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On the Flood Forecasting at the Bulgarian Part Of BALWOIS 2004 Ohrid, FY Republic of Macedonia, 25-29 May 2004 Ultrastructural Features Of The Macrophages Of Testes Of Salmonidae From Ohrid Lake Irena Tavciovska-Vasileva & Katerina Rebok Institute of Biology Skopje, Republic of Macedonia Abstract In this paper the ultrastructural analysis of macrophages of the testes of salmonidae from Ohrid lake has been done. In the seminiferous lobules of Ohrid trout, Salmo letnica (Karaman) and Ohrid belvica (Acantholingua ohridana) where Sertoli degenerated material is present, it is characteristic that there are macrophages located in the wall of the lobules and in their lumen. The macrophages possess a characteristic deepened nucleus. Some macrophages have widened perinuclear space. At ultra structural level we can see that macrophages possess a segmented nucleus, which is characteristic for these cells, and a cytoplasm rich with phagolysosomes. Their presence in the seminiferous lobules when an intensive degeneration (involution) of sertoli cells goes on, can be noticed with their eventual phagocytosis, i.e. elimination of the necrotic material, which originates from the Sertoli cells. It has to be pointed out that presence of macrophages in the postspawning period is much more intensive in Ohrid belvica (Acantholingua ohridana) in contrast to the Ohrid trout, Salmo letnica (Karaman). Introduction In the literature there are the data which point to the participation of the macrophages in elimination of the sperm residues with some species of Teleostei in the postspawning period. This phenomenon with Salvelinus fontinalis was described by Henderson (1962), with Rutilus rutilus dojranensis K a r. by Dimovska (1965), with Oryzias latipes by Gresik et al. (1973), and also was stated with other Vertebrata, for example with swan (Cygnus olor) by Breucker (1978). The presence of macrophages with Perca fluviatilis macedonica K a r. in the postspawning period was pointed out by Dimovska et al. (1986/87), Tavciovska-Vasileva (1992). 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. According to Loir et al. (1995) with trout the interlobular macrophages can participate in the reinitiation of the spermatogonoal proliferation. Material and methods Testes of sexually mature Ohrid trout, Salmo letnica (Karaman) and Ohrid belvica (Acantholingua ohridana) males caught in Ohrid lake in a period of 3 years (1993/96) were analysed. Analyses have been done with electronic microscope. Smal parts of testes 1-2 mm big have been used for electronic microscopy. The material has been fixed by the following procedure: immediately after the tissue sections have been taken, they are fixed in 3% glutaraldehyde and then conserved in 0, 1 M phosphate puffer. After adequate fixation the material has been submitted to post fixation in 1% osmium tetraoxid (OsO4 ). In the further treatment the material has been washed in phosphate puffer, 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 N° 1, Durcopan N° 2, fit in Durcopan N° 2 and polymerised. For the ultra structural analysis, ultra thin sections of 40-60 ηm thickness have been prepared, with the help of glass knives, on Reichert-Yung “Ultracut: ultra microtome, installed on copper nets, contrasted with uranil acetate and lead cytrate. The sections have been observed on Tesla BS 500 and OPTON (Zeiz) EM 109 electronic microscope. The microphotographs 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. Lakes 1 BALWOIS 2004 Ohrid, FY Republic of Macedonia, 25-29 May 2004 Results In the seminiferous lobules of Ohrid trout, Salmo letnica (Karaman) where Sertoli degenerated material is present, it is characteristic that there perinuclear space (Fig. 1). With Ohrid belvica (Acantholingua ohridana) similarly to the trout, the macrophages have been noticed in the wall of the lobules, as well as in the lumen. At ultra structural level we can see that macrophages possess a segmented nucleus, which is characteristic for these cells, and a cytoplasm rich with phagolysosomes (Fig. 2, 3, 4). The presence of macrophages in seminiferous lobules in both species in the period of intensive degeneration, i.e. involution of Sertoli cells shows that these phagocyte elements probably participate in elimination (phagocytosis) of Sertoli necrotic material. Figure 1 Macrophage (MF) with deepened nucleus (N), with widened perinuclear space (arrows). Ultra thin section, 12.000x. Figure 2 Macrophage (MF) with segmented nucleus (N). Ultra thin section, 12.000x. Lakes 2 BALWOIS 2004 Ohrid, FY Republic of Macedonia, 25-29 May 2004 Figure 3 Macrophage (MF) and cytoplasm rich with phagolysosomes (arrows). Ultra thin section, 7.000x. Figure 4 A part of macrophage (MF) with nucleus (N) and cytoplasm (C) with phagolysosomes (arrows). Ultra thin section, 12.000x. It has to be pointed out that the presence of macrophages in the postspawning period is much more intensive in Ohrid belvica (Acantholingua ohridana) in contrast to the Ohrid trout, Salmo letnica (Karaman) in the same period. Discusion In the testes of the both analysed species, Ohrid trtout, salmo letnica (Karaman) and Ohrid belvica (Acantholingua ohridana) the presence of macrophages have been stated, especialy with Ohrid belvica on the level of the interstitium, as well as in the lumen of the seminiferous lobules. Their presence can point to their possible participation in the elimination of Sertoli necrotic material. This phenomenon in salvelinus fontinalis was pointed out by Henderson (1962), Rutilus rutilus dojranensis K a r. (Dimovska, 1965), Oryzias latipes (Gresik et al., 1973), and it was also stated with other Vertebrata, for example with swan (Cygnus olor) by Breucker (1978). In Perca fluviatilis macedonica K a r. (Dimovska et al., 1986/87; Tavciovska-Vasileva, 1992) also pointed to the presence of macrophages in the postspawning period. Lakes 3 BALWOIS 2004 Ohrid, FY Republic of Macedonia, 25-29 May 2004 In our previos examinations the presence of macrophages in the testes of Ohrid trout, Salmo letnica (Karaman) and Ohrid belvica (Acantholingua ohridana) was also stated in the postspawning period (Tavciovska-Vasileva, 1999). Because of the fact that they appear in the interlobular interstitium, as well as in the lumen of the seminiferous lobules when degeneration (involution) of Sertoli cells happens, their presence can be connected with phagocytosis of the residues which originate from Sertoli degenerated (necrotic) material. With the trout according to Loir et al. (1995) the interlobular macrophages can participate in reinitiation of the spermatogonial proliferation. General conclusion With the cytologic analysis based on ultra structural findings of the testes of Ohrid trout, Salmo letnica (Karaman) and Ohrid belvica (Acantholingua ohridana) we can state that macrophages possess a characteristic deepened and segmented nucleus, widened perinuclear space and a cytoplasm rich with phagolysosomes. They can be noticed in the lumen of some seminiferous lobules, and their role can be connected with the elimination of Sertoli necrotic material. Their presence is significantly weaker in Ohrid trout, Salmo letnica (Karaman). Reference Breucker, H. (1978) Macrophages, a normal component in seasonally involuting Testes of Swan. Cell Tiss. Res., 193: 465-471. Dimovska, A. (1965) Prilog kon poznavanjata na poloviot ciklus na dojranskata crvenoperka (Rutilus rutilus dojranensis K a r.). Doktorska disertacija, Skopje, Makedonia. Dimovska, A. (1965) Favor toward knowledge of sex cycle of Dojran robin (Rutilus rutilus dojranensis K a r.). PhD Thesis, Skopje, Macedonia. Dimovska, A., Tavciovska, I. & Karaman, B. (1986/87) Citomorfoloski aspekt na Sertoli kletkite na Dojranskata perkija (Perca fluviatilis macedonica K a r.) vo postmrestitelniot period. God Zbornik na PMF, Biol., Skopje, 39-40: 117-127. Dimovska, A., Tavciovska, I. & Karaman, B. (1986/87) Cytomorphological aspect of sertoli cells of Dojran Perch (Perca fluviatilis macedonica K a r.) in the postspawning period. Annual Biology, 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 teleost Oryzias latipes. Gen. Comp. Endocrinol., 21: 341-352. Henderson, N. E. (1962) The annual cycle in the testis of the Eastern Brook trout, Salvelinus fontinalis (Mitchill). Can. J. Zool., 40: 631-641. Loir, M., Sourdaine, P., Mendishandagama SMLC. & Jegon, B. (1995) Cell-cell interactions in the testis of the teleosts and elasmobranchs. Microscopy Research and Technique, 32 (6): 533-552. Tavciovska-Vasileva, I. (1992) Histoloska struktura na semenikot na Dojranskata perkija (Perca fluviatilis macedonica K a r.) vo periodot po mrestenjeto. Magisterski trud, Skopje, Makedonia. Tavciovska-Vasileva, I. (1992) Histological structure of the testis of Dojran Perch (Perca fluviatilis macedonica K a r.) in the period after the spawning. MSc Thesis, Skopje , Macedonia. Tavciovska-Vasileva, I. (1999) Komparativni strukturni i ultrastrukturni karakteristiki na semenicite na Salmonidae (Pisces: Teleostei) od Ohridskoto Ezero vo postmrestitelniot period. Doktorska disertacija, Skopje, Makedonija. Tavciovska-Vasileva, I. (1999) Comparative structural and ultrastructural characteristics of the testes of Salmonidae (Pisces: Teleostei) from Ohrid Lake in postspawning period. PhD Thesis, Skopje, Macedonia. Lakes 4 .
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