Abstract
Two functionally distinct isoforms of warm-temperature acclimation related 65-kDa protein (Wap65-1 and Wap65-2) with a role in the immune response are present in fish. To our knowledge, contrary to Wap65-1, Wap65-2 has neither been isolated nor functionally characterized in carp especially in reproductive system. The aim of this study was to characterize Wap65-2 and ascertain its functions in immune response and temperature acclimation within reproductive system. Wap65-2 corresponded to one of the most abundant proteins in carp seminal plasma, with a high immunologic similarity to their counterparts in seminal plasma of other fish species and a wide tissue distribution, with predominant expression in the liver. The immunohistochemical localization of Wap65-2 to spermatogonia, Leydig cells, and the epithelium of blood vessels within the testis suggests its role in iron metabolism during spermatogenesis and maintenance of blood-testis barrier integrity. Wap65-2 secretion by the epithelial cells of the spermatic duct and its presence around spermatozoa suggests its involvement in the protection of spermatozoa against damage caused by heme released from erythrocytes following hemorrhage and inflammation. Our results revealed an isoform-specific response of Wap65 to temperature acclimation and Aeromonas salmonicida infection which alters blood-testis barrier integrity. Wap65-2 seems to be related to the immune response against bacteria, while Wap65-1 seems to be involved in temperature acclimation. This study expands the understanding of the mechanism of carp testicular immunity against bacterial challenge and temperature changes, in which Wap65-2 seems to be involved and highlights their potential usefulness as biomarkers of inflammation and temperature acclimation.
Highlights
Fish warm-temperature acclimation related 65 kDa protein (Wap65) is an ortholog of hemopexin (Hpx, alternative name beta-1B-glycoprotein), a protein that has been identified in all vertebrates [1]
Wap65 function is mainly related to warm-temperature acclimation, which is most significant in eurythermal fish such as carp that live over a wide range of temperatures, from near zero to over 30 °C [5,6,7, 9, 10, 14, 15, 20, 22]
To confirm that the detected spots in seminal plasma corresponded to Wap65-2, all spots related to the blot were identified by mass spectrometry MALDI-matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (TOF)/TOF (Additional file 3)
Summary
Fish warm-temperature acclimation related 65 kDa protein (Wap65) is an ortholog of hemopexin (Hpx, alternative name beta-1B-glycoprotein), a protein that has been identified in all vertebrates [1]. In contrast to a single isoform of Hpx in mammals, there are two functional Wap isoforms in fish (Wap and Wap65-2) [10,11,12, 14, 16, 18, 19, 21,22,23,24,25] These isoforms display distinct tissue distribution patterns and physiological functions in response to stimuli. Wap is widely expressed and strongly regulated by an increase in temperature, while Wap has a restricted tissue distribution (mainly in the liver) and is more regulated by bacterial infection rather than warmtemperature stress; that fact indicates its role in immune response in several teleosts [18,19,20, 22,23,24]. The expression patterns of Wap and Wap and response to different stimulatory treatments are variable and contradictory depending on the fish species [10,11,12, 14, 21, 23, 25]
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