Zoomorphology Zoomorphology (1987) 107 : 26-32 © Springer-Verlag 1987 Ultrastructure of the testicular gland of Blennius pavo (Pisces, Teleostei) M. Seiwaid and R.A. Patzner Zoological Institute, University of Salzburg, Hellbrunner Str. 34, A-5020 Salzburg, Austria and Marine Biological Laboratory "Sorgenti di Aurisina", Strada Costiera 336, 1-34010 Trieste, Italy Summary. The testicular gland of Blennius pavo Risso, 1810 is an accessory sex organ situated ventrally, adjacent to the testis. Sperm have to cross it to reach the vas deferens. Changes in the fine structure of the gland were monitored monthly over a period of I year. The annual cycle is closely correlated to the reproductive cycle. Maximum synthetic activity and cell size are reached during late prespawning and early spawning. At that time the number of lipid vacuoles increases markedly. Vesicles of smooth endoplasmic reticulum ER, numerous Golgi cisternae and tubulovesicular mitochondria which are characteristics of steroid producing cells can be observed. Secretory vesicles are synthesized in the apical region of the cells and then released into the ducts of the gland. The content of lipids is maintained until the resting period. The supranuclear region of the cells contains accumulations of lamellar bodies that probably derive from lysosomal vacuoles. Fine structural characteristics suggesting the function of the testicular gland are discussed. A. Introduction In Blenniidae, the structure of the testis differs from that of most other teleosts. It is clearly divided into a seminiferous region and a testicular gland situated ventrally to it. Little has been published about the morphology and function of the gland. It was first described in detail by Champy and Gley (1922). Courrier (1922) supposed it to have an endocrine function. Eggert (1931) investigated the structure of the reproductive organs of blenniid and gobiid fishes and suggested that the function of the testicular gland is similar to mammalian epididymis. Chieffi and Botte (1964) detected 3-fl-hydroxysteroid-dehydrogenase positive cells in the testicular gland of some blenniid fishes. Bliim (1972) reported the testicular gland of Blennius sphinx to be an LH-dependent lipid body. Also in gobiid fishes the testis is separated into two parts. Fine structural and histochemical investigations reveal that the testicular gland of Gobiidae is a site of intensive steroid production (Colombo and Burighel 1974; Colombo et al. 1979; Asahina et al. 1983, 1985). This paper presents a study on fine structure and fine structural seasonal changes in the testicular gland of Blennius pavo Risso, 1810. OJJprint requests to: R.A. Patzner B. Materials and methods During the course of 1 year, adult male Blennius pavo were collected monthly in the Gulf of Trieste (Italy) (Patzner 1983). The specimens were caught in funnel traps or with Chinaldine diluted in isopropanol. Every month, three to four males were used for fine structural studies. Further investigations were made on three juvenile males which had not yet spawned; one was caught in January and two in April. The animals were anaesthetized with MS 222. Then the testes were quickly removed, the testicular gland was cut into pieces on ice and prefixed at room temperature in a fixative containing 4.5% paraformaldehyde, 2.25% glutaraldehyde in 0.1 M cacodylate buffer with 5% saccharose (pH of the fixative 7.5). After 15 rain, pieces smaller than 1 mm 3 were cut and fixation continued for 3 to 4 h at room temperature in fresh fixative. The tissue was postfixed in 1% OsO, in 0.1 M cacodylate buffer on ice for 2 h. After dehydration in ethanol, the pieces were embedded in Spurr's resin (Polaron). Thin sections were made with a Reichert microtome (OM2) and counterstained with uranyl acetate and bismuth. Thick sections (1 I~m) were stained with azur II (1%), methylenblue (1%) in Na-Tetraborat. C. Results The testicular gland of Blennius pavo is situated ventrally adjacent to the testis and is interposed between the seminiferous tubules and the efferent duct (Fig. 1). Therefore spermatozoa have to cross it to reach the vas deferens (Fig. 2). An artery and a large vein run parallel along the ventral side of the gland. The gland is composed of numerous branched tubuli formed by a monolayered epithelium (Fig. 2). The tubules are separated from each other by a basal membrane (Fig. 3). Between the tubules, cells of connective tissue and capillaries can be observed. Fine structural investigations reveal that the testicular gland cells show differentiation into three distinct zones (Fig. 3): an apical pole, a supranuclear region and a basal region. At the apical pole, the luminal cell membrane forms prominent microvilli which often completely fill the lumen of the duct. Characteristics of the supranuclear region are accumulations of myelin-like lamellar bodies, and depending on season, secretory vesicles, Golgi vesicles, and smooth endoplasmatic reticulum (ER). The basal region is characterized by lipid vacuoles; their number also depends on 27 / <' q . ù .................. \T .~[~B~. :~..: :~~ "1 1 Fig. I. Schematic drawing of reproductive system of male Blennius pavo, with the posterior side to the right. T testis; 1 testicular gland; 2 vas deferens; 3 blind pouch Fig. 2. Semithin cross-section of testicular gland and testis (t) of Blennius pavo. Arrows indicate ducts of the testicular gland filled with ripe spermatozoa season. Additional characteristic features of the gland cells are numerous desmosomes linking the cells mainly in the apical region and stacks of membranes formed by interdigitating cell membranes in the more basal region (Fig. 3). The basal lamina is offen heavily interlocked with the cell membranes of connective tissue cells. Smooth endoplasmatic reticulum (ER) was observed mainly in the form of vesicles; the mitochondria are tubulovesicular (Figs. 5, 9). Prespawning period. This lasts from March to the end of May. In the middle of April the height of the testicular gland cells has increased slightly in comparison to the resting period. The ducts through the gland are completely filled with microvilli (Fig. 3). The interlocking of the basal lamina with connective tissue cells and the interlocking of adjacent gland cells are most prominent during that time of the year. The basal cell region contains few lipid vacuoles but many tubulovesicular mitochondria offen with dense granules. Numerous Golgi cisternae and vesicles of smooth ER are observed (Figs. 5, 9). The supranuclear region mainly contains myelin-like lamellar bodies (Fig. 3), few vacuoles and few secretory vesicles. Between the lamellar bodies, Golgi cisternae and vesicles of smooth ER are visible. By the middle of May the number of lipid vacuoles in the basal cell region has increased (Fig. 6). Numerous Golgi cisternae and smooth ER can still be detected. At that time synthesis of secretory vesicles starts in the apical cell region. These secretory vesicles are stored near the lumen of the ducts. Fibrillous structures become visible between them and these are mainly situated concentric to the duct (Fig. 6). At the end of the prespawning period (end of May), the lumina of the ducts become filled with spermatozoa. They are partially surrounded by microvilli of the luminal cell membrane (Fig. 6). Now the basal cell region is densly filled with lipid droplets and tubulovesicular mitochondria. Spawning period. This lasts from June to the middle of August. At the end of prespawning the height of the testicular gland cells is about 20 p,m. The amount of smooth ER starts to diminish. The luminal cell membrane forms secretory blebs containing the secretory vesicles which have been accumulated in the apical cell region during the prespawning period (Figs. 10, 12). They are then released into the ducts in form of these secretory blebs. The secretory granules are bordered by a membrane and have a granular appearance. Their diameter is about 0.23 p~m. In some cases it seems that the membrane of the vesicles is dissolved within the secretory blebs (Fig. 12). By the end of the spawning period cell size starts to diminish again although the ducts are still filled with sperm. 28 Figs. 3-5. Testicular gland cells of Blennius pavo during the prespawning period. Fig. 3. General view of testicular gland cells. 1 lipid vacuoles; m microvilli in the lumen of a duct; mb myelin-like lamellar bodies; n nucleus; open arrows basal lamina; black arrows interdigitating cell membranes; arrowheads desmosomes. Fig. 4, Interlockings of basal lamina with connective tissue cell (prespawning period). Arrows indicate basal lamina. Fig. 5. Detail of interdigitating cell mernbranes. Numerous vesicles of Golgi cisternae (g) and smooth ER are visible Postspawning period. This lasts from the middle o f August to the middle o f November. Until October the testicular gland cells have distinctly decreased in size (Fig. 7). The height o f the testicular gland cells is now a b o u t 14 lam. The lumina o f the ducts are n a r r o w again and completely filled by microvilli (Fig. 7). The apical region does not contain secretory vesicles any more, so that the lamellar bodies are situated next to the duct. Sometimes transitional stages between lamellar bodies and lipid vacuoles can be detected (Fig. 11). The high level o f lipids is maintained, but only a few Golgi cisternae and vesicles o f smooth E R can be observed. Few m i t o c h o n d r i a are visible between the lipid droplets. Resting period. This lasts from the middle o f N o v e m b e r to March. D u r i n g this time the testicular gland cells do not change in appearance and size. They stay 13 txm in 29 Figs. 6, 7. General view of testicular gland cells of B l e n n i u s p a v o at the end of the prespawning (Fig. 6) and during the post-spawning periods (Fig. 7). 1 lipid vacuols; rn microvilli; n nucleus; s ripe sperm in a duct of the testicular gland; sv secretory vesicles stored in the apical region of the cells; open a r r o w s basal lamina; a r r o w h e a d s fibrillous structures. Fixation of tissue in Fig. 6 is 4% glutar aldehyde in 0.1 M cacodylate buffer 30 Fig. 8. Testicular gland cells ofjuvenile male B l e n n i u s p a v o . m microvilli; n nucleus; open a r r o w s basal lamina; a r r o w h e a d s lysosome-like bodies Fig. 9. Detail of testicular gland cell in the prespawning period. Tubulovesicular mitochondria and vesicles of smooth ER. Fixative is 1% OsO» in 0.1 M cacodylate buffer Fig. 10. Detail of apical region of testicular gland cells densely filled with secretory vesicles (sv). m microvilli in the duct; sb secretory bleb. Fixative is 1% OsO4 in 0.1 M cacodylate buffer Fig. 11. Transitional stage of lipid vacuole and lamellar body in a testicular gland cell Fig. 12. Apical region of testicular gland cell, with the luminal cell membrane forming a secretory bleb (sb). lu lumen of a duct with spermatozoa (s); sv secretory vesicles. Fixative is 1% OsO4 in 0.1 M cacodylate buffer 31 height until the next start of spermatogenesis (end of March). Only the number of lipid droplets is reduced. Testicular gland of juvenile males. The general appearance of the testicular gland differs distinctly from that of adult specimens as it does not contain lamellar bodies and lipid droplets (Fig. 8). The testicular gland appears very uniform in structure; only one cell type can be detected apart from connective tissue cells. The ducts of the tubuli are narrower than in adults but are distinct (Fig. 8). The height of the gland cells is 8.3 pm. The cytoplasm is filled with Golgi cisternae and vesicles of smooth ER. Some lysosome-like bodies can be detected (Fig. 8). Just as in adult specimens the mitochondria are tubulovesicular. D. Discussion Within the Teleostei only the Gobiidae posses an accessory sex organ similar to the testicular gland of Blenniidae (Eggert 1931). In contrast to the blenniids, where the testicular gland is composed of tubules formed by a monolayered epithelium, the testicular gland of gobiid consists of cords of polyhedral cells (Colombo and Burighel 1974). In blenniid fishes, the spermatozoa have to pass through the testicular gland by numerous ducts to reach the vas deferens which is situated ventrally to the gland. However in most gobiids the testicular gland is surrounded by the seminiferous tubules and the vas deferens is situated between testis and the glandular tissue so that the spermatozoa need not cross it. In some gobiids it is located in the centre of the gland. Then spennatozoa reach the vas deferens through a narrow duct (Asahina et al. 1983, 1985; Colombo and Burighel 1974). The fact that in bleniids spermatozoa taust cross the gland may indicate that the function of their testicular gland is at least partly different from that of gobiid fishes. L Fine structure The testicular gland cells of Blennius pavo show smooth ER and tubulovesicular mitochondria which generally are characteristics of steroid producing cells (Gorbman et al. 1983). As only very few interstitial cells could be detected in the testis of Blennius pavo, it seems that the testicular gland represents the interstitial tissue of the testis. However, the amount of smooth ER is rather small in comparison to the interstitial cells of other teleosts (Nicholls and Graharn 1972; Hoar and Nagahama 1978; Cruz-Höfiing and Cruz-Landim 1984). In gobiid testicular gland cells which also synthesize steroids, huge areas are filled by smooth ER (Colombo and Burighel 1974; Asahina et al. 1983). Colombo et al. (1979) report that the steroids produced in the testicular gland of Gobiusjozo are mainly steroid glucuronides which are used as pheromones in reproduction. In Blennius pavo, histochemical investigations also prove the ability of the gland cells to synthesize steroids (Seiwald and Patzner 1986; Seiwald and Patzner, in prep). Till now we could not find out whether they are also used as pheromones or only for spermatogenesis. Biochemical and ethological investigations are necessary to obtain information on this question. The general appearance of the testicular gland cells closely resembles that of cells of the rat epididymis (Brandes 1974), which also fall into three distinct zones. Like Blennius pavo they contain accumulations of lamellar bodies in the supranuclear region and secretory vesicles which look like those of the testicular gland (Brandes 1974). Storage of sperm can be excluded from being the function of the testicular gland because the ducts are too narrow to contain the large amounts of spermatozoa which are necessary for spawning. It is supposed that they cross the gland and then are stored in the upper part of the vas deferens. Light microscope investigations proved that the vas deferens is densely filled with sperm in the morning before spawning begins (unpublished observation). In Blennius pavo the luminal cell membrane of the testicular gland cells forms prominent microvilli that often fill the lumen of the ducts. As there is no morphological evidence for the absorbance of materials from the ducts, it is supposed that orte of the main functions of the gland is to produce constituents of the seminal fluid. In the testicular gland of gobiid fishes microvilli are never observed (Colombo and Burighel 1974; Asahina et al. 1983). In some respects the gland cells resemble the cells of the "glandular tissue" or "interstitial tissue", the main steroid producing part of the testis of Salamandra salamandra. This probably indicates a similar function. Adjacent cells are interlocked in a similar way and bound together by desmosomes. In both species the number of lipid vacuoles is larger than in most other interstitial cells. In gobiid testicular gland cells only a few lipid droplets are observed (Colombo and Burighel 1974). One of the most obvious characteristics of the testicular gland cells of Blennius pavo are the lamellar bodies situated in the supranuclear region of the cells. There are no reports of similar structures in other teleosts but they are also found in the Leydig cells of the dromedar (Friedländer et al. 1984), in rat epididymis (Brandes 1974), in the glandular tissue of Salamandra salamandra (Bergmann et al. 1983) and in the interstitial cells of the newt Necturus maculosus (Pudney et al. 1983). Brandes (1974) considers them as lysosomal vacuoles filled with membranes of lipids and lipoproteins that have not been digested completely. In Blennius pavo transitional stages between lipid vacuoles and lamellar bodies often occur. It is possible that the lamellar bodies of the testicular gland cells might have a similar function (Seiwald and Patzner 1986). In anaphibians and Dromedarius, the myelin-like bodies derive from degenerating smooth ER (Pudney et al 1983; Friedländer et al. 1984). It is interesting that the testicular gland cells of juvenile Blennius pavo posses neither lipid vacuoles nor lamellar bodies. This suggests that both are correlated to reproduction and therefore are only formed in adult males. II. Annual changes Until now there have been no fine structural investigations on annual cycles of accessory sex organs in teleosts. All changes taking place in the testicular gland cells of Blennius pavo are closely correlated to the reproductive cycle (Patzner and Seiwald 1986). Their activity and size increases as soon as spermatogenesis begins. Maximum size is reached during the spawning period (Seiwald and Patzner 1986). Smooth ER and Golgi cisternae are best developed during prespawning and spawning and are mainly observed in the basal and supranuclear zones. In postspawning period they are only rarely visible. This correlates with the annual changes of Leydig cells which mainly produce most 32 steroids during spermatogenesis (Wiebe 1969; van den Hurk et al. 1978; Leceta et al. 1982; Pudney et al. 1983). F r o m the middle of May until June secretory vesicles are synthesized and stored in the apical region of the cells and are released during the spawning period. The formation of secretory vesicles by the luminal cell m e m b r a n e and their structure resemble those of rat epididymis (Brandes 1974). There are no reports of similar observations in teleosts. The n u m b e r of lipid vacuoles also shows a distinct a n n u al cycle. They increase towards the spawning season and then are maintained until the resting period. They probably provide energy for steroidogenesis during the spawning season and for the resting period. Blüm (1972) reports that the structure of the testicular gland of Blennius sphinx differs from that of other blenniids. At times of full activity the testicular gland of Blennius pavo contains large a m o u n t s of lipids similar to the glandular tissue of Salamandra (Bergm a n n et al. 1983). In contrast to this, G u r a y a (1979) reports that lipids in interstitial cells of teleosts disappear with the increase of synthesis of steroids. The lamellar bodies do not change during the a n n u a l cycle. The present investigations on the fine structure of the testicular gland indicate that steroidogenesis, storage of lipids and synthesis of a part of the seminal fluid are functions of the gland. The a n n u a l changes that take place are closely correlated with the reproductive cycle. Aeknowledgements. This study was supported by the Austrian ùFonds zur Förderung der wissenschaftlichen Forschung" (grant no. 5338) and by the Austrian National Bank. The authors are grateful to the director of the Marine Biological Station of Aurisina (University of Trieste), Dr. G. 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