1959 423

Spermatogenesis of the Gastropod , globosa, with S pecial Reference to the Cytoplasmic Organelles

G. P. Sharma, Brij L. Gupta , and O. P. Mittal

Department of Zoology, Panjab University , Hoshiarpur, Punjab, India Received December 22, 1958

Introduction The gastropods constitute the classical material for the study of sperma togenesis. The two most important aspects which have been the subject of controversy for the cytologists are the acrosome formation and the dimorphic sperms. Whereas the disagreement regarding the acrosome formation has been in the pulmonates, the problem of dimorphic sperms is restricted to the order prosobranchia of the class . According to Wilson (1925), von Siebold (1837) was the first worker to report dispermy in the prosobranch, Paludina (now called Viviparus). He described two types of sperms, viz., worm-shaped or oligopyrene, and the hair-shaped or eupyrene. This preliminary report of von Siebold was later on confirmed and ex tended by a number of subsequent workers like Meves (1902), Gatenby (1919), Ankel (1924), Alexenko (1926), Tuzet (1930), Woodard (1940), Pol lister and Pollister (1943) etc., in Viviparus (Paludina) vivipara, and a number of other prosobranchs. All of these workers have based their obser vations on the fixed and sectioned material. Pollister and Pollister (1943) have, however, studied only the chromosomes and centrosomes in both the eupyrene and the oligopyrene sperms of Viviparus vivipara. Recently Hanson et al. (1952) have worked out the detailed structure of the eupyrene and the oligopyrene sperms in the prosobranch, Viviparus. These authors examined the living cells under the phase-contrast microscope and the fixed material with the electron microscope and the various cyto chemical techniques. They have, however, confined themselves mainly to the structure of the ripe sperm and the fate of the mitochondria. They did not pay much attention to the phenomenon of the acrosome formation. Almost all the aforementioned workers have described an oligopyrene sperm which is much longer than the eupyrene one, but has only a very small head. Franzen (1955) has also given a survey of spermiogenesis in the eupyrene sperm of a number of prosobranchs studied in the living material with the phase-contrast microscope. Some of his interpretations, however, seem er roneous to us. In the present paper are recorded the observations made on the living 424 G. P. Sharma, B. L. Gupta, and O. P. Mittal Cytologia 24

male germ cells of an Indian fresh water prosobranch, Pila globosa, with the phase-contrast microscope. While the formation of the eupyrene sperm in this material is described in detail with particular reference to the cyto plasmic organelles, only a brief account of the oligopyrene sperm has been given. The latter is, however, shorter than the former in this material and it appears that it is devoid of a mitochondrial middle-piece. Further details regarding its formation are being worked out.

Material and technique

The living specimens of the gastropod, Pila globosa Swainson (Apple

- Snail) were procured from Patna (Bihar) as they are not available in the

Punjab. The were placed in a glass aquarium containing some algae

and other green vegetation. They could be kept in this condition for months

without showing any ill effect.

The sexes in P. globosa are separate. The single testis lies closely at

tached to the upper surface of the digestive gland in the 2nd and 3rd coils

of the shell.

The testicular material was fixed in the various fixatives like Lewitsky,

Champy and Bouin etc., and embedded in paraffin. The sections, 5p in

thickness, were subsequently stained with 0.5% iron-haematoxylin.

The living material was studied in Baker's (1944) calcium-saline and

Carl Zeiss stand 'W' microscope with phase-contrast optics was used for

this purpose. The photomicrographs have been taken with a Carl Zeiss

micro-reflex attachment camera, fitted with a Contax adapter. 'Kodak,'

35mm Pan-X Film was used for the same. Only Ph 1.25/100 oil immersion

objective and a compensating K 8•~ocular were employed for photomicro

graphs. Blue green light (Zeiss filter) was used for both the fresh study and

photographic work.

Observations Dimorphism of spermatozoa The present investigation on the spermatogenesis of Pila globosa Swainson shows that there are two distinct types of spermatozoa in this material, both occurring equally frequently. One of them has a structure typical of the uniflagellate sperm of the other gastropods (see Nath 1957), while the other is short and stumpy, bearing 4 to 8 flagella. The former, which is the normal type of sperm, may be called as 'eupyrene' and the latter, the abnormal one, as 'oligopyrene'. As the detailed processes of spermatogenesis, related to the fate of the cytoplasmic organelles, also differ considerably in these two types of sperms, it will be best to describe them separately. In the present paper, however, the genesis of only the eupyrene sperm has been given in detail and for the oligopyrene just a brief preliminary note has been included. 1959 Spermatogenesis of the Gastropod , Pila globosa 425

Further details regarding the latter type of sperm are being worked out and will be published as soon as they are ready . Both the eupyrene and the oligopyrene sperms are motile . The latter nevertheless moves much more slowly than the former , when observed in a d rop of physiological saline under the phase-contrast microscope .

Figs. 1-14. Diagrammatic representations of the various stages of spermatogenesis of Pila globosa based upon a study of the fixed as well as the living material under the phase contrast microscope. 1, germinal epithelium. 2 to 4, spermatogonia. 5, gonial telophase. 6-8, primary spermatocytes . 9, meiosis I, telophase. 10, secondary spermatocyte. 11, meiosis II, telophase. 12, schematic figure of a ' duplex sphere' showing its growth and division into two. 13, 14, spermatids.

The germinal epithelium in P. globosa is a syncytium, unlike that of aspersa (Gatenby 1917). The nuclei of the germinal layer are generally rounded,. but sometimes oval in shape and rest upon a fibrous layer-the Ancel 426 G. P. Sharma, B. L. Gupta, and O. P. Mittal Cytologia 24 layer (Gatenby 1917). The cytoplasm, which is scarce, is hyaline and devoid of any granulation whatsoever (Fig. 1). Some of the cells in the germinal layer are quite large and have distinct cell boundaries. These are the precursors of the oligopyrene sperms. Genesis of the eupyrene sperm Spermatogonia:-There are more than one gonial generations. The cells of the early gonial generations are small, with a thin layer of cytoplasm around their nuclei. The cytoplasm, however, does not show any granulation at this stage (Fig. 2). The cells of the last gonial generation have more of the cytoplasm around a centric nucleus. The cytoplasm also reveals a juxta-nuclear mass of granules in early stages. In the living cells, examined under the phase-contrast, some of these granules give a higher phase-change than the rest (Fig. 3). Gradually, these granules become circumnuclear, appearing greyish in Lewitsky/iron haematoxylin preparations. A comparatively larger and deeply staining granule now makes its appearance in the cytoplasm, presumably arising by the fusion of the discrete dark granules of the early stages. This large chromophil granule or sphere is homologous to the so-called 'Golgi body' of other workers (Nath 1957), while the rest of the granules are the mitochondria. Soon, the single chromophil sphere grows and develops a duplex structure with a darkly staining complete or incomplete cortex, enclosing a chromophobic medulla (Fig. 4). This duplex sphere is homologous to the 'duplex Golgi spheroid' described earlier (Nath 1957, Sharma and Gupta 1957). When the living cells are examined with the phase-contrast microscope the duplex nature of the sphere is very clear. However, the difference in the phase-change produced by the cortex and the medulla is not very marked. The sphere may appear as a 'ring' or a 'crescent' in optical sections, depending on whether the sheath or the cortex is complete and uniform in thickness, or it is incomplete and variable in thickness. The mitochondria in the living cells appear as fine granules and also as long filaments of low contrast with one dark granule at one or both of their tips (Fig. 4). It may be mentioned that this disparity in the appearance of the mito chondria in Lewitsky/iron-haematoxylin preparations and in the living cells examined with the phase-contrast microscope exists in all the subsequent stages of spermatogenesis except in the spermatid when the mitochondria begin to form the middle-piece. It has also been observed previously by Sharma and Gupta (1957) in ticks and by Gupta (unpublished) in a variety of cells of different animals. It seems that the fine mitochondrial filaments of the living cell are broken up into granules by the strong precipitating effect of the chromic acid in Lewitsky on the proteins (Baker 1945). Lewitsky, therefore, does not seem to be a good fixative for the mitochondria. Mitosis:-The dividing spermatogonia present a very interesting feature 1959 Spermatogenesis of the Gastropod, Pila globosa 427

inasmuch as the single duplex sphere of the early prophase stages divides into two during the late prophase, so that each of the two daughter cells receives one of them. During division the single duplex spheroid first assumes a ' ringed' appearance. Gradually it becomes oval and a constriction begins to appear in its middle to give it a dumb-bell-shaped appearance. At this stage the dividing sphere consists of two 'crescentic' spheres with their concavities facing each other and their medullary or chromophobic material joined together by a small curved canaliculus (Figs. 12). Ultimately the two daughter spheres become separated. During anaphase each of these two spheres moves with the respective mass of chromosomes (Fig. 5). Hence each daughter cell gets its own share of the parent sphere . This process is repeated in both the meiotic divisions also and in this way the sphere is passed on to all the four spermatids, where it secretes the acrosome (vide infra). The mitochondria are also distributed equally between the two daughter cells. They always occupy the periphery of the cell during metaphase, anaphase and telophase stages. Spermatocytes:-The primary spermatocytes are definitely the largest cells in the testis, otherwise both the primary and the secondary spermatocytes have a similar structure, with eccentric nuclei. The cytoplasmic organelles also do not change in their morphology. The mitochondria continue to be filamentous as well as granular, in form. A single duplex sphere is always present in the prophase stages, occupying the paranuclear zone of the cyto plasm which is free from the mitochondria (Figs. 26 to 29). The duplex sphere seems to grow during the early prophase stages (growth period) of the primary spermatocyte (Figs. 6 and 7). In the diplotene or diakinesis of meiosis I and late prophase of meiosis II the single duplex sphere divides into two, just as in the gonial stages described already (Figs. 8, 10 and 30). At telophase, therefore, each daughter cell gets one duplex sphere (Figs. 9, 11 and 31). Besides the single duplex sphere, the cytoplasm of the spermatocytes sometimes reveals one, two or even three spherical or oval bodies of different sizes. They appear homogeneously dark-grey in the living cells studied under the phase-contrast microscope. In Lewitsky/iron-haematoxylin preparations they are stained deeply. They are not destroyed or corroded even in Bouin/iron haematoxylin preparations, in which again they take up a deep stain. They can be homologized with the chromatoid bodies described by Gupta (1955) and Sud (1955). The chromatoid bodies are occasionally met with even in the actively dividing cells. However, unlike the duplex sphere they do not divide and pass on as such only to one of the two daughter cells in both the divisions. Spermateleosis:-The early spermatid, which emerges after meiosis II, has more or less the same structure as the spermatocytes, with uniformly

Cytologia 24, 1959 29 428 G. P. Sharma, B. L. Gupta, and O. P. Mittal Cytologia 24 1959 Spermatogenesis of the Gastropod , Pila globosa 429

distributed mitochondria and a single duplex spheroid . The latter has a complete sheath of uniform thickness during the early st ages, consequently, appearing as a dark ring in optical sections (Fig . 13). Soon the process of spermateleosis sets in. The first change appears in the nucleus of the spermatid. Its entire chromatin contents be come cond ensed in the form of a kidney-shaped mass in its poste rior half leaving the anterior half as a clear vesicle . This mass of chromatin appears dark grey under the phase-contrast microscope and is highly chromophilic i n Lewitsky or Bouin/iron-haematoxylin preparations . The rest of the nucleus is perfectly clear (Fig. 14). A single granular centrosome can always be observed at the posterior pole of the nucleus in the living cells (Fig . 14), but it is not discernible in iron-haematoxylin preparations due to deep straining of this region. An axial filament soon springs up from it and the nucleus becomes slightly oval (Fig. 15). A small dark rod going into the nucleus can also be seen arising from the centrosome (Fig . 16). This is the intra-nuclear rod or filament (see Nath and Gupta 1956, Kanwar 1958). In the meantime the single duplex sphere also changes with the result that it no longer appears as a ring . Its sheath becomes thick on one side leaving a bit of the medulla exposed on the other , thus appearing as a typical crescent in optical section. As this sphere will ultimately secrete the acrosome , i t may now be termed as the acroblast . First it migrates to the anterior pole of the nucleus (Fig. 14). It then slowly approaches it, till the exposed part of its medulla comes in contact with the nuclear membrane at a point which is exactly opposite the centrosome (Figs. 15, 16 and 32). The nucleus of the spermatid now becomes pyriform , its posterior end being narrower (Fig. 17). The mass of the chromatin which was, to begin with, relegated to its posterior part gradually encroaches upon its clear anterior part till the latter is completely obliterated (Figs. 17 and 18). After this stage the nucleus loses its staining capacity . It, thus, appears uniformly grey in iron-haematoxylin preparations. Under the phase-contrast microscope it gives a phase-change lower than that of the surrounding cytoplasm . The shape of the nucleus, however, again changes. Though remaining pear-shaped,

Figs. 15-25. Diagrammatic figures showing the process of spermateleosis of the eupyrene sperm in Pila globosa based upon a study of the living material under the phase-contrast microscope as well as the fixed preparations. 15, 16, early stages showing the transformation of the mitochondria into spheres and the appearance of the axial and the intranuclear fila ments. 17, 18, the nucleus has become pear-shaped . The mitochondrial spheres have ag gregated at the posterior end. The acroblast has secreted a granular acrosome. Also note a chromatoid body. 19, the mitochondria have become rod-like and have started forming the sheath of the middle-piece. 20, polar-view showing the arrangement of the mitochondrial rods, the centrosome and the axial-filament . 21, 22, the elongating spermatids showing the spiral twisting of the mitochondrial ribbons, the appearance of the distal centrosome, the backward movement of the acroblast , and the conical acrosome. 23-25, further elong ation of the spermatids showing the process of nuclear spiralization. The mitochondrial sheath of the middle-piece has become smooth.

29* 430 G. P. Sharma, B. L. Gupta, and O. P. Mittal Cytologia 24

its anterior pole becomes conical, while the posterior end is broadened (Fig. 19). The acroblast deposits a small granular acrosome at the point of its contact with the nuclear membrane (Figs. 17, 18 and 33). A little later the acrosome takes up its position at the pointed tip of the nucleus and the acroblast begins to move back (Fig. 19). A single chromatoid body may also be seen in some of the spermatids (Figs. 17 and 18). Occasionally it may divide into two or more smaller granules (Fig. 21). The mitochondria undergo the most interesting change during these stages. To begin with, they start aggregating at the posterior pole of the nucleus and at the same time they begin to lose their filamentous appearance (Figs. 15 and 16). Gradually they are transformed into a number of spheres situated around the axial filament just below the narrow posterior end of the pyriform nucleus (Figs. 17, 32 and 33). The smaller spheres gradually fuse to form larger spheres, till ultimately there are formed about 6 to 8 fairly large spheres arranged in a ringlet around the axial filament, and lying closely apposed to the nuclear membrane (Figs. 18 and 33). These mitochondrial spheres take up a deep stain in Lewitsky/iron-haematoxylin preparations. Soon the mitochondrial spheres resolve themselve into 6 to 8 small rodlets. Each of these mitochondrial rodlets has a broad anterior and a tapering posterior end. The anterior ends of these rodlets are closely ap posed to the nuclear membrane, but they do not meet each other. On the posterior side, however, these rodlets are joined together around the axial filament (Figs. 19 and 34). If viewed from the posterior pole, the spermatid at this stage gives a very characteristic appearance. The mitochondrial rod lets are arranged in a ring around the centrosome from which comes out an axial filament. Each rodlet appears flame-like, with the tip facing the centrosome. In the living cells, examined under the phase-contrast micro scope, they give a duplex appearance with a very dark thick externum, enclosing a highly refractile medulla (Fig. 20). This is obviously an optical artifact produced as a result of the great thickness and high phase-change of these rodlets (Gupta 1958). In Lewitsky/iron-haematoxylin preparations these rodlets are stained uniformly dark. With the beginning of the process of elongation, and the maturation of the sperm, the acrosome becomes conical and pointed. It does not, however, grow. The nucleus also becomes elongated and cylindrical. The mitochondrial rodlets in the meanwhile get packed up tightly around the axial filament. Consequently the gaps between the individual rodlets disappear and they no longer appear to be arranged in the form of a ring in polar views. Gradually they begin to wind spirally around the axial filament (Figs. 20 to 22 and 35). The acroblast has, by this stage, drifted back into the tail region along with the cytoplasm. Simultaneously, the single centrosome at the posterior end of the nucleus 1959 Spermatogenesis of the Gastropod, Pila globosa 431 seems to divide into two-a proximal and a distal one. The latter moves to the tip of the mitochondrial sheath and marks the posterior limit of the middle-piece (Figs. 21 to 23). It is difficult to say whether the proximal centrosome becomes ring-shaped or it again divides into two. In the living cells, examined under the phase-contrast microscope, it appears in the form of two granules lying just at the point of contact of the nuclear membrane with the mitochondrial rodlets (Figs. 21 to 23). Whether these structures are two individual granules or they just represent the optical section of a ring-shaped centrosome, it is difficult to say. They cannot be identified in Lewitsky or Bouin/iron-haematoxylin preparations due to the deep staining of the mitochondrial middle-piece. The axial filament seems to become con tinuous with the intra-nuclear rod. Gradually the nuclear contents become denser and chromatic. The nucleus itself now becomes cylindrical and starts twisting spirally like those of the other gastropod sperms (Nath 1957, Kanwar 1958). A thin filament has often been observed traversing the whole length of the nucleus from the proximal centrosome to the acrosome (Fig. 24). This seems to be the ex tension of the small nuclear filament of the earlier spermatids. The mito chondrial sheath becomes attenuated by the tightening of the spirals of the rodlets. The proximal centrosome(s) is no longer discernible. The distal centrosome also, which is quite prominent in the fixed preparations, is hardly perceptible in the living material. Finally, most of the cytoplasm along with the acroblastic remnants and the chromatoid bodies (if any) is also sloughed off in the form of blebs, leaving only a thin film around the entire sperm excepting a small end-piece (Figs. 21 to 25 and 36 to 38). The ripe eupyrene sperm of P. globosa is not a very long structure. It has a cork-screw like head, followed by a long middle-piece. The latter indistinguishably merges into the main-piece. Only a small part of the axial filament is naked and that forms the end-piece (Fig. 39). The sperm is very actively motile. It moves by a spiral rotation of the head, which has been observed to be both clockwise and anti-clockwise under the phase-contrast optics. Very often a thin and very long filament, which is sometimes even longer than the sperm itself, has been observed coming out of the acrosome of the living maturing spermatids or even sperms under the phase-contrast optics (Fig. 22). This seems to be cytoplasmic and, according to Franzen (1955, 1956), it helps in the attachment of the spermatids to the follicular wall. Oligopyrene sperm It has not been possible to make out clearly the early cells which give rise to the oligopyrene sperms. It has already been mentioned, however, that some of the cells in the germinal layer appear quite large with distinct cell boundaries. Their cytoplasm, though comparatively abundant, does not show any granulation. The oligopyrene spermatocytes show a paranuclear 432 G. P. Sharma, B. L. Gupta, and O. P. Mittal Cytologia 24

Figs. 26-39. The photomicrographs of the living male germ cells of Pila globosa, showing

the genesis of the eupyrene sperm. •~1600 approximately. 26-28, primary spermatocytes showing the mitochondria and 'duplex spheres' in various forms. 29, secondary sperma tocyte. 30, secondary spermatocyte showing the division of the 'sphere' into two. 31, two early spermatids still connected by the remnants of the spindle-fibres. 32, 33, spermatids showing the condensation of the nuclear material in the posterior half, the aggregation of the mitochondria and the beginning of the acrosome formation. 34, 35, spermatids showing 1959 Spermatogenesis of the Gastropod , Pila globosa 433

collection of filaments and granules which cannot be categorized properly in L ewitsky/iron-haematoxylin preparations . No dividing cells in the oligopyrene series were encountered in our preparations . The spermatid which forms the oligopyrene sperm can always be dis tinguished from the normal one both in the living cells and the fixed mate rial.

Figs. 40-45. The photomicrographs of the living male germ cells of Pila globosa showing some stages of the genesis of the oligopyrene sperm. •~1600 approximately. 40, early spermatid showing the spherical mitochondria and some 'Golgi bodies'. 41, 42, spermatids showing the appearance of a number of axial filaments in each. The acroblast is also

discernible in some of the cells. 43 to 45, maturation of the oligopyrene sperm.

An early spermatid of the oligopyrene series reveals globular mitochondria and more than one duplex spheres in its cytoplasm (Fig. 40). The nucleus is clear and gives a lower phase-change than that of the ground cytoplasm. Very soon there arises a tuft of flagella from its base. This tuft may com prise 4 to 8 flagella (Figs. 41 and 42). the conical acrosome, intra-nuclear filament, and spiral twisting of the mitochondrial rib bons. 36 to 38, elongating spermatids showing the spiral twisting of the nucleus and the sloughing off of the residual cytoplasm with the acroblast. 39, mature eupyrene sperm lying near an oligopyrene sperm showing slight movement. 434 G. P. Sharma, B. L. Gupta, and O. P. Mittal Cytologia 24

Soon the nucleus elongates and becomes cylindrical. The duplex spheres fuse to form one, which seems to fit over the proximal part of the nucleus to give rise finally to a cap-like acrosome. The mitochondria never form any balls or rodlets, as observed in the eupyrene type (Figs. 43 to 45). The ripe oligopyrene sperm of P. globosa is shorter and much stouter than the eupyrene type (Fig. 39). The nucleus is long and cylindrical which sometimes reveals granulation and a central filament (Fig. 45). It is headed by a cap-like acrosome and is followed by a short middle-piece, which seems to be devoid of any mitochondria (Figs. 44 and 45). The middle-piece is terminated by a ring-like distal centrosome. There is no main-piece and the flagella come out directly from the distal centrosome.

Discussion There exists a rich literature on the morphogenesis of the eupyrene and oligopyrene sperms in the prosobranchs (Franzen 1955). Excepting, however, the latest works of Hanson et al. (1952) and Franzen (1955), who have mainly studied the living material under the phase-contrast microscope, all the other workers examined only the fixed and sectioned material or the living sperms under the ordinary microscopes. They have, therefore, given a diversified account of the various cytoplasmic inclusions according to the techniques employed by them. Many of them have confined themselves to a study of only the structure of the sperms or the centrosomes etc. It is, therefore, the work of Hanson et al. (1952) on Viviparus vivipara and of Franzen (1955) on the eupyrene sperms of a number of prosobranchs which needs comparison with the observations presented in this paper. Hanson et al. (1952) have studied the process of spermatogenesis in the living material under the phase-contrast microscope and in the fixed material by the electron micrography and cytochemical techniques. The most salient features of their observations are as follows: -1. The head of the eupyrene sperm is spirally twisted. It is followed by a long middle-piece and a tail. 2. The proximal centrosome is ring-like which surrounds a short conical structure entering the nucleus of the sperm. Neither of these two structures can be made out in an intact ripe sperm. 3. The middle-piece is ensheathed by a superficial membrane and by four tape-like fibres spirally wound around the axial-fibres. These tapes arise from the four mitochondrial balls present in the spermatid. 4. The oligopyrene sperm has a thimble-shaped head surrounding the end of the thick and long middle-piece. The number of tails is commonly 10. The proximal centriole lies under the apex of the head and the distal at the base of the tail brush. In spite of the fact that we have worked on an altogether different proso branch (i.e. P. globosa) and we did not use an electron microscope, our 1959 Spermatogenesis of the Gastropod, Pita globosa 435 observations on the eupyrene sperm are in full conformity with those of Hanson et al. However, the middle-piece in P. globosa is ensheathed by six mitochondrial tapes as against 4 in V. vivipara . Franzen (1955), who studied the process of spermiogenesis in about 16 prosobranchs, has divided the order into two groups, viz. Diotocardia and Monotocardia. The former possesses a primitive type of sperm, while that of the latter is more typical. The dispermy occurs in monotocardia only. Franzen's account of 10 of monotocardia is very general and frag mentary, and he has confined himself mainly to the late stages of sperma teleosis. His account of acrosome formation is more or less the same as given by us for P. globosa. The sperm in the present material is more or less like that of Crepidula fornicata and velutina. In the latter Franzen also observed a thin filament traversing the whole length of the nucleus and reaching the acrosome. He has, however, shown that a centro some is present at the top of the nucleus just below the acrosome in this and some other species. On the other hand, our own observations and also those of Hanson et al. (1952) indicate that the proximal centrosome is ring like and it lies at the base of the nucleus. The centrosome observed by Franzen (1955) at the top of the nucleus, therefore, seems to us to be the acrosome which might have retained a bit of its granular nature. The fila ment in the nucleus is only a prolongation of the rod-like (or triangular) projection present at the base of the nucleus and is not a part of the axial filament of the tail. Franzen (1955) has not described any spirals in the sperm nucleus of any of his prosobranchs. Nor did he make out the ribbon like and spirally twisted nature of the mitochondrial sheath of the middle piece. We have, on the other hand, clearly observed the mitochondrial balls (6 to 8) in the spermatid gradually resolving into ribbons which are wound spirally around the axial-filament forming the core of the middle-piece. Though the middle-piece in the ripe-sperm exhibits an optically homogeneous sheath, Hanson et al. (1952) also state that the electron microscopy clearly reveals its ribbon-like characteristic. The single duplex spheroid, observed by us in all the stages of spermatogenesis, excepting the very early spermatogonia of P. globosa, is homologous to the 'Golgi element', 'Golgi bodies' or 'Golgi dictyosomes' of the early workers on gastropod sperm (see Roque 1954; and Nath 1957 for references). In its morphology it resembles one of the duplex spheres described by Roque (1954) and Nath and Gupta (1956) in pulmonates; and by Sharma and Gupta (1957) in ticks. In the spermatid it deposits a granular acrosome in the same way as recorded much earlier by Gatenby (1919) in Paludina (Viviparus) .A very characteristic feature of the single duplex spheroid in P. globosa is its remarkable persistence in the dividing cells. In such cells it has also been seen to divide into two in a very characteristic way with the result that each of the daughter cells, thus formed, receives a part of it. It is 436 G. P. Sharma, B. L. Gupta, and O. P. Mittal Cytologia 24 never obliterated from view. It neither gives any indication of a change in its appearance, nor does it fragment into the so-called dictyosomal granules as described in many insects (Moriber 1956). This behaviour is in direct contrast with that of the so-called Golgi dictyosomes in the pulmonates where they are never visible in the dividing cells (Roque 1954, Nath 1957). A similar behaviour of the single duplex sphere has also been observed in the male germ-cells of the earth worm, Pheretima posthuma by Nath, Gupta and Dewan (unpublished). The oligopyrene sperm in P. globosa differs considerably from that of V. vivipara as described by Hanson et al. (1952). It is shorter than the eupyrene sperm and seems to have a cylindrical nucleus with a cap-like acrosome as compared to a very small thimble-shaped head in V. vivipara. The middle-piece of an oligopyrene sperm in P. globosa appears to be very small and is devoid of the mitochondria, while in V. vivipara it is very long and contains the mitochondrial granules, giving the whole sperm a worm-like appearance. Further details of the oligopyrene sperm in P. globosa are being worked out histochemically.

Summary 1. The spermatogenesis of the Indian apple-snail, Pila globosa Swainson has been described in this paper. 2. The spermatozoa are of two types, viz., the uniflagellate eupyrene and the multiflagellate oligopyrene. Only the eupyrene sperm has been described in detail. 3. Late spermatogonia and the spermatocytes reveal filamentous and granular mitochondria and a single duplex sphere. 4. The single duplex sphere divides into two by binary fission both during mitosis and meiosis. 5. In the spermatid the single duplex sphere secretes a small granular acrosome which later becomes conical. The duplex sphere is sloughed off. 6. The mitochondria of the spermatid fuse to form 6 to 8 balls. These later on elongate and form ribbon-like structures which give rise ultimately to the mitochondrial sheath of the middle-piece. 7. The head of the eupyrene sperm is also spirally twisted. 8. The oligopyrene sperm in this prosobranch is stumpy and short bearing 4 to 8 flagella. The cylindrical nucleus bears a small cap-like acro some. The small middle-piece seems to be devoid of the mitochondria.

Literature cited Alexenko, B. 1926. Plasmatische Bildungen bei der Spermatogenese der Paludina vivipara. Z. Zellforsch. 4: 413 (As quoted by Hanson et al. 1952). Ankel, W. E. 1924. Der Spermatozoendimorphismus bei Bythinia tentaculata L. und Viviparus vivipara L. Z. Zellenbhre 1: 85-166. 1959 Spermatogenesis of the Gastropod , Pila globosa 437

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