Abascal et al.: Testicular development in Thunnus thynnus 



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of electron-dense perinuclear material ("nuages") that 

 indicate nucleocytoplasmic transport (Fig. 5, A and B). 

 Such chromatoid bodies persist throughout spermatogen- 

 esis until the spermatid stage, but their size and number 

 is far higher in primary spermatogonia. Spermatogonia 

 B are grouped in clusters of a few cells. They are per- 

 ceptibly smaller (6.75 ±0.37 um) than spermatogonia A 

 and their nucleus contains patchy chromatin (Fig. 5, A 

 and B). 



Spermatocytes form clusters in which the cells are 

 interconnected by cytoplasmic bridges. Primary sperma- 

 tocytes (4.84 ±0.45 Jim) show a heterochromatic nucleus 

 (-3.5 um in diameter) that varies in appearance depend- 

 ing on the prophase-I stage. The cytoplasm contains 

 free ribosomes (mostly polysomes), mitochondria, clear 

 vesicles, and the diplosome (Figs. 5A, 6A). Synapton- 

 emal complexes are clearly recognizable at pachytene 

 (Fig. 6A). Secondary spermatocytes are apparently 

 short-lived cells because they are rare in histological 

 samples — a finding that suggests that the second meiotic 

 division is triggered shortly after completion of the first 

 division. Spermatocytes II are difficult to distinguish 

 morphologically from early spermatids, although they 

 are slightly larger (3.31 ±0.47 /jm). The cytoplasm is 

 more reduced than in spermatocytes I and the nucleus 

 shows diffuse chromatin forming moderately electron- 

 dense patches (Fig. 6B). 



During spermiogenesis, the spermatid nucleus changes 

 in shape and decreases in volume as the chromatin con- 

 denses. In early spermatids (2.39 ±0.28 /Jm) the spheri- 

 cal nucleus shows a dense chromatin with some elec- 

 tron-lucent areas (Fig. 6C). Then the chromatin becomes 

 more homogeneous in mid spermatids (2.56 ±0.21 fim) 

 (Figs. 5A, 6D), and eventually in late spermatids (1.81 

 ±0.32 /iml condenses into a coarse granular pattern, 

 whereas the nucleus assumes an ovoid shape and forms 

 a basal indentation over the proximal segment of the 

 axoneme (Fig. 6E). Cytoplasmic changes involve elonga- 

 tion of the flagellum, reduction of the cytoplasmic mass, 

 and coalescence of the mitochondria into a few large 

 spherical units located around the proximal portion 

 of the axoneme. Rotation of the nucleus does not take 

 place during spermiogenesis, therefore the flagellum 

 axis remains parallel to the base of the nucleus and 

 the spermatozoon shows the typical ultrastructure of 

 teleostean type-II sperm (Fig. 6F). 



Discussion 



Histologically, the bluefin tuna testis is of the unre- 

 stricted spermatogonial testicular type found in most 

 teleosts, where spermatogonia occur along the greater 

 part of the testicular tubules. In the restricted sper- 

 matogonial testicular type of the atheriniforms, on the 

 other hand, the spermatogonia are confined to the distal 

 end of the tubules, and spermatogenesis proceeds as the 

 germ cells approach the efferent ducts (Grier et al., 1980; 

 Grier, 1981). Efferent ducts are generally absent in unre- 

 stricted spermatogonial testes, so that germinal cysts 



form along the testicular tubule length (Grier et al., 

 1980; Grier, 1981; Lahnsteiner et al., 1994). However, 

 in maturing and spawning bluefin tuna a well-developed 

 network of ducts collects the sperm produced by the ger- 

 minal epithelium and voids them into the main sperm 

 duct. The central ducts of the testis are continuous with 

 the proliferative segment of the testicular lobules, which 

 lose the germinal epithelium in the innermost region 

 of the testis and function as sperm storage structures. 

 This process has been documented in the common snook 

 iCentropomus undecimalis) (Grier and Taylor, 1998), the 

 cobia (Rachycentrum canadum) (Brown-Peterson et al.. 

 2002), and the swamp eel (Synbranchus marmoratus) 

 (Lo Nostro et al., 2003). Grier et al. (1980) showed that 

 in the atheriniform Fundulus grandis the efferent duct 

 wall cells derive from Sertoli cells. A system of efferent 

 ducts has been described in other species of teleosts pos- 

 sessing testes of the unrestricted spermatogonial type 

 (Rasotto and Sadovy, 1995; Manni and Rasotto, 1997). 

 As has been shown in other species of the genus (Ratty 

 et al., 1990; Schaefer, 1996; 1998), the main sperm duct 

 of T. thynnus has a thick wall and is located near the 

 center of the testis, whereas in many other teleosts the 

 main duct is dorsal (Grier et al., 1980). 



Ultrastructural features of bluefin tuna spermatogen- 

 esis are comparable to those described extensively in 

 teleosts (for examples of recent literature see Gwo and 

 Gwo, 1993; Stoumboudi and Abraham, 1996; Quagio- 

 Grassiotto et al., 2001; Huang et al., 2002; Koulish et 

 al., 2002; Fishelson, 2003). The primary spermatogonia 

 are the largest male germ cells and exhibit several 

 conspicuous perinuclear ("nuage") bodies. After several 

 divisions they give rise to cysts of secondary spermato- 

 gonia that enter meiosis to produce successively primary 

 and secondary spermatocytes. Primary spermatocytes 

 are abundant, particularly at the pachytene phase, and 

 are therefore thought to be of long duration. In contrast, 

 the spermatocyte-II stage is thought to be the shortest 

 spermatogenetic step, because, as occurs in teleosts in 

 general, it is the least frequent in histological samples. 

 Spermiogenesis develops without the occurrence of rotation 

 of the spermatid nucleus, resulting in a teleostean type-II 

 spermatozoon (Mattei, 1970), in which the flagellar axis 

 lies tangential to the nucleus instead of being inserted 

 perpendicular to its base (Abascal et al., 2002). 



Santamaria et al. (2003) divided the testicular cycle of 

 T. thynnus caught in Mediterranean waters from Febru- 

 ary to September into five periods. Those developmental 

 stages are similar to stages 2-6 classified by Grier (1981) 

 for a generalized teleost annual reproductive cycle. Most 

 probably, stage 1 (spermatogonial proliferation) occurs 

 in Mediterranean bluefin tuna between October and 

 January. More recently, annual histological changes in 

 the germinal epithelium have been used to identify five 

 distinct reproductive classes in males of several teleost 

 species (Grier and Taylor, 1998; Taylor et al., 1998: 

 Brown-Peterson et al., 2002; Lo Nostro et al., 2003). It 

 is assumed that the most advanced maturation classes in 

 males are characterized by the presence of a discontinu- 

 ous germinal epithelium. According to this criterion, all 



