U radu je data uporedna ultrastrukturalna analiza makrofaga testisa Ohridske pastrmke (Salmo letnica Kar.) i Ohridske belvice (Acantholingua ohridana) u periodu posle mresta. U semenifernim režnjevima ovih vrsta sa degenerisanim Sertolijevim materijalom može se opaziti prisustvo makrofaga. Oni se nalaze ili u zidu režnjeva, ili u njihovoj šupljini. Makrofagi poseduju karakterično produbljeno jedro. Neki makrofagi imaju proširen perinuklearni prostor. Makrofagi imaju segmentirano jedro, što je karakteristično za sve ćelije i citoplazmu bogatu fagolizozomima. Njihovo prisustvo u semenim režnjevima kada teče intenzivna involucija tj. degeneracija Sertolijevih ćelija može se opaziti po njihovoj eventualnoj fagocitozi, odnosno eliminaciji nekrotičnog materijala koji potiče iz Sertolijevih ćelija

Jordanova, Maja

Rebok, Katreina

Tavchiovska-Vasileva, Irena

English

CaSA NaRA

V MEĐUNARODNA KONFERENCIJA ”AKVAKULTURA I RIBARSTVO” - ZBORNIK PREDAVANJA   223ultrastructural vieW of tHe  MacropHaGes of testes of tWo oHriD  salMons, oHriD trout (SalMO lETniCa Kar.) anD oHriD belvica (aCanTHOlingua OHriDana) IRENA TAVCHIOVSKA-VASILEVA, KATREINA REBOK,  MAJA JORDANOVAInstitute of Biology, Faculty of Natural sciences and Mathematics, 1000 Skopje,  Republic of MacedoniaULTRASTRUKTURNI IZGLED MAKROFAGA U TESTISIMA DvE  OHRIDSKE PASTRMKE, OHRIDSKE PASTRMKE (SAlMO lETNIcA KAR.) I OHRIDSKE BELvIcE (AcANTHOlINguA OHRIDANA)AbstraktU radu je data uporedna ultrastrukturalna analiza makrofaga testisa Ohridske pa-strmke (Salmo letnica Kar.) i Ohridske belvice (Acantholingua ohridana) u periodu posle mresta. U semenifernim režnjevima ovih vrsta sa degenerisanim Sertolijevim ma-terijalom može se opaziti prisustvo makrofaga. Oni se nalaze ili u zidu režnjeva, ili u njihovoj šupljini. Makrofagi poseduju karakterično produbljeno jedro. Neki makrofagi imaju proširen perinuklearni prostor. Makrofagi imaju segmentirano jedro, što je ka-rakteristično za sve ćelije i citoplazmu bogatu fagolizozomima. Njihovo prisustvo u semenim režnjevima kada teče intenzivna involucija tj. degeneracija Sertolijevih ćelija može se opaziti po njihovoj eventualnoj fagocitozi, odnosno eliminaciji nekrotičnog materijala koji potiče iz Sertolijevih ćelija.Ključne reči: Ohridska pastrmka (Salmo letnica Kar.), Ohridska belvica (Acantho-lingua ohridana), testis, makrofagi, period posle mrestaINTRODUcTIONIn literature there are data wich describe macrophages as phagocytotic elements of testes in many Teleost fish (Chavez-Pozo et al., 2005, 2007;Hales, 2002; Hedger, 2002; Lahnsteiner & Patzner, 1990; Liarte et al., 2007; McClusky, 2005; Micale et al, 1987; 224 V INTERNATIONAL CONFERENCE “AQUACULTURE & FISHERY” - CONFERENCE PROCEEDINGSSepulcre et al., 2002; Young, 2001). Also macrophages was noticed in Chondroichtyes, in the spotted ray (Torpedo marmorata) by Prisco et al. (2003). There are literature data which poin to the participation of the macrophages (besides Sertoli cells) in the elimina-tion of the sperm residues with some species of Teleostea in the period after the spawn-ing.This phenomenon with Salvelinus fontinalis was described by Henderson (1962), with Rutilus rutilus dojranensis K a r . by Dimovska (1965), with Oryzias latipes by Gresek et al. (1973) and also was stated with other Vertebrata, for example with swan (Cygnus olor) by Breucker (1978). The presence of macrophages with Preca fluviatilis macedonica K a r. in the period after the spawning was pointed out by Dimovska et al. (1986/87), Tavciovska-Vasileva (1992). According to Loir et al. (1995) with trout the interlobular macrophages can participate in the reinitiation of the spermatogonial pro-liferation. Macrophages was noticed in Salmonidae, in rainbow trout (Salmo gairdneri) by Billard et al. (1983); Scott & Sumpter (1989); Van den Hurk et al. (1978). The pres-ence of these phagocytotic elements also was noticed with Ohrid trout (Salmo letnica Kar.) and Ohrid belvica (Acantholingua ohridana) in the period after the spawinig by Tavciovska-Vasileva (1999, 2001), Tavciovska-Vasileva & Rebok (2004). The presence of macrophages is connected with the phagocytosis of Sertoli cells, because they are present in interstitium, as well as in the seminiferous lobules.MATERIAL AND METHODSTestes of sexually mature Ohrid trout (Salmo letnica Kar.) and Ohrid belvica (Acan-tholingua ohridana) males caught in and Ohrid Lake were analysed. Analyses have been done with electronic microscope. Small parts of testes 1-2 mm big have been used for electronic microscopy. The material has been fixed by the following procedure: im-mediately after the tissue sections have been taken, they are fixed in 3%  glutaraldehyde and then conserved in 0, 1 M phosphate buffer. After adequate fixation the material has been submitted to postfixation in 1% osmium tetraoxid (OsO4). In the further treatment the material has been washed in phosphate buffer, dehydrated in series of acetone and uranil acetate, and after that it has been dehydrated in dry acetone. The tissue sections have been infiltrated with Durcopan ACM mixture, a mixture of acetone-Durcopan, Durcopan No. 1, Durcopan No. 2, fit in Durcopan No. 2 and polymerised. For the ultra-structural analysis, ultrathin sections of 40-60 ηm tickness have been prepared, with the help of galss knives, on Reichert-Yung “Ultacut” ultramicrotome, installed on copper nets, contrasted with uranil acetate and lead cytrate. The sections have been observed on Tesla BS 500 and OPTON (Zeis) EM 109 electronic microscope. The microphoto-graphs for electronic microscopy have been photographed with Agfa Scientia EM Film 23056/6, 5 x 9 cm, ORWO NP 20 panchromatic 120, KODAK 120 and made on Agfa Papirtone Paper P1-3.RESULTSIn the seminiferous lobules of Ohrid trout (Salmo letnica Kar.) where Sertoli degen-erated material is present, it is characteristic that there are macrophages located in the wall of the lobules, in their lumen or in the interstitium (Fig. 1). The macrophages pos-sess a characteristic deepened nucleus. Some macrophages have widened perinuclear space (Fig. 2).V MEĐUNARODNA KONFERENCIJA ”AKVAKULTURA I RIBARSTVO” - ZBORNIK PREDAVANJA   225Figure 1. A part of the cytoplasm of Sertoli cell (SK) in degeneration. A part of the lumen (L) of the lobulus. Developed interstitium (I) with macrophage (MF) and blood vessel (KS). Well seen basal lamina (arrows) of the lobulus. Ultrathin section, x 4.400.Figure 2. Macrophafe MF with deepened nucleus (N) with widened perinuclear space (arrows). Ultrathin section, x 12.000The presence of macrophages in seminiferous lobules in the period of intensive de-genetation, i. e. involution od Sertoli cells shows that these phagocyte elements prob-ably participate in elimination (phagocytosis) of Sertoli necrotic material. Macrophages can be noticed in the lobules with the Sertoli degenerated material. With Ohrid belvica (Acantholingua ohridana) the macrophages have been noticed in the wall of the lobules, as well as in the lumen. At ultrastructural level we can see that macrophages possess a segmented nucleus (Fig. 3) which is characteristic for these cells, and a cytoplasm reach with phagolysosomes (Fig. 4, 5). Their presence in the seminiferous lobules when intensive involution, i. e degeneration of Sertoli cells goes on, can be connected with their eventual phagocytosis, i. e. elimination of the necrotic material, which originates from the Sertoli cells. 226 V INTERNATIONAL CONFERENCE “AQUACULTURE & FISHERY” - CONFERENCE PROCEEDINGSFigure 3. Macrophage (MF) with segmented nucleus (N). Ultrathin section, x 12.000.Figure 4. Macrophage (MF) and cytoplasm rich with phagolysosomes (arrows). Ul-trathin section, x 7.000.V MEĐUNARODNA KONFERENCIJA ”AKVAKULTURA I RIBARSTVO” - ZBORNIK PREDAVANJA   227Figure 5. A part of macrophage (MF) with nucleus (N) and cytoplasm (C) with phagolysosomes (arrows). Ultrathin section, x 12.000.DIScUSSIONIn the testes of these two analysed species Ohrid trout (Salmo letnica Kar.) and Ohrid belvica (Acantholingua ohridana ) the presence of macrophages have been stated on the level of interstitium, as well as in the wall or in the lumen of the seminiferous lobules by Tavciovska-Vasileva (1999, 2001), Tavciovska-Vasileva & Rebok (2004). Macrophages was noticed in Salmonidae, in rainbow trout (Salmo gairdneri) by Billard et al. (1983); Scott & Sumpter (1989); Van den Hurk et al. (1978). Their presence can point to their possible participation in the elimination of the Sertoli necrotic material. This phenomenon in Salvenilus fontinalis was pointed out by Henderson (1962), Rutilus rutilus dojranen-sis Kar. (Dimovska, 1965), Oryzias latipes (Gresek et al., 1973), Perca fluviatilis mace-donica Kar. (Dimovska et al, 1986/87; Tavciovska-Vasileva, 1992). In literature there are data wich describe macrophages as phagocytotic elements of testes in many other Teleost fish (Chavez-Pozo et al., 2005, 2007;Hales, 2002; Hedger, 2002; Lahnsteiner & Patzner, 1990; Liarte et al., 2007; McClusky, 2005; Micale et al, 1987; Sepulcre et al., 2002; Young, 2001). Also macrophages was noticed in Chondroichtyes, in the spotted ray (Torpedo marmorata) by Prisco et al. (2003). It is also stated with other Vertebrata, for example with swan (Cygnus olor) by Breucker (1978). Because of the fact that they ap-pear in the interlobular interstitium, as well as in the wall and in the lumen of the seminif-erous lobules when degeneration, i. e. involution of Sertoli cells happenes, their presence can be connected with phagocytosis of the residues which originate from Sertoli necrotic (degenerated) material. With the trout, according to Loir et al. (1995) the interlobular macrophages can participate in reinitiation of the spermatogonial proliferation.cONcLUSIONWith the cytological analysis based on ultrastructural findings of the testes of Ohrid trout (Salmo letnica Kar.) and Ohrid belvica (Acantholingua ohridana) we can state that macrophages can be noticed in the wall and in the lumen  of  some seminiferous lobules, and their role can be connected with the elimination of Sertoli necrotic material.228 V INTERNATIONAL CONFERENCE “AQUACULTURE & FISHERY” - CONFERENCE PROCEEDINGSREFERENcESBillard R, Takashima F. (1983): Resorption of spermatozoa in the sperm duct of rain-bow trout during the post-spawning period. Bull Japan Soc Sci Fish. 49, 387–392.Breucker, H. (1978): Macrophages, a normal component in seasonally involuting Testes of Swan, Cygnus olor. Cell Tiss. Res. 193, 465-471.Chaves-Pozo E, Liarte S, Vargas-Chacoff L, García-Lopez A, Mulero V, Meseguer J, Mancera JM, García-Ayala A. (2007):17β-estradiol triggers postspawning in spermato-genically active gilthead seabream (Sparus aurata L.) males. Biol Reprod.76,142–148.Chaves-Pozo E, Mulero V, Meseguer J, García-Ayala A. (2005): Professional phago-cytic granulocytes of the bony fish gilthead seabream display functional adaptation to testicular microenvironment. J Leukoc Biol. 78, 345–351.Dimovska, A. (1965): Prilog kon poznavanjata na poloviot ciklus na dojranskata crvenoperka (Rutilus rutilus dojranensis K a r.). Doktorska disertacija, Skopje.Dimovska, A., Tavciovska, I., Karaman, B. (1986/87): Citomorfoloski aspekt na Ser-toli kletkite na Dojranskata perkija (Perca fluviatilis macedonica K a r.) vo postmres-titelniot period. God. Zbornik na PMF, Biol., Skopje, 39-40, 117-127.Gresik, E. W., Quirk, J. K., Hamilton, J. B. (1973): Fine structure of the Sertoli cells of the testis of the teleost Oryzias latipes. Gen. Comp. Endocrinol. 21, 341-352.Hales DB. ( 2002): Testicular macrophages modulation of Leydig cell steroidogen-esis. J Reprod Immunol.  57, 3–18.Hedger MP. (2002): Macrophages and the immune responsiveness of the testis. J Reprod Immunol. 57, 19–34.Henderson, N. E. (1962): The annual cycle in the testis of Eastern Brook trout, Salvelinus fontinalis (Mitchill). Can. J. Zool. 40, 631-641.Lahnsteiner F, Patzner RA. (1990): The mode of male germ cell renewal and ultra-structure of early spermatogenesis in Salaria (= Blennius) pavo (Teleostei: Blenniidae) Zool Anz. 224, 129–139.Liarte, S., Chaves-Pozo, E., Garcia-Aleazar, A., Mulero, V., Meseguer, J., Garcia-Ayala, A. (2007): Testicular involution prior to sexchange in gilthead seabream is char-acterized by decrease in DMRT1 gene expression and by massive leukocyte infiltration. Reprod. Biol. Endocrinol. 5, 20. Loir, M., Sourdaine, P., Mendishandagama SMLC., Jegon, B (1995): Cell-cell in-teractions in the testis of the teleosts and elasmobranchs. Microscopy Research and Technique 32, 6, 533-552.McClusky LM. (2005): Stage and season effects on cell cycle and apoptotic activi-ties of germ cells and Sertoli cells during spermatogenesis in the spiny dogfish (Squalus acanthias) Reproduction, 129, 89–102.Micale V, Perdichizzi F, Santangelo G. (1987): The gonadal cycle of captive white bream, Diplodus sargus (L.) J Fish Biol. 31, 435–440.Prisco M, Liguoro A, Comitato R, Cardone A, D’Onghia B, Ricchiari L, Angelini F, Andreuccetti P. (2003): Apoptosis during spermatogenesis in the spotted ray Torpedo marmorata . Mol Reprod Dev. 64, 341–348.Scott AP, Sumpter JP. (1989): Seasonal variations in testicular germ cell stages and in plasma concentrations of sex steroids in male rainbow trout (Salmo gairdneri) matur-ing at 2 years old. Gen Comp Endocrinol. 73, 46–58.Sepulcre MP, Pelegrín P, Mulero V, Meseguer J. (2002): Characterisation of gilthead seabream acidophilic granulocytes by a monoclonal antibody unequivocally points to V MEĐUNARODNA KONFERENCIJA ”AKVAKULTURA I RIBARSTVO” - ZBORNIK PREDAVANJA   229their involvement in fish phagocytic response. Cell Tissue Res. 308, 97–102.Tavciovska-Vasileva, I. (1992): Histoloska struktura na semenikot na Dojranskata perkija (Perca fluviatilis macedonica K a r.) vo periodot po mrestenjeto. Magisterski trud, Skopje.Tavciovska-Vasileva, I. (1999): Komparativni strukturni i ultrastrukturni karakteris-tiki na semenicite kaj Salmonidae (Pisces: Teleostei) od Ohridskoto Ezero vo postmres-titelniot period. Doktorska disertacija, Skopje.Tavciovska-Vasileva, I. (2001): The role of macrophages on the level of seminiferous lobules of Ohrid trout (Salmo letnica K a r.) and Ohrid belvica (Salmothymus ochri-danus Steind.) in the postspawning period. Veterinary Medicine VII, 1-2, 61-64.Tavciovska-Vasileva, I., Rebok, K. (2004): Ultrastructural features of the macrophag-es of testes od Salmonidae from Ohrid Lake. Conference of Water Observation and Information Sistem for Decision Support (BALWOIS). Proceedings, Topic 6: Lakes, p. 1-4. Van den Hurk R, Peute J, Vermeij JAJ. (1978): Morphological and enzyme cy-tochemical aspects of the testis and vas deferens of the rainbow trout, Salmo gairdneri. Cell Tissue Res.186, 309–325.Young KA, Nelson RJ. (2001): Mediation of seasonal testicular regression by apop-tosis. Reproduction 122,  677–685.

Ultrastrukturni izgled makrofaga u testisima dve ohridske pastrmke, ohridske pastrmke (salmo letnica kar.) i ohridske belvice (acantholingua ohridana)

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Ultrastrukturni izgled makrofaga u testisima dve ohridske pastrmke, ohridske pastrmke (salmo letnica kar.) i ohridske belvice (acantholingua ohridana)

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