Alfonsa García Ayala

Early innate immune response and redistribution of inflammatory cells in the bony fish gilthead seabream experimentally infected with Vibrio anguillarum.

An obvious difference between the immune system of fish and mammals is that fish lack both bone marrow and lymph nodes; in their place, the head-kidney acts as a major haematopoietic and lymphoid organ in adult fish, whereas the thymus, spleen and mucosa-associated lymphoid tissues are common to both fish and mammals. This suggests that differences exist in antigen presentation and naïve lymphocyte stimulation, a prerequisite for the initiation of adaptive immune responses. Intraperitoneal injection of the bony fish gilthead seabream (Sparus aurata L.) with intact Vibrio anguillarum, as a particulate bacterial antigen, results in the mobilisation of head-kidney leucocytes to the peritoneal cavity and priming of their respiratory burst activity. We have also observed the rapid infiltration of acidophilic granulocytes, which are leucocytes functionally equivalent to mammalian neutrophils, into the spleen. These cells may be involved in antigen capture and transport to the spleen, since an apparent association between mobilised acidophilic granulocytes, bacterial antigens and proliferating lymphocytes has been seen in this organ. Collectively, these results suggest that, in addition to being actively involved in bacterial clearance, fish phagocytic granulocytes play a role in the initiation and support of the adaptive immune response.

An Overview of Cell Renewal in the Testis Throughout the Reproductive Cycle of a Seasonal Breeding Teleost, the Gilthead Seabream (Sparus aurata L)

Abstract The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost with a bisexual gonad that offers an interesting model for studying the testicular regression process that occurs in both seasonal testicular involution and sex change. Insofar as fish reproduction is concerned, little is known about cell renewal and elimination during the reproductive cycle of seasonal breeding teleosts with asynchronous spermatogenesis. We have previously described how acidophilic granulocytes infiltrate the testis during postspawning where, surprisingly, they produce interleukin-1β, a known growth factor for mammalian spermatogonia, rather than being directly involved in the elimination of degenerative germ cells. In this study, we are able to discriminate between spermatogonia stem cells and primary spermatogonia according to their nuclear and cytoplasmic diameters and location in the germinal epithelium, finding that these two cell types, together with Sertoli cells, proliferate throughout the reproductive cycle with a rate that depends on the reproductive stage. Thus, during spermatogenesis the spermatogonia stem cells, the Sertoli cells, and the developing germ cells (primary spermatogonia, A and B spermatogonia, and spermatocytes) in the germinal compartment, and cells with fibroblast-shaped nuclei in the interstitial tissue proliferate. However, during spawning, the testis shows few proliferating cells. During postspawning, the resumption of proliferation, the occurrence of apoptotic spermatogonia, and the phagocytosis of nonshed spermatozoa by Sertoli cells lead to a reorganization of both the germinal compartment and the interstitial tissue. Finally, the proliferation of spermatogonia increases during resting when, unexpectedly, both oogonia and oocytes also proliferate. This proliferative pattern was correlated with the gonadosomatic index, testicular morphology, and testicular and gonad areas, suggesting that complex mechanisms operate in the regulation of gonocyte proliferation in hermaphrodite fish.

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

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‐1β, 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.

Investigation into the duality of gonadotropic cells of Mediterranean yellowtail (Seriola dumerilii, Risso 1810): immunocytochemical and ultrastructural studies.

Two antisera against the follicle-stimulating hormone-like gonadotropin (FSH) of Mediterranean (M.) yellowtail, anti-My FSHa and anti-My FSHb, were obtained. Anti-My FSHa serum specifically recognized FSH cells and did not react with any other pituitary cell type, while anti-My FSHb serum recognized the alpha-subunit of the pituitary glycoprotein hormones and immunostained FSH, luteinizing hormone-like gonadotropin (LH), and thyrotropin (TSH) cells. Anti-My FSHa serum, together with a previously obtained anti-My LHbeta serum, were used to further investigate the duality of gonadotropic cells in M. yellowtail by light and electron microscopic immunocytochemistry; three immunologically different gonadotropic cell populations expressing FSH, LH, or both hormones, were revealed. The three cell populations had the same regional distribution in the pituitary gland: the proximal pars distalis, including the thin ring surrounding the pars intermedia. However, while FSH cells were found isolated or forming small clusters, LH cells formed strands or compact groups, and were more numerous than FSH cells. FSH/LH cells were scarce. At the ultrastructural level, vesicular, granular, and intermediate FSH, LH, and FSH/LH cells were found; secretory granules and globules, on the one hand, or conspicuous dilated cisternae of rough endoplasmic reticulum (or both) predominated, respectively, in these cell types. The production of either FSH or LH, or both hormones, was not reflected in the ultrastructural features of gonadotropic cells. Thus, a single morphological cell type of varying ultrastructure depending on the functional stage seemed to encompass all gonadotropic cells in M. yellowtail. All forms of FSH, LH, and FSH/LH cells were found in involution.

Flow cytometry based techniques to study testicular acidophilic granulocytes from the protandrous fish gilthead seabream (Sparus aurata L.).

The gilthead seabream is a protandrous seasonal breeding teleost that is an excellent model for studying the testicular regression process which occurs in both seasonal testicular involution and sex reversion. Little is known about the cell types and the molecular mechanisms involved in such processes, mainly because of the lack of appropriate methods for testis dissociation, and testicular cell isolation, culture and functional characterization. We have previously reported that gilthead seabream acidophilic granulocytes infiltrate the testis at post-spawning stage, settle close to the spermatogonia and accumulate intracellular interleukin-1β. In this paper, we report several flow cytometry based assays which allow to establish the role played by gilthead seabream testicular acidophilic granulocytes and permits their quantification.