Seasonal changes in the ovary of a freshwater crab, Potamon koolooense (Rathbun)

Transcription

1 Proc, Indian Acad, Sci. (Anini- ScL), Vol. 91, Number 5, September 1982, pp, Printed in India. Seasonal changes in the ovary of a freshwater crab, Potamon koolooense (Rathbun) P C JOSHI and S S KHANNA* Department of zoology, Government P G college, Pithoragarh , India * Joint Secretary, Ministry of Education, Lucknow, India MS received 25 November 1981 ; revised 22 July 1982 Abstract. The ovaries of P. koolooense which are paired H-shaped structures undergo seasonal morphometric and histological changes. A minute oviduct leads into the seminal receptacle which receives sperms during breeding season. Oogonia and young oocytes develop in the genninal zone, present in the centre of the ovary. The resting or residual oogonia which occur throughout the year divide Shortly after Ovulation and supply new crop of germ cells for the next breeding season. Five maturational stages of ova have been described on the basis of changes that occur in their nuclei and cytoplasm. They are oogonium, premeiotic oocyte, previtellogenic oocyte, vitellogenic oocyte and the ripe ovum. Spawning occurs during May or June, The weight of the ovaries, gonad index and ova diameter were minimum in June and reached a maximum value in April. Keywords, Potamon ; ovarian histology; ovarian cycle; vitellogenesis. 1. Introduction The structure of female reproductive organs has been desc ribed in some species of crabs (Weitzman 1966 ; Rouquette 1970 ; Chiba and Honma 1971 ; Laulier and Demeusy 1974). The process of yolk formation differs in various species of crustaceans (Harvey 1929; Bhatia and Nath 1931 ; Hinsch and Cone 1969; Hinch 1970). Most of the studies On the female reproductive cycle are based on the morphometric characters of the ovaries only. Nevertheless, relatively little emphasis has been laid on the seasonal histological changes in the ovaries of the crabs and other decapods (Weitzman 1966 ; Chiba and Honma 1972 ; Laulier and Demeusy 1974; Badawi 1975; Goldstein and Lauria 1975 ; Rao et al 1981). In the present investigation the structure and seasonal histomorphological changes in the ovaries of a hill stream crab, Potamon koolooense were studied. 2. Material and methods 15 to 20 live specimens of adult female P. koolooense (carapace width 3'8 to 4'5 cm) were collected locally from a stream each month during The 451

2 452 P C Joshi and S S Khanna weight of each specimen was recorded immediately before dissection. Ovaries along with oviducts and seminal receptacles were removed and placed in fixative. Ovarian weight was recorded after fixation. The gonosomatic index (GSI) was calculated using the formula (Giese 1959): _ weight of the gonad 100 GSI- izh fth. IX' weig toe aruma For histology, different regions of ovaries were fixed in Bouin's or Helly's fluid. Paraffin sections of 5-6 micra thickness were cut and stained with Delafield's haematoxylin or Mayer's haemalum, using eosin as counterstain. Heidenhain's Azan or Mallory's triple stain was also used. The average oocyte diameter was calculated by measuring rounded oocytes having complete nuclei. The moulting stages of the crabs are not determined in this paper. 3. Observations 3.1. Morphology of reproductive organs The female reproductive organs include paired ovaries, oviducts and seminal receptacles. Ovaries are elongated 'H'-shaped structures, situated between hypodermis of carapace and the hepatopancreas. Just behind the pyloric stomach, the two ovaries are connected together by a cross connection. Ovary leads into a very minute oviduct. In sections the ovarian wall appears to continue as the oviduct (figure 2), which opens into a large thick walled pouch, the seminal receptacle. Each receptacle leads into a narrow vaginal tube which further opens outside through a small circular gonopore situated on the 6th sternal segment of the cephalothorax. Ovaries show morphological changes associated with their degree of maturity, as reflected in their size, shape, colour and weight Histology of ovary The ovarian wall is continuous with ovarian stroma (figure 1) and is thicker during post-spawning period but becomes thin at maturing and mature stages of the ovary. Ovarian stroma consists of connective tissue, muscle fibres and blood vessels, and is abundant during post-spawning period but greatly reduced in mature ovaries (figure 1). A germinal zone is present all along the centre of the ovary (figures 1, 3). During early phases of maturation, the germinal zone consists of oogonia and young oocytes whereas the developing oocytes are displaced towards outer region of the ovary (figure 1). As maturity advances the germinal zones become greatly reduced consisting of a few residual oogonia, the rest of the ovary is filled with maturing oocytes Stages of developing ova The oogonium passes through different maturational stages before it becomes the ripe ovum. This process involves changes in the nucleus and cytoplasm. According to the classification of Raven (1961) and Laulier (1974), the following developmental stages have been observed :

3 Seasonal changes in the ovary of crab 453 Figures Structure of ovary in transverse section, June specimen x Section passing through oviduct (avo) x LoS. of ovary showing germinal zone (cz) x Oogonium (OG) showing mitotic figure (MF), yolk droplets (YD) appear in peripheral ooplasm of primary vitellogenic oocyte (pya) x Showing premeiotic oocyte in germinal ZOne X 380. (OF, discharge follicle; OZ, germinal zone; 00, oogonia ; as, ovarian stroma; OY, ovary; ovw, ovarian wall; PMO, prerneiotic oocytes ; Po, previtellogenic oocytes; PVo, primary vitellogenic oocytes; SK, synezesis knot; SR, seminal receptacle).

4 454 P C Joshi and S S Khanna Figures Primary vitellogenic oocyte showing yolk droplets (YO) extended towards perinuclear region (PR). Displacement of nucleolus is due to mechanical disturbance during section cutting x An enlarged portion of above showing vacuoles (v) inside yolk droplets (YD) x 380, 8. Yolk granules (Yo) and yolk vesicles (rv) in periphery of secondary vitellogenic oocyte x Vacuolation of yolk dropkts (YD) to form yolk vesicles (YV) x Portion of secondary vitellogenic oocyte showing light yolk granules (yo), dark yolk droplets (YD) and yolk vesicles (YV) x 380, (OF, discharged follicle ; FL, follicular layer; N, nucleus, YM, vitelline membrane.)

5 Seasonal changes in the ovary of crab 455 Figures Secondary vitellogenic oocyte showing vacuolation of yolk. droplets (arrow). Perinuclear region is yolkless x Showing vacuolation (v) and eccentric position of nucleolus (NL) x Secondary vitellogenic oocyte showing yolk globules (YOL) and yolk vesicles (YV). Yolk droplets (vo) are in the background x Tertiary vitellogenic oocyte filled with yolk. globules. (VGL) and yolk vesicles (vv), Perinuclear region (PR) is occupied by yolk. x Ripe ovum filled with yolk globules (YGL) and yolk vesicles (YV) X 40. (FL, follicular layer; NM, nuclear membrane; PR, perinuclear region; YG, yolk granules; YV, yolk vesicles).

6 Seasonal changes in the ovary of crab a. Oogonium (figures 3, 4, 5): A few primary or residual oogonia which occur throughout the year in the germinal zone, divide mitotically (figure 4) shortly after ovulation and give rise to additional oogonia to provide for further growth of ovary. Oogonium is a small spherical cell with a pale nucleus and thin rim of poorly basophilic cytoplasm. Oogonia develop into premeiotic oocytes but a few remain undifferentiated (residual oogonia) till the ovulation. 3.3b. Premeiotic oocyte (figures 3, 5): The oogonia which enter into prophasic activities are termed as premeiotic oocytes or primary oocytes, The chromosomes condense at one side of the nucleoplasm and form synezesis knot (figure 5) which corresponds with zygotene or synapsis stage of meiotic prophase. At the final developmental stage (diakinesis) the chromatin appears in the form of discrete clumps lying close to nuclear wall (figure 5) and the nucleolus is not visible. The oocyte measures 20 micra in diameter. 3.3c. Previtellogenic oocyte (figure 3): As the premeiotic activities come to an end, the nucleus increases in volume and oocyte acquires a large amount of basophilic cytoplasm. The nucleus appears vesicular containing peripherally arranged chromatin clumps and a centrally placed nucleolus which appear solids and stain blue-black with haematoxylin or red with Mallory's triple or Azan stains. The yolk formation has not yet begun and oocyte attains a diameter of 95 micra. 3.3d. Vitellogenic oocyte: The oocyte now enters a synthetic or vegetative phase resulting in the formation of yolk. The nucleus, nucleolus and ooplasm undergo marked changes in their cytology as described below : (a) Primary vitellogenic oocyte (figures 4,6, 7): Further increase in amount of basophilic ooplasm and the volume of nucleus and nucleolus accompanies the appearance of few small yolk droplets in peripheral ooplasm (figure 4). The yolk droplets stain purple to black with haematoxylin or blue with Mallory's triple or Azan stains. Chromatin clumps become finely granular arranged in a network in nucleoplasm. Nucleus is solid and central in position. A thin layer of follicular cells forms around the oocyte (figures 6, 7). Further increase in oocyte diameter is accompanied with increase in amount of yolk droplets which progressively extends towards the yolkless and homogeneous perinuclear region (figures 6, 7). The yolk droplets swell and minute unstainable vacuoles begin to appear in them (figure 7). Average diameter of oocyte is 250 micra. (b) Secondary vitellogenic oocyte (figures 8,9, 11, 13): The oocyte shows a rapid growth and the un stainable vacuoles in yolk droplets increase in number and size (figures 8, 11). This results in marked decrease in stainable contents of yolk droplets which thus appear reticulated or spongy. Vacuoles fuse with each other and ultimately form large unstainable yolk vesicles, initially in peripheral ooplasm but later on in other regions (figures 9, 11, 13). This is followed by the appearance of small eosinophilic yolk granules in the extravesicular ooplasm (figures 9, 11). In Mallory's triple or Azan stains the yolk granules stain red. They increase in size probably by their fusion and thus gives rise to large oval yolk globules

7 458 P C Joshi and S S Khanna (figure 13). Oolemma and follicular layers are well differentiated (figure 10). While the yolk granules are appearing in the ooplasm, the nucleolus enlarges and becomes eccentric and vacuolated without any change in its tinctorial properties (figures 12, 13). Yolk droplets gradually disappear. Oocytes measure 600 micra in diameter. (c) Tertiary vitellogenic oocyte (figure 14): With further growth of oocyte, the amount of yolk globules increases. Yolk granules still continue to appear in peripheral ooplasm. Yolk droplets are absent because of their conversion into yolk vesicles. Ooplasm thus becomes entirely acidophilic. Yolk globules become polygonal and occupy most of the ooplasm including the perinuclear region. Nucleolus is eccentric in position and poorly stainable due to its extensive vacualization. Follicular layer is stretched to a thin membrane and its nuclei become spindle shaped, probably due to the turgidity of oocyte. Vitelline membrane is distinct. Oocyte is 950 micra in diameter. 3.3e. Ripe ovum (figure 15): It is largest in size measuring upto 1400 micra in diameter. Ooplasm is heavily impregnated with large yolk globules and yolk vesicles. J.4. Seasonal changes in ovaries The annual ovarian cycle can be conveniently divided into following four stages on the basis of histomorphological features of ovaries : 3 4a. Spawning and spent stage (May-June): During this period females carry their spawn attached onto their pleopods. Ovaries are small, smooth and cream coloured. Seminal receptacles contain sperms. Discharged follicles are found in stroma, indicating that the spawning has taken place (figure 1). Ovarian wall becomes thick and the germinal zone consists of numerous oogonia, premeiotic and previtellogenic oocytes (figures 1, 3). A few primary vitellogenic oocytes also develop at peripheral region of ovary. 3 Ab. Early maturing stage (July-October): Ovaries increase in size and are coloured deep yellow. Externally they appear granular owing to the preponderance of primary and secondary vitellogenic oocytes in them. Germinal zone contains a few residual oogonia and previtellogenic oocytes. 3.4c. Advanced maturing stage (November-February): Ovaries become enlarged, convoluted and orange in colour. Large orange ova bulge out on the surface of ovaries. Tertiary vitellogenic oocytes are commonly present but a few secondary ones also occur. Premeiotic and primary vitellogenic oocytes are rare in the ovaries. 3 Ad. Mature stage (March-April): Ovaries attain a maximum size and deep orange mature ova are visible through the thin ovarian wall. The gonosomatic index is minimum in June and reaches a maximum value in April and is in conformity with the average ova diameter (figure 16).

8 Seasonal changes in the ovary of crab G> GSI 0-.<0 OVA DIAMETER 3 2, O'---~----I'---'--~_...L.---I- JON F M A M J J A S 0 MONTHS ~~_",,",-...L.IO Figure 16. Seasonal changes in the gonosomatic index (081) and ova diameter. 4. Discussion The female reproductive organs of P. koolooense are built on the same general plan as observed in other crabs by Hartnoll (1968), Vasisht and Relan (1971) and Chiba and Honma (1971). Several decapods possess a germinal zone in the centre of ovary (Kessel 1968 ; Hinsch and Cone 1969 ; Rouquette 1970 ; Laulier and Demeusy 1974; Rao et al 1981) whereas in others the germinal zone is peripheral (Cronin 1942 ; King 1948) or is in the form of nests of germ cells distributed throughout the ovary (Weitzman 1966). In P. koolooense the germinal zone occurs at the centre of ovary. Little is known about the origin of new crop of germ cells in adult decapods It is generally agreed that the new crop of germ cells arise by division of existing oogonia. In Gecarcinus lateralis (Weitzman 1966) and Pachygrapsus mormoratus (Rouquette 1970) oogonial mitosis occurs throughout the year. In P. koolooense the resting oogonia or residual oogonia occur throughout the year but they divide shortly after ovulation and produce a new drop of oogonial cells for further growth of ovaries, as observed by Aoto (1952) and Laulier and Demeusy (1974) in other decapods. The process of yolk formation varies considerably in different decapods. The yolk vesicles and yolk globules in the oocyte of P. koolooense correspond with the fatty yolk vacuoles and protei nous yolk bodies of Carcinus maenas (Harvey 1929), Palaemon lamarrei and Paratelphusa spinigera (Bhatia and Nath 1931) and Paratelphusa hydrodromous (Vasisht and Relan 1971). Bhatia and Nath (1931) observed that the fatty yolk vacuoles appear initially at the peripheral ooplasm while the protein yolk bodies near perinuclear region. In shrimp, Chirocephalus bundyi (Linder 1959) the fatty yolk droplets and proteinous yolk granules first appear in the central region of ooplasm. In P. koolooense both yolk vesicles

9 460 P C Joshi and S S Khanna and yolk granules first appear at peripheral ooplasm and then extend progressively towards perinuclear region as has also been observed by Harvey (1929) in Carcinus maenas. Besides the structural changes, the yolk bodies and nucleolus in some crabs undergo parallel changes in their cytochemical nature and staining affinity (Otsu 1963 ; Carmignani et ai 1973). In P. koolooense, neither the yolk globules nor the nucleolus undergo changes in their staining reactions. The source of yolk material differs in various species of crustaceans according to several authors. Generally the yolk is formed from both extra oocyte sources (perinuclear endoplasmic reticulum and golgi bodies in collaboration with nucleolar extrusions (Kessel 1968 ; Hinsch and Cone 1969 ; Hinsch 1970 ; Dhainaut and Leersynder 1976) and extra oocyte sources (yolk precursors are incorporated from haemolymph into ooplasm) by diffusion through follicular cell layer (Linder 1959; Beams and Kessel 1963) or by micropinocytosis at oocyte surface (Hinsch and Cone 1969; Dhainaut and Leersynder 1976). The nucleolus during vitellogenesis undergo progressive increase in size and shows vacuolation (Harvey 1929 ; Hinsch 1970 ; Dhainaut and Leersynder 1976). The nucleolar extrusions or granules which pass into ooplasm are believed to take part in yolk formation (endogenous yolk) in crabs (Harvey 1929 ; Bhatia and Nath 1931 ; Hinsch 1970). In P. koolooense, neither the nucleolar granules were seen nor the nucleolus was found moved into the ooplasm. Although the increase in the size of nucleolus, its eccentric position and progressive vacuolization during vitellogenesis in this species indicates that it has some role in yolk ormation, nothing can be said about its functions in the absence of direct evidence. Seasonal morphometric studies revealed that the breeding cycle in crabs and other decapods varies widely, even in species having close taxonomic relationships or similar ecological niches, i.e., (i) continuous breeder around the year (Boolootian et al 1959; Knudsen 1964; Rahman 1967; Badawi 1975), (ii) Seasonal breeders having one spawning season (Boolootian et al 1959 ; Hartnoll 1963 ; Otsu 1963 ; Knudsen 1964 ; Diwan and Nagabhushanam 1974 ; Bomirski and Klek 1974; Badawi 1975) or two distinct spawning seasons (Knudsen 1964; Chandran 1968; Adiyodi 1968, Goldstein and Lauria 1975). P. koolooense is a seasonal breeder, as all the oocytes in the ovaries become fully mature at the onset of breeding season and are laid simultaneously during May or June, followed by the next ovarian cycle. The gonad index begins to decline in May, reaching a minimum level in June and increases slowly during the following months with a peak in April (figure 16). Such variations in the breeding season among decapods may be due to the genetic differences and the local ecological conditions. Studies on the seasonal histological changes in the ovaries revealed that in post spawning period the vitellogenesis takes place late and is preceded either by a long resting stage (Weitzman 1966; Diwan and Nagabhushanam 1974) or by recovery or previtellogenic stages (Chiba and Honma 1972; Badawi 1975). In biannual breeders, the vitellogenesis of second ovarian cycle begins soon after the completion of the preceding cycle while in first ovarian cycle the vitellogenesis is preceded either by long resting phase (Adiyodi 1968) or a brief resting stage

10 Seasonal changes in the ovary of crab 461 (Bomirski and Klek 1974). In P. koolooense vitellogenesis occurs shortly after spawning and it appears that the resting stage in this species is of very short duration probably of a few days only. Acknowledgements One of the authors (PCJ) is thankful to the UGC, New Delhi, for the financial assistance. Thanks are due to Miss Maya Deb and Dr M Koshy, ZSI, Calcutta, for the identification of the species. References Adiyodi R G 1963 On the reproduction and molting ill the crab, Paratelphusa hydrodromous; Physwl. Zoot Aoto T 1952 Sexual phases in the prawn, Pandulus kessleri (Zern.) with special reference to the reversal of Sex. ; J. Fae. Sci. Hokkaido Univ Badawi H K 1915 On maturation and spawning in some penaeid prawns of Arabian Gulf; Mar. Biol. (Berlin) Beams H Wand Kessel R G 1963 Electron microscopic studies On developing crayfish oocytes with special reference to the origin of yolk; J. Cell Bioi Bhatia D Rand Nath V 1931 Studies Onthe origin of yolk. VI. The crustacean oogenesis; Quart. J. Micro. Sci Bornirski A and Klek E 1914 Action of eyestalks on the ovary in Rhithropanopeus harrisii and Cragon cragon (Crustacea, Decapoda); Mar. Bio/ Boolootian R A, Giese A C, Farmanfarmaian A and Tucker J 1959 Reproductive cycle of five west coast Crabs; Physiol. Zoot Carmignani M. Albanese P, Bolognari A and Zaccove G 1913 Morphological stud) On the yolk globules in the growing oocyte of Maja verrucosa (Edw.) (Crustacea, Brachyura); Acta Histochem Chandran M R 196& Studies Oil marine crab, Charybdis variegata. I. Reproductive and nutritional cycle in relation to breeding periodicities; PrOC. Indian Acad. sci. B Chiba A and HOuma Y 1911 Studies On gonad maturity in some marine invertebrates. II. Structure of leproductive organs of the lined shore Crab; Bull. Jpn. Soc. Sci. Fish Chiba A and Houma Y 1912 Studies on gonad maturity in sorne marine invertebrates. VII. Seasonal changes in the OVary of the lined shore crab; Bull. Jpn. Soc. Sci. Fish. 38 3Z3-329 "Cronin L E 1942 A histological study of the development of ovary and accessory reproductive organs in the blue crab, Callinedes sapidus (Rathbun) ; M.S. Thesis Univ. Mary/and Dhainaut A and Leersynder De M 1916 Cytochemical study of the oocyte development in crab, Eriocheir Sinensis. I. Natural oogenesis: Arch. Biol Diwan A D and Nagabhushanam R 1914 Reproductive cycle and bioch(,mical changes in gonads of freshwater crab, Barytelphusa cunicularis (Westwood); Indian J. Fish Giese A C 1959 Annual reproductive cycle of marine invertebrates; Ann. Rev. Cvtol, Goldstein B and Lauria. L 1915 Cycle of sexual maturation and preliminary studies on spawning of the freshwater shrimp. Palaemonetes argentinus, 1. Female; Physis. Agnas Cont. Org. Sect. B Hartnoll R G 1963 The biology of manx spider crabs; Proc. Zoo/. Soc. London Hartnell R G 1968 Morphology of the genital ducts in female crabs; J. Linn. Soc. (Zoo/.) Harvey L A 1929 The oogenesis of Carcinus maenas (Penn.) with special reference to yolk formation; Trans. Roy. Soc. Edinburgh

11 462 P C Joshi and S S Khanna Hinsch G W 1970 Possible role of intranuclear membranes in nuclear cytoplasmic exchange in spider Crab oocytes ; J. Cell Bioi Hinsch G Wand Cone M V 1969 Ultrastructural observations of the vitellogenesis in spider crab, Libinia emarginata ; J. Cell stot Kessel R G 1968 Mechanism of protein yolk synthesis and deposition in cruhacean oocyte; Z. Zellforsch King J E 1948 A Study of the reproductive organs of the common marine shrimp, Pe1lJJeUs setiferous (Linn.) ; BioI. Bull Knudsen J W 1964 Observations of the reproductive cycle and ecology of the common brachyura and crab like anomura of Puget Sound coast, Washington; Pac, Sci Laulier M 1974 Caraeteres cytologique de la cellule sexuele female du crabe, Carcinus maenas L. au cours de la gametogenese ; Cah. Biol, Mar Laulier M and Demeusy N 1974 Etude histologique du fonctionment ovarian an cours d'une maturation de ponte chez le crabe, Carcinus maenas L. (Crustacea, Decapode) ; Coho Biol. Mar Linder H J 1959 Studies On freshwater fairy shrimp, Chirocepha!us bundyi (Forbes). 1. Structure and histochemistry of ovary and accessory reproductive tissue ; J. Morphnl Otsu T 1963 Bihormonal control of sexual rycle in the freshwate, crab, Potamon dehaani ; Embryolngica Rahman A A 1967 Reproductive and nutritional cycle of the 'Tab, Portunus pelagicus (Linnaeus) (Decapods, Brachyura) of Madras coast; Pror, Indian Acad. SCi. B Rao Ch Narasimha, Shakuntala K and Reddy S R 1981 Moult-reproduction relationship in the freshwater prawn, Machrobrachium lanchesteri (de Man); Proc. Indian Acad. Sci Raven C P 1961 Oogenesis: The storage of developmental information (London and New York: Pergamon Press) Rouquette M 1970 Etude du tissu evarien chez Ie crabe Pachygrapsus marmoratus (Fabricius) Premiers resultats concernant Ies role de la temprature et des pedoncules oculaires : Bull. Soc. Zoo!' France Vasisht H Sand Relan U 1971 Anatomy of Paratelphusa masoniana (Henderson). V. Reploductive system; Res. Bull. Punjab Univ, Weitzman M C 1966 Oogenesis in tropical land Crab, Gecarcinus lateralis (Freminville) ; Z. Zellforsch, * Not consclted in original.