The events of spermatogenesis in two species of brachyuran crabs (Barytelphusa jacquemonti (Rathbun) and Oziotelphusa senex (Fabricius) were studied by light microscopy. Four different cell types were categorised in the testis, namely 1. Spermatogonia, 2. Spermatocytes, 3. Spermatids and 4. Spermatozoa. Each cell type has been studied in detail, for its structure, morphometry and the process of transformation into the next stage. Comparisons are drawn with the observations on the spermatogenesis of other decapods

Rao, Geetha K.

Shakuntala, Katre

ePrints@Bangalore University

I. Indian Inn Sci, Nov.—Dec. 1995, 75, 693-698. C Indian Institute of Science. Short Communication Studies on the spermatogenesis of two Indian freshwater brachyurans GEETHA K. RAO AND KATRE SHAKUNTALA Department of Zoology, Bangalore University, Jnana Bharathi, Bangalore 560 056, India. Received on August 10, 1994; Revised on August 16, 1995. Abstract The events of spermatogenesis in two species of brachyuran crabs [(Barytelphusa jacquemonti (Rathbun) and Oziotelphusa senex (Fabricius)1 were studied by light microscopy. Four different cell types were categorised in the testis, namely 1. Spermatogonia, 2. Spermatocytes, 3. Spermatids and 4. Spermatozoa. Each cell type has been studied in detail, for its structure, morphometry and the process of transformation into the next stage. Comparisons are drawn with the observaitons on the spermatogenesis of other decapods. Keywords: Spermatogenesis, light microscopy, freshwater brachyuran crabs. 1. Introduction The class Crustacea is a remarkably successful group, as evidenced by the number of ex- tant species and the diverse habitats colonised by them 1 . This diversity is also reflected in the life history patterns and reproductive strategies 2 . On account of their commercial and/or consumptional importance, over the years, considerable information has been gathered on the biology of crustaceans in general and decapods in particular 3-11 . How- ever, with particular reference to decapods of the Indian sub-continent, much work re- mains to be done, especially for the freshwater brachyurans--Barytelphusa jacquemonti and Oziotelphusa senex. As both the species contribute substantially to the local fresh- water crustacean biomass, and are of notable commercial value, an understanding of the species-specific events and patterns of spermatogenesis would permit a clearer knowl- edge of their reproductive cyclicities, a pre-requisite for developing techniques of con- trolled breeding of the species, in closed aquaculture systems. 2. Material and methods Specimens of B. jacquemonti (Rathbun) were hand-picked from the Byramangala canal System, a perennial reservoir in the Bangalore south taluk 12 . Individuals of 0. senex (Fabricius) which prefer shallow, lentic habitats, were collected from the paddy fields, near Whitefield, south-east of Bangalore 13 . About 15-20 males were randomly selected and the sampling was done on a fortnightly basis. Testes and vasa deferentia, excised from either species, were fixed overnight in aque- ous Bouin's fluid (18-20 hours). After washing the tissues in distilled water and dehy- 694 	 GEETHA K. RAO AND KATRE SHAKUNTALA drating in progressive grades of alcohol, the tissues were cleared in xylene and embed- ded in paraffin (melting point: 58-60°C). Subsequently, they were sectioned at 7-8 p thickness and stained with hematoxylin-eosin. DPX was used as a mountant and stained sections were observed for morphometric and histological details. Such histological ob- servations on the testis were made over one annual cycle during 1991-92. 3. Results and discussion Each testis was composite, consisting of many lobules and was covered by a discrete limiting membrane made up of cells with flattened nuclei. A muscle layer, as described by Reger" for the testis of the isopod, Asellus militaris, was not discernible in the testis of the crabs under study. As the adjacent testicular lobules were closely packed, the inter-lobular space was considerably reduced. Loose connective tissue and distinct blood- sinuses were found in apparent inter-lobular spaces. There was considerable asynchrony in the spermatogenic activity when the different lobules of the same testis were observed, although the spermatogenic activity was fairly well defined with regard to any one lobule (Fig. 1). This was true in the histological observations made throughout the annual cy- cle. From these observations it can be adduced that males of B. jacquemonti and 0. senex exhibit an extended reproductive activity in natural populations, throughout the year. Such an asynchronous activity with respect to spermatogenesis has also been ob- served in the testis of another crab, Menippe mercenaria" and the freshwater prawn Macrobrachium lanchesteri 16 . However, in both the species under study, heightened spermatogenic activity was evident between March and October of each calendar year. The testis of either species of crabs was made up of two kinds of cells, namely, so- matic and germinal cells. Somatic cells were large, each with a single voluminous nu- cleus, and one to several nucleoli. In Decapoda reptantia, such somatic cells are gen- erally designated as the `intercalaryi cells 2 . Four distinct cell types of the spermatogenic cycle have been categorised in the testis of the two brachyurans. These are spermatogonia, spermatocytes, spermatids and sper- matozoa. The term `spermatogonia' here refers only to secondary spermatogonia, as primary spermatogonia were not encountered in the histological sections of the testis. Perhaps this stage is rather short-lived and passes off quickly into the next phase. Such a Table I Morphometry of different cell types during the spermatogenic cycle Cell type Morphometry (p) ± S. E. Longest axes Widest axes B. jacquemonti 0. senex B. jacquemonti 0. senex 1. Spermatogonia 2. Spermatocytes 3. Spermatids 4. Spermatozoa 12.20 ± 0.32 9.37 ± 0.30 6.59 ± 0.23 4.22 ± 0.07 9.06 ± 0.27 5.34 ± 0.15 5.12 ± 0.17 3.17 ± 0.06 8.52 ± 0.21 7.93 ± 0.30 6.20 ± 0.16 	 a" 3.47 ± 0.07 7.41 ± 0.25 4.54 ± 0.10 4.82 ± 0.18 2.50 ± 0.07 rieraag STUDIES ON THE SPERMATOGENESIS OF TWO INDIAN FRESHWATER BRACHYURANS 695 situation has also been described for another crab, Potamon koolooense l7 and the prawn Macrobrachium lanchesteri16 . In contrast, distinct primary spermatogonia have been described in the testis of Squilla oratoria2 4 18 . Table I presents the morphometry of all the cell types a the spermatogenic cycle of B. jacquemonti and O. senex, measured along the longest and the widest axes. Spermatogonia were the largest of all cell types encountered in the histological sections of the testis. Outlines of these cells were not distinct as the adjacent cells were compacted (Fig. 2). The large, more or less elliptical, nucleus occupied almost the entire cell area. The number of nucleoli per nucleus ranged from one to two. Only one nucleo- lus has been reported for the spermatogonial cell of another crab, Lophopanopeus bellus I9  . The uniformly distributed chromatin was positive to hematoxylin. As is characteristic of the process of spermatogenesis in natantians 16 ' 20, in these reptantians also, cytoplasmic reduction was evident. In the final stages of spermatogonial morphol- ogy, the cytoplasm was restricted to a narrow, indistinct rim around the nucleus. Such spermatogonial cells were mostly distributed towards the periphery of each testicular lobule. The spermatogonia, as above, underwent spermatogonial division to give rise to pri- mary spermatocytes. A distinct cell outline could be noted at this stage and the cells were rather loosely arranged in the matrix of the testicular lobule. Metaphase I was the most commonly encountered stage (Fig. 3). Spindle fibres were clearly visible under the light microscope. Displacement of the diads to the respective poles was also observed. Primary spermatocytes underwent mitosis, to give rise to secondary spermatocytes, which were neary half the size of the former cell type, owing to further cytoplasmic re- duction 19 . Golgi granules were fewer in number, as perhaps most of them had undergone disintegration. FIG. 1. Cross-section of the testis (x 490). Note the MYnchrony in the events of spermatogenesis in the adjacent testicular lobules, TL = testicular lobule, FCr--  flattened cells of the lining of the testicular lob- ule,  a = secondary spermatogonia, b = metaphase Plates of spermatocytes, c = spermatids, d = sperms. FIG. 2. Higher magnification of Fig. I to show the spermatocytes (x 1220). G = apparent golgi material. 696 	 GEETHA K. RAO AND KATRE SHAKUNTALA FIG. 3. Higher magnification of Fig. 1 to show the spermatocytes (b) and spermatids (c) (x 1250). Note the metaphase plates. FIG. 4. Higher magnification of Fig. 1 to show the nature of sperms (x 1200). AC = acrosome. Spermatids were formed following the second meiotic division of the secondary spermatocytes and were accompanied by further cytoplasmic condensation (Fig. 3). Gen- erally, among brachyurans, the mitochondrial vesicle or `nebenkern' is said to gradually fit into the cavity of the nucleus 21 . Spermatids underwent transformation to give rise to the characteristic, atypical, non-motile, reptantian type of spermatozoa. In the fully formed sperm, the acrosome having fused completely with the lips of the nuclear cup, could not be made out as a separate structure (Fig. 4). No stellae or rays as described for other reptantians 2. 15, 19, 21-25 could be discerned in the sperm of B. jacquemonti and 0.senex. As compared to the structure of the sperm of Carcinus maenas26, Libinia emarginata and other reptantians 24 ' 25 , in their external morphology; the spermatozoa of B. jacquemonti and 0.senex bear a close resemblance to that of Pinnixia sp27 . During the process of spermatogenesis, the acroblast of the spermatid increased in size to get transformed into a ring-like acrosome, which finally fused with the margins of the nuclear cup of the sperm. In many of the decapods studied, the origin of the acro- some is a much debated topic. The formation of the acrc some is variously attributed to golgi elements21, 28-30, 34-37 or even to the nuclear derivatives 28 ' 38 . In the species under study also, the origin of the acrosome could not be discerned under light microscopy. References I. DOROTHY, B. E. (ED.) Biology of crustacea, Vol. 1 (Systematics, fossil records and bio- geography), p. 319, 1982, Academic Press. 2. POCHON-MASSON, J. Reproductive biology of invertebrates, Vol. 2 (K. G. Adiyodi and R. G. Adiyodi, eds) pp. 407-449, 1983. 3. DowirHY, B. E. (ED.) Biology of crustacea, Vol. 2 (Embryology, morphology and genet- ics), p. 440, 1982, Academic Press. 4. DoRoTHY, B. E. (ED.) Biology of crustacea, Vol. 3 (Neurobiology: structure and func- tion), p. 479, 1982, Academic Press. 5. DOROTHY, B. E. (ED.) Biology of crustacea, Vol. 4 (Neural integration and behaviour) p. 327, 1982, Academic Press. STUDIES ON THE SPERMATOGENESIS OF TWO INDIAN FRESHWATER BRACHYURANS 697 Biology of crustacea, Vol. 5 (Internal anatomy and physiological 6. DOROTHY, B. E. (ED.) regulation), p. 471, 1983, Academic Press. 7. DOROTHY, B. E. (ED.) 	 Biology of crustacea, Vol. 6 (Pathobiology), p. 290, 1983, Aca- demic Press. 8. DOROTHY B. E. (ED.) 	 Biology of crustacea, Vol. 7 (Behaviour and ecology), p. 338, ,  1983, Academic Press. 9. DoRoTHY, B. E. (ED.) 	 Biology of crustacea, Vol. 8 (Environmental adaptations), p. 383, 1982, Academic Press. 10. DOROTHY, B. E. (ED.) 	 Biology of crustacea, Vol. 9 (Integument, pigments and hormonal processes), p. 550, 1985, Academic Press. 11, DOROTHY, B. E. (ED.) 	 Biology of crustacea, Vol. 10 (Economic aspect, fisheries and culture), p. 331, 1985, Academic Press. 12. SRIVATHSA, S. Ecophysiological studies on a freshwater brachyuran crab of Bangalore, Ph. D. Thesis, Bangalore University, 1992, p. 152. 13. MEERA, B. E. 	 Histomorphological studies on the reproductive system of a brachyuran crab, M. Phil. Dissertation, Bangalore University, 1991, p. 70. 14. REGER, J. F. 	 The fine structure of spermatozoa from the isopod, Asellus milita- ris (Hay), J. Ultrastruct. Res., 1964, 11, 181-192. 15. BINFORD, R. 	 The germ cells and the process of fertilization in the crab, Menippe mercenaria, J. Morph., 1913, 24, 147-182. 16. RAO, CH. N. 	 Investigations on the ecophysiology of reproduction in a fresh- water prawn, Ph. D. Thesis, Bangalore University, 1983. p. 199. 17. JOSH!, P. C. AND KHANNA, S. S. 	 Structure and seasonal changes in the testes of a freshwater crab, Potamon koolooense (Rathbun), Proc. Indian Acad. Sci. (Anim. Sci.), 1982, 91, 439-450. 18. KOMAI, T. 	 Spermatogenesis of Squilla oratorio de Haan, J. Morph., 1920, 34, 307-333. 19. FASTEN, N. 20. ARSENAULT, A. L., CLATTENBURG, R. E. Spermatogenesis 	 in 	 the 	 black 	 clawed 	 crab, 	 Lophopanopeus be//us (Stimpson) Rathbun, Biol. Bull., 1926, 30, 277-293. Spermiogenesis 	 in 	 the 	 shrimp, 	 Crangon 	 septemspinosa, 	 say, AND ODENSE, P. H. Can. J. Zool., 1979, 57, 486-498. 21. MATH, V. The decapod sperm, 	 Vol. 	 II, National Institute of Science of India, 1937, pp. 87-119. 22. FASTEN, N. Comparative 	 stages 	 in 	 the 	 spermatogenesis of various 	 cancer crabs, J. Morph., 1924, 39, 47-61. 23. RINSCH, G. W. Penetration of oocyte envelope by spermatozoa in the spider crab, J. VItrastruct. Res., 1971, 35, 86-97. 24. VAUGHN, 3. C. AND H1NSCH, G. W. Isolation and characterization of chromatin and DNA from the sperm of the spider crab, Libinia emarginata, J. Cell. Sci., 1972, 11, 131-152. 25. IlittiscH, G. W. Sperm structure of Oxyrhycha, Can. J. Zool., 1973, 51, 421-429. 698 	 GEETHA K. RAO AND KATRE SHALUNTALA 26. REGER, J. F., FRANCOIS ESCAIG, J. a 	 Observations on crab_ Caroni maenas, spermatozoa following POCHON-MASSON, J. AND 	 rapid freeze and anninatinaal fixation technique, J. Ultrastruct. FITZGERALD, M. E. C. 	 Res., 1984, 89, 12-21 27. REGER, J. F 	 Studies on the spennaliiis and spernitozoa of the crab, Pinni.via J. Morph., 1970, 131 IM-100. 28. BOWEN, R. H. 	 Further notes on *be 	 of the animal sperm: The homolo- gies of non-flagella spa, Anat. Rec_ 1925, 31, 201. 29. GRABOWSKA, Z. 	 L. appareil de galgi orni les spermatozoides des crustaces (Astacus fluviatilis et Attacks koptodactybss), C. k Soc. Biol., 1927, 97, 3. 30. GRABOWSKA, Z. 	 Uber die plasmacceposemen (Golgi-apparat u.a) in dem mannli- chen geschlectsuilen 	 Potamovius astacus L. Polska Akad. limiejetnosci. Krakow Wydzial. Mat_ Pryzyord. Bull. Int. B. Sci. Natn II Zoo., 1929_ 2_ 1C7. 31. NATH, V. 	 Cytology of spennasoresesis, ha. Rev. Cytol., 1956, 5,395. 32. KOLTZOFF, N. K. 	 Studien uber die gestalt der zellei. unter suchungen uber die spa- mien der decapoden, Arch. Atikr. Anat., 1906, 67, 364. 33. MCCROAN, J. E. 	 Spermatogenesis af dee crayfish Cambarus virilis, with special reference to the ?pip material and mitochondria, Cytologia, 1940, 11, 36. 34. NA'n-t, V. 	 The spermatid and sperm of the crab Paratelphusa spinigera Quat, J. Micratc_ Sci_, 1932, 75, 543. 35. NATH, V. 	 The decapod sperm. Tim Nam Inst. Sci. India, 1972, 2, 87. 36. NATH, V. 	 Animal gametes (.ak)_ A morphological and cytochemical ac- count of spennatoraesis, 1965, p. 78, Asia Publishing House, Bombay, 37. GROBBEN, C. 	 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Studies on the spermatogenesis of two Indian freshwater brachyurans

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Studies on the spermatogenesis of two Indian freshwater brachyurans

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