional phagocytic granulocytes also display functional adaptation to different microenvironments and strongly suggest a role for these cells in the reorganization of the testis during post-spawning. J. Leukoc. Biol. 78: 345-351; 2005.

Immunocytochemical Tools Reveal a New Research Field Between the Boundaries of Immunology and Reproductive Biology in Teleosts

“…The G7 also allowed applying the MACS techniques using micromagnetic-beads to specific purified acidophilic granulocytes guaranteeing the viability and functional characteristics of these cells as occurred in mammals (Miltenyi et al, 1990) (Figure 7). Thus, we demonstrated that the main functional activities of acidophilic granulocytes are inhibited once they migrated into the testis (Chaves-Pozo et al, 2005b). As previously discussed (see point 4) each antibody allowed a fixed number of techniques that depended on its ability to react with its specific epitope under native or denatured conditions.…”

“…In that sense we have first performed the phagocytic assay (review in Esteban et al, 1998), and then the immunostaining keeping the cells at 4ºC to avoid digestion of the phagocyted-fluorescence-bacteria and internalization of the antigen-antibody complex at the membrane. Thus, we have observed that the 95% of the head-kidney acidophilic granulocytes had phagocytic capability in contrast with the 1% of the testicular acidophilic granulocytes (Chaves-Pozo et al, 2005b;Sepulcre et al, 2002). However, most of the functional activities of the immune response can not be combine with a posterior antibody immunostaining, so the purification of the testicular acidophilic granulocytes were mandatory to really understand the role of the acidophilic granulocytes once they migrated into the gonad.…”

“…Moreover, Sertoli cells also presented this molecule in their membrane, as other tissue specific macrophages such as those located in the spleen, liver or gills . These data together with the fact that the testicular cells which were not recognized by the G7 antibody (G7-cells) have a huge phagocytic capability as revealed by an in vitro challenge with bacteria and subsequent electron microscopy analysis (Chaves-Pozo et al, 2004b, 2005b, support the hypothesis that Sertoli cells are the main phagocytic cells in the testis and in contrast to what occurs in mammals, teleosts Sertoli cells have proliferative capability (Chaves-Pozo et al, 2005a;Schulz et al, 2010). The origin of the new Sertoli cells that restart spermatogenesis during the following reproductive cycle has been widely discussed and is still a matter of controversy.…”

“…Since the antibody was able to bind to the epitope in the native conformation, a fixative was not needed, although the staining must be performed at 4ºC to avoid internalization of the protein-antibody complex (see Table 3). Analyzing the fluorescence of the immunostained cells by flow cytometry, we have determined that there was an increase in the amount of acidophilic granulocytes present in the testis at specific stages of the reproductive cycle (post-spawning and testicular involution stages) reaching the 6 and 10% of the total cells, respectively (Chaves-Pozo et al, 2005b;Liarte et al, 2007). This data, together with the fact that the acidophilic granulocytes did not proliferate in the testis, demonstrated the infiltration of this cell type from the blood stream to the testis at these specific stages of the reproductive cycle, suggesting a role on fish reproduction of these immune cells , 2005a.…”

“…Those enriched cell suspensions together with total testicular and head-kidney cell suspensions have been used in flow cytometry assays to asses phagocytosis (Esteban et al, 1998), reactive oxygen intermediates (ROI) production (Banati et al, 1994) and metalloproteinases activity (Leber and Balkwill, 1997). Combining those techniques, it has been demonstrated that headkidney acidophilic granulocytes infiltrate the gonad in response to gonadal and hormonal stimulus and showed completely different functional capabilities than their head-kidney counterparts (Chaves-Pozo et al, 2005b, 2008a: impaired phagocytic and ROI production activities although they were the majority of cells which were able to produce ROIs in the testis upon phorbol myristate acetate (PMA) stimulation (Chaves-Pozo et al, 2005b). Moreover, testicular acidophilic granulocytes also showed a completely different array of metalloproteinases activity than their head-kidney counterparts (Chaves-Pozo et al, 2008b).…”