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Notice: ©1981 John Wiley and Sons Inc. This manuscript is an author version with the final publication available and may be cited as: Eckelbarger, K. J., & Eyster, L. S. (1981). An ultrastructural study of spermatogenesis in the nudibranch mollusk Spurilla neapolitana. Journal of Morphology, 170 (3), 283-299. doi: 10.1002/jmor.1051700303

JOURNAL OF MORPHOLOGY 170:283-299 (19811

An Ultrastructural Study of Spermatogenesis in the Nudibranch Mollusc Spurilla neapolitana

KEVIN J. ECKELBARGER AND LINDA S. EYSTER Harbor Branch Foundation. Inc., Fort Pierce, Florida 33450 (KJ.E.), and Marine Science Institute, Northeastern University, Nahant, Massachusetts 01908 (L.S.E.)

ABSTRACT The ultrastructural features of spermatogenesis were investiga­ ted in the nudibranch mollusc Spurilla neapolitana. Sperm develop in the proximal half of numerous sac-like acini which are radially arranged within about ten ovo­ testis lobes. Accessory cells line the inner wall of the testicular portion of each acinus and are connected to developing sperm by numerous desmosomes. Stages of spermatid development have been divided into precup, cup, postcup, and elon­ gate stages depending on the general shape of the nucleus. Nuclear differentiation includes the formation of anterior and posterior nuclear plaques, condensation of chromatin fibrils into nuclear lamellae, the insertion of the developing flagellar ax­ oneme into a shallow, nuclear implantation fossa, and eventual formation of an elongated sperm head with a terminal twist. Spermiogenesis also includes the dif­ ferentiation of an anterior, perinuclear structure having the characteristics of an acrosome, the appearance of peculiar arrays of ER cisternae, and the fusion of mi­ tochondria into a large mitochondrial derivative which eventually encircles the ax­ oneme, forming a crystalline-like periaxonemal sheath.

Although studies on nudibranch molluscs Spurilla neapolitana (Fig. 1) is a widely dis­ have provided information on the ultrastruc­ tributed nudibranch that feeds on anemones ture of the reproductive system (Schmekel, '71) and is found on both eastern and western and the mature spermatozoa (Thompson, '66, shores of the Atlantic Ocean (Marcus, '57). '73; Holman, '72), only light microscopic re­ Like other nudibranchs, S. neapolitana is a ports are available on nudibranch sperm differ­ simultaneous hermaphrodite and undergoes entiation (Beeman, '77) and structural features internal fertilization after exchanging sperm of the ovotestis (Thompson, '61), Franzen ('55) during reciprocal copulation. Comments on examined spermiogenesis in several nudi­ other aspects of the reproductive system and branch species using light microscopy but no reproduction of S. neapolitana have been pre­ one has yet reported on the ultrastructural fea­ sented by Marcus ('57), Schmekel ('71), and tures of this process. Only recently have as­ Eyster ('80). pects of spermatogenesis been described on a fine structural level for any opisthobranch mol­ MATERIALS AND METHODS lusc (Thomas, '75; Beeman, '77) and still not in Sexually mature Spurilla neapolitana were great detail. Since nudibranchs represent such collected subtidally on oyster shell rubble and a large and important invertebrate group, a on the under surfaces of large rocks at Sebas­ clearer understanding of gametogenesis would tian Inlet (Brevard Co.) and at Little Jim Is­ be of value. The purpose of this paper, there­ land (St. Lucie Co.), Ft. Pierce, Florida, in Oc­ fore, is to describe spermatogenesis in the aeo­ tober 1978, June 1979, and October 1979. lid nudibranch Spurilla neapolitana (Delle Specimens were fixed the day after collection Chiaje, 1824) and to report developmental fea­ by dropping them whole and unrelaxed into tures of the sperm not previously recorded in cold (4°-5°C) primary fixative for a few min­ the molluscs or metazoan sperm in general. utes before dissecting out the individual lobes Oogenesis and the role of the accessory cells in of the ovotestis. The primary fixative was pre­ S. neapolitana will be considered in a forthcom­ pared just before use and contained 3% glutar­ ing paper. aldehyde, 1% formaldehyde made from para-

0362-2525/81/1703-0283$05.00 © 1981 ALAN R. LISS, INC. 284 K.J. ECKELBARGER AND L.S. EYSTER formaldehyde (Karnovsky, '65), 0.1 M phos­ Ovotestis structure phate buffer (pH 7.4), 3% NaCI, and 4.5% su­ Each ovotestis lobe is composed of a large crose. Dimethylsulfoxide (0.2%) was added to number of radially arranged, elongate, sac-like aid penetration. acini or follicles (Fig. 2). Each acinus contains Ovotestes were fixed whole or cut into sev­ both developing eggs and sperm. The ovarian eral small pieces, depending on their size. Tis­ portion is located in the distal half of the acinus sue was fixed for 2 hours in cold (4°-5°C) pri­ while the testicular portion lies in the proximal mary fixative and then rinsed for 1 hour in 3 half. The wall of each acinus consists of an in­ changes of cold 0.1 M Millonig's phosphate ner, thin (0.3 j.tm), fibrillar basal lamina con­ buffer containing 8% sucrose. The tissue was taining a few collagen fibers and an ou~er d!s­ then post-fixed for 1 hour at room temperature continuous layer of squamous myoepithelial using 1% OsO. in 0.1 M Millonig's phosphate cells. In the testicular portion, a third layer of buffer, and dehydrated for 2 hours in increas­ accessory cells lies subj acent to the basallami­ ing concentrations of cold ethanol. The final na (Fig. 3). Large follicle cells surrounding the dehydrations with 100% ethanol and two ex­ ovary, and the accessory cells which line the changes with propylene oxide (3 min. each) testes, form a two-layered somatic cell barrier were done at room temperature, and followed which separates the two regions of the acinus. by embedding in Epon after Luft ('61). The accessory cells contain large, irregularly Using light microscopy for orientation, 0,5­ shaped nuclei with prominent nucleoli, abun­ 1.0 j.tm thick Epon sections were cut with glass dant polymorphic electron dense granules mor­ knives and stained with Richardson's stain phologically resembling lysosomes (Fig. 4), (Richardson, et al., '60). For transmission elec­ spherical and bacilliform mitochondria, granu­ tron microscopy, thin sections were cut on a lar endoplasmic reticulum, and clusters of Sorvall MT-2B ultramicrotome with a dia­ a-and (3-glycogen granules (Figs. 4, 5). The mond knife, stained 30-60 min. each with satu­ lysosomal-like bodies are often closely rated aqueous uranyl acetate and lead citrate associated with numerous Golgi complexes and examined with a Zeiss EM9S-2 transmis­ (Fig. 5). Accessory cells often have endocytotic sion electron microscope. vesicles along the plasmalemma and For scanning electron microscopy, sperm heterophagic vacuoles in the cytoplasm. Adja­ were obtained by separating a copulating pair cent accessory cells are attached at their of nudibranchs and pipetting up the opaque lateral margins by junctional complexes con­ white fluid discharged from the penis. This sisting of zonulae adhaerentes and septate sperm-containing fluid was fixed during gent.le desmosomes (Fig. 6). centrifugation in cold 2.5% glutaraldehyde m Late stage spermatids are frequently ob­ seawater for a total of 1 hour. The sperm were served in close association with the accessory then spun down into a pellet, rinsed in 10% eth­ cells (Fig. 7). In most instances, the tip of each anol (15 min.) and 30% ethanol (35 min.) and developing spermatid is embedded in the held in 70% ethanol overnight. The sperm pel­ accessory cell cytoplasm (Fig. 8), and a number let was then pipetted onto a carrying stub and of small desmosomes are often observed be­ allowed to air dry. The stub was sputter-coated tween early and late stage spermatids and as­ with gold-palladium and examined with a Zeiss sociated accessory cells (Figs. 9, 10). Although Novascan scanning electron microscope. the accessory cells line the testis, they often ex­ RESULTS tend into the lumen and may have filiform pro­ jections. The ovotestis of Spurilla neapolitana is com­ posed of about ten rounded lobes which ?C~ur alternately to the right and left of the midline Fig. 1. Adult Spurilla neapolitana. X 4. in the posterior two thirds of the body. The lobes become swollen and closely opposed as Fig. 2. Transverse I I'm-thick Epon section through an the gametes develop and mature. Morphologi­ ovotestis lobe showing separate acini (between arrowsI com­ cally mature eggs and sperm exit each lobe posed of a distal ovary (0) and proximal testis ('1'). X 100. through a small hermaphroditic duct which Fig. 3. Section through the wall of the testicular portion joins the main hermaphroditic duct. Upon of an acinus showing the outer layer of myoepithelial cells leaving the ovotestis, and prior to copulation, (My), inner layer of accessory cells (Ac), and an intermediate the sperm are stored in the ampulla, a portion basal lamina (arrow). X 19,000. of the hermaphroditic duct that functions as a Fig. 4. Nucleus (N) and large nucleolus (Nu) of an acces­ seminal vesicle. sory cell. X 10,665.

286 K.J. ECKELBARGER AND 1,.S. EYSTER

nuclear surface of the envelope. The nuclear en­ Spermatogonia and spermatocytes velope is noticeably dilated where the nuclear No distinct germinal epithelium and no mito­ plaques are absent. The nucleus is eccentrical­ tic figures have been observed in the testicular ly positioned with its posterior edge nearest region. Spermatogonia were never positively the plasma membrane. identified and apparently are quite rare. Devel­ The cytoplasm contains numerous clusters oping sperm are somewhat randomly distribu­ of free ribosomes and abundant, dilated cister­ ted although, generally, primary and second­ nae of granular ER. Although several parallel, ary spermatocytes lie near the periphery and perinuclear cisternae may be present in the developing spermatids in the lumen. posterior cytoplasm, a single ER cisterna char­ Primary spermatocytes (Fig. 11) are spheri­ acteristically conforms to the contour of the cal to irregular in shape and range from about anterior and lateral margins of the nuclear en­ 9-12 Jim in diameter. Their nuclei range in size velope (Fig. 15). This consistent feature further from about 6-9 Jim and often possess a promi­ identified the anterior-posterior axis of the nu­ nent, eccentric nucleolus. Synaptonemal com­ cleus at this and later stages. plexes (chromosome cores) representing the zy­ The mitochondria have increased in size and gotene or pachytene stages of meiosis are oc­ have become more irregular in shape. The cris­ casionally observed. The cytoplasm contains tae have elongated and become more electron abundant free ribosomes, scattered small mito­ dense than the surrounding matrix (Fig. 16). A chondria, and a few tubular cisternae of granu­ number of cristae may lie parallel to each other lar ER. and dense intramitochondrial granules appear. Secondary spermatocytes are smaller (5-7 The mitochondria typically aggregate on the Jim) and less commonly encountered than pri­ anterior side of the nucleus at this stage (Figs. mary spermatocytes. Their nuclei are approxi­ 14,17). mately 4-6 Jim in diameter and contain scatter­ The nucleus changes from a spherical to an ed clumps of chromatin and one or two large, ir­ ellipsoidal shape prior to the cup stage with a regularly shaped nucleoli (Fig. 12). From one to noticeable flattening of its anterior face (Fig. several clumps of mottled fibrogranular mate­ 17). Although the event· is rarely observed in rial are also characteristic at this stage (Fig. sections, the axoneme of the future tail forms 12, insert). Secondary spermatocytes are con­ from a centriole near the posterior margin of nected by intracellular bridges resulting from the cell during this stage. incomplete cytokinesis (Fig. 13) and usually contain several perinuclear Golgi complexes Cup stage closely associated with one or two centrioles The nucleus flattens along the anterior-pos­ (Fig. 13). terior axis and forms a shallow, hollow cup on Spermatids For convenience, spermiogenesis has been Fig. 5. Accessory cell containing lysosome-like bodies (arrowsI and associated Golgi complexes (G) and rougb divided arbitrarily into four stages based on endoplasmic reticulum (ER). X 22.560. the general shape of the nucleus: the precup stage, cup stage, postcup stage, and elongate Fig. 6. Junctional complex between two accessory cells. stage. Cytoplasmic bridges are infrequently X 46.750. encountered in spermatids but they have been Fig. 7. Spermatids (SI in close association with an acces­ observed as late as the elongate nucleus stage. sory cell (Ac]. X 5.280.

Precup stage Fig. 8. Spermatid nucleus (SIembedded in accessory cell. The nucleus at this stage is subspherical and G. Golgi complex. X 26.600. contains a scattered flocullum and clusters of Figs. 9 and 10. Desmosomes (arrows] connecting early perichromatin material (Fig. 14). The anterior­ (Fig. 9) and late (Fig. 101 spermatids (S) to adjacent acces­ posterior axis of the cell is established early sory cells (Acl, X 52,400 (Fig. 91. X 47.800 (Fig. 10). with the appearance of two plaques of electron­ Fig. II. Primary spermatocyte with large nucleus (NI opaque material on opposite sides of the nucle­ containing a nucleolus (Nul and several synaptonemal com­ us. The anterior plaque forms first, is thicker plexes (arrows). X 7.900. (60-70 nm), and is deposited on both the nu­ Fig. 12. Secondary spermatocyte containing nucleus (N). clear and cytoplasmic sides of the nuclear en­ nucleolus (Nu), and clumps of fibrogranular material in cyto­ velope (Fig. 15). The posterior plaque is about plasm (arrow). X 7.000. Insert: higher magnification of fi­ 30-40 nm thick and is deposited solely on the brogranular material. X 23.750.

SPERMATOGENESIS IN AN AEOLID NUDIBRANCH 289 the anterior side (Figs. 18,19). The nucleus is small gap (30 nm) remains between the cap and about 5 /Lm wide at this stage and still posses­ the nuclear envelope. The small electron-dense ses the anterior and posterior plaques plaque, which was described earlier near the although they have lengthened. The nuclear mid-anterior surface of the nucleus (Fig. 21), is chromatin begins to condense and form pat­ now dome-shaped (Figs. 23,24). Since this ches along the posterior and lateral surfaces of structure persists and further differentiates the inner nuclear envelope (Fig. 19). An oblique during later stages of spermiogenesis, we have section through the anterior rim of the nucleus termed it the "putative" acrosome to reflect our reveals a banded pattern to these patches re­ uncertainty as to its origin and fate. sulting from the absence of chromatin at Spermatid mitochondria undergo extensive points where the nuclear pores occur (Fig. 20). differentiation during spermiogenesis and The patch of mottled, fibrogranular material playa major role in the morphogenesis of the which appeared in the perinuclear cytoplasm spermatozoan. Following insertion of the new­ in the precup stage is still present, in addition ly formed tail into the nuclear implantation to a single, prominent Golgi complex whose fossa, mitochondria begin to fuse and become maturing face closely opposes the posterior closely associated with the axoneme (Figs. surface of the nucleus (Fig. 19). The Golgi com­ 23, 25), eventually forming a periaxonemal plex appears to produce a variety of secretions sheath (Fig. 26). The mitochondrial cristae fol­ including from one to several multivesicular low the inner contour of the outer mitochon­ bodies (Fig. 19, insert) and membrane-limited, drial membrane and they remain more elec­ electron-dense rods and ring-like structures. tron-dense than the surrounding flocullant ma­ The large mitochondria are unchanged since trix. At various points, one observes swollen the previous stage but some are now observed regions of the cristae containing a homogene­ for the first time on the posterior side of the nu­ ous, electron-dense material (Fig. 27). As mito­ cleus. In addition, a small (0.2 by 0.3 /Lm) but chondrial fusion continues, one also commonly distinctive electron-dense plaque appears near encounters a large polymorphic mitochondrial the mid-anterior face of the nucleus in close as­ derivative with stacks of parallel or highly con­ sociation with the perinuclear ER cisterna voluted cristae (Fig. 28) resulting from the fu­ (Fig. 21). sion of smaller mitochondria. The mottled, fi­ The developing flagellum with its dense cen­ brogranular material observed in the cyto­ triolar cap inserts into a small implantation plasm of earlier spermatids, differentiates into fossa in the center of the posterior surface of a compact mass of fine, fibrillar material which the nucleus. The axoneme initially lies oblique­ is usually closely associated with (Fig. 29), or ly to the surface of the nucleus (Fig. 22) but is in many instances surrounded by (Fig. 30), this later oriented perpendicular to it, along the an­ mitochondrial derivative. terior-posterior axis of the cell (Fig. 23). A The cytoplasm of all spermatids at this stage characteristically contains symmetrical arrays of parallel, undulating cisternae exhibiting a variety of different profiles, depending on the Fig. 13. Secondary spermatocyte containing nucleus INI, Golgi complexes (G), and closely associated centriole (CI. plane of section (Fig. 3ll. These peculiar arrays Note the intracellular bridges (arrows) between adjacent are often associated with the Golgi complex of spermatocytes. X 17,500. the spermatid. Closer examination reveals that they are paired cytomembranes continuous Fig. 14. Spermatid in precup stage with nuclear plaques on the anterior Idouble arrowsI and posterior side (single ar­ with the walls of parallel ER cisternae. row) of nucleus. M, mitochondria. X 15,800. Postcup stage Fig. 15. Higher magnification of anterior nuclear enve­ The nucleus loses its cup shape and becomes lope INEI showing dense plaques deposited on both sides of membrane and closely associated cisterna of smooth endo­ ellipsoid in form (Fig. 32) with a slight concav­ plasmic reticulum (ERI. X 51,000. ity on its posterior surface. In the lateral and posterior regions of the nucleus the chromatin Fig. 16. Spermatid mitochondrion with electron dense condenses into fibers about 20-30 nm in diam­ cristae. X 25,500. eter while the dome-shaped putative acrosome Fig. 17. Flattened anterior surface of nucleus Idouble ar­ enlarges and develops an electron-dense core row] in precup stage spermatid. Single arrow indicates pos­ (Fig. 33). The chromatin fibers become more terior surface of nucleus. X 16,000. numerous and because of their anterior-pos­ Fig. 18. Four spermatids in the cup stage of develop­ terior orientation form nuclear lamellae (Figs. ment. X 4,300. 34,35). A cross section through the nucleus at

SPERMATOGENESIS IN AN AEOLID NUDIBRANCH 291 this stage reveals the somewhat radial sym­ oneme remains inserted into the shallow im­ metry of the lamellae as well as their apparent plantation fossa (Fig. 39) but the small gap be­ attachment to the inner surface of the nuclear tween it and the nuclear envelope is now ab­ envelope between the nuclear pores (Fig. 35). sent. A subnuclear vesicle, formed by anterior The putative acrosome continues to differenti­ extensions of the mitochondrial derivative ate into a more complex structure which now composing the periaxonemal sheath, sur­ contains a membrane-bound, centrally placed, rounds the anterior portion of the axoneme oval-shaped, dense body (Fig. 36) adjacent to a (Figs. 39, 42). Scattered electron-dense de­ basal and lateral complex of fibrous, extra­ posits are also evident on the inner walls of this vesicular material. The cytoplasmic mass of fi­ structure (Fig. 42). brogranular material has disappeared by this Two sheaths are formed by the periaxonemal stage and is no longer encountered. mitochondria: a primary sheath around the ax­ A centriolar adjunct resembling two equilat­ oneme and a secondary sheath enclosing the eral triangles in longitudinal section (Fig. 34), axoneme and a helical keel (Fig. 43). The keel is appears at the junction of the axoneme and its a continuous, uncompartmentalized structure dense, terminal centriolar cap. A cross section arising at the base of the nucleus and encirc­ through the axoneme just below this level re­ ling the axoneme in a clockwise direction when veals the typical 9 + 2 arrangement of the mi­ viewed anteriorly. The keel formed by the crotubules and peripheral, coarse accessory fi­ mitochondrial sheath is surrounded by a row of bers (Fig. 37). Spermatids at this stage are still microtubules and contains electron-dense joined by intercellular bridges and are closely granules with the ultrastructural characteris­ associated with accessory cells (Fig. 38). tics of glycogen particles (Figs. 43, 44). In fa­ vorable sections at this stage, one observes Elongate stages that the mitochondrial sheath now has a para­ The nucleus reaches about 4.2 /lm in length crystalline appearance due to the presence of at this stage and the nuclear lamellae are more three rows of parallel, closely packed tubules. tightly packed (Figs. 39, 40). Slightly oblique Figure 44 illustrates this feature in both sections give a misleading impression that the longitudinal and cross section. Although the chromatin fibers have condensed into a homo­ mitochondrial sheath appears to continue the geneous mass of chromatin (Fig. 41). A man­ entire length of the axoneme, the keel does not. chette composed of evenly spaced microtu­ A transverse section near the tip of the bules appears for the first time and surrounds flagellum shows only the axoneme, periax­ the nucleus. Itis most easily observed in trans­ onemal sheath and ring of microtubules (Fig. verse section (Fig. 41). Nuclear pores are still 45). prominent (Fig. 40). The terminal cap of the ax- Despite extensive ultrastructural observa­ tions of thin sections of a large number of ovo­ testes from a variety of specimens, we never observed spermatozoa in a more advanced Fig. 19. Spermatid in cup stage showing large Golgi state of maturation than that just described. complex IG)and associated multivesicular body IMv) on pos­ terior side of nucleus. FG. fibrogranular material. X 20.000. The most advanced spermatid nuclei were ob­ Insert. multivesicular body. X 32.570. served in the nuclear lamellae stage and never in a homogeneous stage of complete chromatin Fig. 20. Nuclear envelope on the anterior side of the nu­ condensation. cleus showing chromatin fibrils attached to the inner enve­ lope between the nuclear pores (arrows). X 29.150. A small number of sperm collected with some difficulty from the seminal fluid of two Fig. 21. Electron-dense plaque (arrow) adjacent to the copulating Spurilla neapolitana were exam­ anterior. cytoplasmic side of the nuclear envelope. Note the ined with the scanning electron microscope. closely associated cisterna of endoplasmic reticulum (ER). These sperm are presumed to be morphologi­ N. nucleus. X 51.000. cally mature. Unfortunately, we were unable to Fig. 22. Developing flagellar axoneme (A) and terminal examine them with transmission electron mi­ centriolar cup inserted into implantation fossa of spermatid crosopy. Figure 46 shows the anterior portion nucleus (N). X 26.900. of one sperm illustrating the helical keel form­ Fig. 23. Mitochondrion 1M)associated with axoneme of ed by the mitochondrial sheath. Figure 47 spermatid. Arrow indicates putative acrosome near outer shows a close-up of the head region of the surface of nucleus. N. nucleus. X 21.700. sperm illustrating the distinctive terminal twist. This was a constant feature observed in Fig. 24. Higher magnification of putative acrosome shown in Figure 23. ER. endoplasmic reticulum; NE. nu­ all fixed and live sperm examined and is pre­ clear envelope. X 71.550. sumed, therefore, not to be an artifact.

SPERMATOGENESIS IN AN AEOLID NUDIBRANCH 293

DISCUSSION Thomas ('75) described "supporting cells" oc­ curring alternately between the spermato­ Ovotestis structure gonia along the basal lamina but he did not re­ This paper reports the first ultrastructural port the association of later sperm cell stages details of a nudibranch ovotestis. In species with somatic cells, with a lobate ovotestis, each lobe is composed In Spurilla neapolitana, maturing sperrna­ of numerous acini consisting of individual tids are associated with cells lining the testis pockets enclosed by a basal lamina. Although region of the acinus. We have called these cells Huebner and Anderson ('76l clearly indicated "accessory cells," due to our uncertainty of that the eggs and sperm develop in adjacent their precise function. However, the presence but separate acini in Cratena pilata, in most of lipid droplets, 0'- and {3-glycogen, granular species, including Spurilla neapolitana, several ER, Golgi complexes, lysosome-like bodies, female acini are said to surround and communi­ heterophagic vacuoles, and endocytotic vesi­ cate with one or more male acini (Marcus, '57; cles suggests that these cells may serve both a Hyman, '67; Miller, '77). However, the present nutritive and a phagocytic function. If the ac­ ultrastructural study of S. neapolitana reveals cessory cells have a phagocytic function, it pre­ this to be incorrect. Each acinus in this species sumably involves the digestion of exogenous contains both eggs and sperm but the develop­ cellular components released into the testis lu­ ing germ cells are isolated from each other by a men during sperm maturation, waIl of follicle and accessory cells within each The function of the numerous desmosomes acinus. This observation leads us to suggest observed between the accessory cells and sper­ that interpretation of gross ovotestis morphol­ matids is problematical. Desmosomes such as ogy in other species warrants reexamination zonulae adhaerentes clearly have been shown using electron microscopy. not to be involved as permeability sites for ionic coupling between cells (Gilula, '74). Sea­ Accessory cells lariform junctions such as those reported be­ Nudibranch spermatids have been observed tween stored sperm and spermatothecal cells in association with "nutritive cells" (Chambers, of the polychaete Spirorbis (Daly and Golding, '34), a central mass of "plasm" (Franzen, '55), '77) have been more strongly implicated in in­ and with "nurse cells" (DeVries, '63). Cells pre­ tercellular transport. The junctional com­ sumed to be similar have been reported in plexes of Spurilla neapolitana appear to func­ aplysiid ovotestes and have been referred to as tion more as simple anchoring sites during de­ "nurse cells" (Thompson and Bebbington, '69) velopment. However, this does not preclude and as "phagocytic cells" (Beeman, '70). the possibility that sperm are receiving nutri­ ents from the accessory cells via tissue fluid within the lumen of the testes. A similar meth­ od of nutrient transport has been suggested Fig. 25. Cross section through axoneme of spermatid (ar­ between developing sperm and closely associa­ row] showing surrounding mitochondrion. X 23.000. ted pseudopods of nutritive cells in the pond Fig. 26. Longitudinal section through axoneme (AI snail Cipangopaludina (Yasuzumi et al., '60), showing formation of periaxonemal sheath by fusing mito­ Various membrane specializations, includ­ chondria. Arrows indicate connections between peri axone­ ing zonulae adhaerentes, gap junctions, and mal sheath and mitochondria in cytoplasm. X 18,500. septate junctions, have been found between Fig. 27. Electron dense material in swollen regions of mi­ the follicular epithelial cells of a number of mol­ tochondrial cristae (arrows) of fusing spermatid mitochon­ luscs (Anderson, '74), Hill ('77) determined dria. X 23,300. through the use of a variety of electron opaque Fig. 28. Large mitochondrial derivative 1M) resulting tracers that the intercellular junctions of the from fusion of smaller spermatid mitochondria. X 20,050. follicular epithelial cells of the pulmonate Agri­ olimax reticulatus formed a selectively perme­ Fig. 29. Fibrillar material 1*) in close association with able barrier, thereby regulating the transport mitochondrial derivative 1M). X 46,750. of ions and molecules into and out from the Fig. 30. Mitochondrial derivative 1M) partially ovotestes. The accessory cells in Spurilla nea­ surrounding fibrillar material 1*). X 2,800. politana may serve a similar function by con­ trolling or mediating the intratesticular micro­ Fig. 31. Parallel arrays of ER cisternae in close associa­ tion with a Golgi complex IG). Arrows indicate ER cisternae environment during gamete maturation. How­ which appear in the process of forming the peculiar loop-like ever, the present study did not address this structures. X 36,000. problem.

SPERMATOGENESIS IN AN AEOLID NUDIBRANCH 295

Spermatogenesis forms first and the material of the anterior plaque lies inside the nuclear envelope. In S. This paper represents the first ultrastruc­ neapolitana the anterior plaque forms first and tural description of spermatogenesis in a nudi­ is deposited on both sides of the nuclear enve­ branch mollusc. However, Franzen ('55), using lope. Material at the posterior plaque is de­ light microscopy to examine spermiogenesis in posited on the outer nuclear membrane in both the nudibranchs Tritonia homergi and Onchi­ species. Nuclear plaques are common in devel­ doris muricata, did describe early spermatid oping mollusc sperm but their function is un­ nuclei which were superficially similar to those known. of Spurilla neapolitana. The only ultrastruc­ Condensation of chromatin into nuclear lam­ tural studies on opisthobranch spermiogenesis ellae during spermiogenesis as reported in this are brief descriptions for the aplysiids Bursa­ study has been observed in both prosobran­ tella leachi plei (Thomas, '75) and Phyllaplysia chs (Rebhun, '57; Henley, '73; Buckland-Nicks taylori (Beeman, '77). Both Thomas and Bee­ and Chia, '76; West, '78) and pulmonates (Yasu­ man observed condensation of the chromatin zumi et al., '74; Takaichi, '78j, However, in into nuclear lamellae as in S. neapolitana but most molluscs nuclear condensation usually neither author reported a cup-shaped nucleus, continues, resulting in a highly condensed, clustering of mitochondria at the anterior side homogeneous chromatin (Rebhun, '57; Kaye, of the nucleus prior to axoneme implantation, '58; Buckland-Nicks and Chia, '76; West, '78). the peculiar undulating ER cisternae, or for­ The absence of such a condition in the most ad­ mation of a centriolar adjunct. vanced sperm examined in Spurilla leads us to Spermiogenesis in Spurilla neapolitana re­ believe that these were not morphologically sembles that of other nonopisthobranch mol­ mature cells. If this assumption is correct, it is luscs although there are several unusual fea­ possible that further differentiation occurs tures of the nucleus, mitochondria, and endo­ elsewhere in the reproductive system following plasmic reticulum. Some nuclear changes dur­ release from the ovotestes or in the seminal ing spermiogenesis in S. neapolitana closely re­ receptacle of the copulating partner. semble those in the sperm of the pulmonate The clustering of mitochondria on the ante­ gastropod Euhadra hickonis (Takaichi and rior side of the nucleus during early spermio­ Dan, '77; Takaichi, '78; Dan and Takaichi, '79). genesis in S. neapolitana and later occurrence In both species the axoneme has a dense cen­ of these organelles in the posterior region has triolar cap which initially implants in a nuclear not been reported previously in molluscs. In fossa at an angle oblique to the nucleus and the most molluscs studied, the mitochondria are nuclei develop anterior and posterior plaques. positioned early in the posterior cytoplasm of However, in E. hickonis the posterior plaque developing spermatids (Franzen, '55; Takaichi and Dan, '77; West, '78), The peculiar undulating ER cisternae of mid­ Fig. 32. Spermatid nucleus in postcup stage. Arrow in· dle to late spermatids in Spurilla neapolitana dicates putative acrosome. X 20,000. have been observed in photoreceptors of poly­ Fig. 33. Putative acrosome adjacent to nuclear envelope chaetes and a pulmonate mollusc, in oligo­ (NE) of spermatid. X 56,700. chaete testicular (somatic) cells, and in several vertebrate tissues (for reviews, see Eakin and Fig. 34. Spermatid nucleus showing putative acrosome Brandenburger, '75; Arsenault et al., '80), but (PAl, centriolar adjunct (arrows) associated with the termi­ nal centriolar cap of the axoneme, and the surrounding mito­ to the authors' knowledge not in metazoan sex chondrial derivative (MI. X 23,450. cells. These membrane arrays are transitory, however, being present only during the mid­ Fig. 35. Cross section through the spermatid nucleus at spermatid stage. They appear to originate the stage illustrated by Figure 34 showing the nuclear lamellae and nuclear pores (arrows). X 23,750. from the endoplasmic reticulum in S, neapoli­ tana but their function is unknown. Although Fig. 36. Higher magnification of putative acrosome a number of suggested functions have been ad­ shown in Figure 34. Arrow indicates membrane-bound, vanced (see reviews above), Bassot and Nicolas dense body. NE, nuclear envelope. X 90,200. ('78) have shown that the "paracrystals," to use Fig. 37. Cross section through the flagellar axoneme just their term, are the source of luminescence and below the level of the terminal centriolar cap. N, nucleus: M, fluorescence in the elytra of polynoid polychae­ mitochondrial derivative. X 42,750. tes. Fig. 38. Spermatids (SI closely associated with an acces­ The coarse accessory fibers seen in the pe­ sory cell (Ac). X 3,800. riphery of the distal axoneme of Spurilla nea-

SPERMATOGENESIS IN AN AEOLID NUDIBRANCH 297 politana have been reported from many phyla, iance with that reported for most sperm in that including the mollusca, and may have a con­ the Golgi complex appears not to be involved tractile function (Baccetti and Afzelius, '76). in its secretion. Despite extensive ultrastruc­ Among the gastropod molluscs, these struc­ tural observations of Golgi activity in the sper­ tures have been recorded in the pulmonates matids of S. neapolitana, we were unable to de­ (Anderson and Personna, '69; Dan and Takai­ tect the formation of proacrosomal granules. chi, '79) and prosobranchs (Giusti, '71). This is In addition, the Golgi complex and its copious the first report of these structures in an opis­ secretions consistently remained on the poste­ thobranch although their presence in this rior side of the nucleus, well away from the site taxon is not surprising, since they are reported of putative acrosome appearance. It should be to be typical of higher molluscs with internal noted, however, that Garreau de Loubresse fertilization (Baccetti and Afzelius, '76). ('71) reported that the acrosome of the pulmon­ Acrosomes have been reported in mature ate Nerita senegalensis was formed without nudibranch sperm based on both light micro­ the assistance of the Golgi complex. scopic (Franzen, '55) and electron microscopic The fibrous extravesicular component of the observations (Thompson, '66, '73; Holman, '72). putative acrosome and indeed the entire aero­ The putative acrosome described in the cup somal complex of Spurilla neapolitana is very and postcup nuclear stages are interpreted as similar to that described in late stage sperma­ successive stages in the differentiation of a tids of the pulmonate Euhadra hickonis by true acrosome although we were not successful Takaichi and Dan ('77). However, the latter in identifying such a structure in the latest authors clearly demonstrated that the vesicu­ spermatid stages examined. The structure we lar component of the mature acrosome, at have described fits the usual morphological least, was produced by the Golgi complex. Dan definition of an acrosome by being a terminal, and Sirakami ('71) presented evidence that perinuclear, membrane-bound structure. How­ similar material in starfish spermatids, which ever, if it is an acrosome, its formation is at var- was later identified as actin precursor by Til­ ney and co-workers ('73), is synthesized in the cytoplasm in association with a system of smooth ER vesicles lying close to the face of Fig. 39. Longitudinal section through the nuclear region the acrosomal vesicle. A second extravesicular of a spermatid in the elongate stage. N, nucleus: SV, subnu­ component, making up the fibrous base of the clear vesicle: M, mitochondrial derivative: arrows indicate centriolar adjunct. X 23,750. acrosomal process, was regarded as a product of polyribosomes. Takaichi and Dan ('77) add Fig. 40. Cross section through the nucleus showing nu­ that the fibrous component composing a por­ clear lamellae. X 21,000. tion of the acrosomal complex in Euhadra Fig. 41. Oblique section through base of nucleus (NI and spermatids must be produced by some orga­ centrally placed centriolar cap of axoneme. Arrow indicates nelle other than the Golgi complex because of microtubular manchette surrounding nucleus. X 32,900. the absence of a limiting membrane. They fur­ ther presented evidence to suggest that the cy­ Fig. 42. Cross section through subnuclear vesicle show­ ing dense deposits on the inner surface (arrowsI and the cen­ toplasmic portion of the nuclear plaque should trally placed axoneme. X 32,300. be regarded as a constituent of the acrosome. The origin of the extravesicular portion of the Fig. 43. Cross section through the central region of the putative aerosomal complex in Spurilla neapol­ tail showing the periaxonemal sheath surrounding the ax­ oneme and the keel containing glycogen-like granules (ar­ itana remains unknown based on the limited row). X 56,000. evidence gathered in this study. The centriolar adjunct described in the pres­ Fig. 44. Arrow heads indicate both longitudinal and ent investigation has not been reported previ­ cross section views of the keel portion of the tail showing tu­ bules which produce the paracrystalline appearance of the ously in molluscs. However, Yasuzumi and his periaxonemal sheath. Small arrow indicates glycogen-like co-workers ('74) clearly illustrate such a struc­ granules in keel. X 53,850. ture in their paper on sperm ultrastructure in the pulmonate Limax flavus but refer to it as Fig. 45. Cross section near the tip of the sperm flagellum showing the absence of the keel. X 73,100. the "chromatoid body." Takahashi et al. ('77) published similar micrographs of a centriolar Fig. 46. SEM view of anterior region of sperm illustrat­ adjunct in the same species but make no men­ ing the helical keel formed by the mitochondrial sheath (ar­ tion of it in their description of spermiogenesis. rows). X 7,100. The centriolar adjunct has been widely Fig. 47. SEM view of head region of sperm illustrating reported in insects and mammals (see Baccetti distinctive terminal twist (arrow). X 10,450. and Afzelius, '76, for review). 298 K.J. ECKELBARGER AND L.S. EYSTER

Among the gastropods, thepulmonatesprob­ (Eddy, '75), and the "chrornatoid body" of inver­ ably represent the climax in mitochondrial re­ tebrate male germ cells (see Reger, et aI., '77, modeling (Favard and Andre, '70). The com­ for review). The chromatoid body is most often plex process of metamorphosis observed in the described in association with mitochondria or mitochondria of Spurilla neapolitana is typical the Golgi complex (see Reger et al., '77, for re­ of most gastropods and has been described in view). Its nature, origin, and possible function detail by Kaye ('58) and Andre ('62). In the pres­ is conjectural although Yasuzumi and col­ ent study, numerous small mitochondria fuse leagues ('70) suggested it contributed to forma­ to form a giant mitochondrial derivative which tion of the centriolar adjunct in the grasshop­ in many spermatids resembles the "nebenkern" per Acrida. In addition, Reger et al. ('77) de­ reported during spermiogenesis in euthyneu­ scribed the formation of a subacrosomal rod­ ran gastropods by Walker and MacGregor like structure from the chromatoid body in the ('68), Yasuzumi ('62), and Giusti ('71). The ne­ spermatozoa of two crustaceans. No direct benkern is a common feature of spermiogene­ function can be attributed to this structure in sis in many insect species, but is apparently Spurilla neapolitana. unusual among marine invertebrates. The gi­ ant mitochondrial derivative of S. neapolitana ACKNOWLEDGMENTS differs from a classical nebenkern by having a The authors wish to thank Paul Mikkelsen different position in the spermatid (posterior for assistance with field work, Ms. P.A. Linley to the nucleus rather than lateral), by being for assistance with tissue preparation for TEM highly branched rather than "onion shaped" and SEM, and Dr. Robert Burke and Ms. Lin­ and lacking the multilayered or concentric­ ley for making suggestions on the manuscript. shelled form generally reported from other spe­ Contribution No. 233, Harbor Branch Founda­ cies (Baccetti and Afzelius, '76). tion. The complex paracrystalline sheath derived from the mitochondria is characteristic of both LITERATURE CITED pulmonates and opisthobranchs (Anderson Anderson, E. (19741 Comparative aspects of the ultrastruc and Personne, '76) although crystalline mito­ ture of the female gamete. In G.H. Bourne, J.F. Danielli, chondrial derivatives also occur in other taxa and K.W. Jeans Ieds]: International Review of Cytology, (Andre, '62; Favard and Andre, '70). The para­ Supp!. 4: Aspects of Nuclear Structure and Function. New crystalline sheath of gastropods is made of a York: Academic Press. Anderson, W.A., and P. Per sonne 11969) Structure and his­ proteinaceous crystal composed of globules or tochemistry of the basal body derivative, neck and axon­ hollow rodlets 90 A in diameter (Favard and eme of spermatozoa of Helix aspera. J. Microsc. 8:87~96. Andre, '70). Glycogen storage within one re­ Anderson, W.A .. and P. Per sonne 11976)The molluscan sper­ gion of the paracrystalline sheath (the keel) matozoan: Dynamic aspects of its structure and function. Am. Zoo!' 16: 293~313. seems to be a widespread characteristic of gas­ Andre, J. (1962) Contribution it la connaissance du chon­ tropod sperm (Anderson and Personne, '76). driome. Etude de ses modifications ultrastructurales pen­ We presume the granular material observed in dent la spermatogenese, J. Ultrastruct. Res. Supp!. 3, 185 the keel of Spurilla sperm is also glycogen al­ pp. Arsenault, A.L., RE. Clattenburg, and P.H. Odense (1980) though no tests were performed to confirm Further observations on spermiogenesis in the shrimp, this. The importance of glycogen storage is dis­ Crangon septemspinosa: A mechanism for cytoplasmic re­ cussed by Anderson and Personne ('76). duction. Can. J. Zoo!' 58:497~506. In the cup stage of spermiogenesis in Spurilla Baccetti, 8., and B.A. Afzelius (1976) The Biology of the Sperm Cell. Basel: S. Karger. neapolitana, prominent patches of fibrogranu­ Bassot, J.M., and M.T. Nicolas (1978) Similar paracrystals lar material, which later become intimately as­ of endoplasmic reticulum in the photoemitters and the sociated with the mitochondrial derivative, are photoreceptors of scale-worms. Experientia 34:726~728. Beeman, RD. (1970) An autoradiographic and phase con­ observed in the perinuclear region. To the au­ trast study of spermatogenesis in the anaspidean opistho­ thors' knowledge, no studies on molluscan branch Phylloplysia taylori Dall, 1900. Arch. Zoo!' Exp. spermatogenesis have commented on the pres­ Genet. 111(1):5~22. ence of such material in developing spermato­ Beeman, RD. 119771 Gastropoda: Opisthobranchia. In A.C. Giese and J .S. Pearse (edsl; Reproduction of Marine cytes. However, material of a similar nature Invertebrates, Vo!. 4, Molluscs: Gastropods and has been reported in the developing oocytes of Cephalopods. New York: Academic Press. a cephalopod, a neogastropod and two species Buckland-Nicks, J.A., and F.S. Chia 11976)Spermatogenesis of amphineurons (see Eddy, '75, for review). of a marine snail, Littorina sit kana. Cell Tissue Res. 170: 455~475. This fibrogranular material appears morpho­ Chambers, L.A. 119341 Studies on the organs of reproduc­ logically equivalent to the "nuage" so common­ tion in the nudibranchiata molluska. Bull. Am. Mus. Nat. ly reported in developing female germ cells Hist. 66(6):599~641. SPERMATOGENESIS IN AN AEOLID NUDIBRANCH 299

Daly, J.M., and D.W. Golding (1977) A description of the Plasm) during spermatogenesis and spermiogenesis in spermatheca of Spirorbis spirorbis (L.) IPolychaeta: Ser­ two Peracaridae. J. Ultrastruct. Res. 60:84~94. pulidae) and evidence for a novel mode of sperm transmis­ Rebhun, L.1. 11957) Nuclear changes during spermiogenesis sion. J. Mar. BioI. Assoc. U.K 57:219~227. in a pulmonate snail. J. Biophys. Biochem. Cytology 3: Dan, J.C., and A. Sirakami 11971) Studies on the acrosome. 509~527. X. Differentiation of the starfish acrosome. Dev. Growth Richardson, KC., L. Jarett, and E.H. Finke 119601 Embed­ Diff. 13:37 ~52. ding in epoxy resins for ultrathin sectioning in electron Dan, J.C., and S. Takaichi 119791 Spermiogenesis in the pul­ microscopy. Stain Technol. 35:313~323. monate snail, Euhadra hickonis. III. Flagellum forma­ Schmekel, L. (19711 Histologie und Feinstruktur der Geni­ tion. Dev. Growth Diff. 21111:71 ~86. talorgane von Nudibranchiern IGastropoda, Euthynerv­ DeVries, J 11963) Contribution to the morphology and his­ ia). Z. Morphol. Tiere. 69:115~183. tology of the nudibranch Melibe roseo. Rang. Ann. Univ. Takahashi, Y., Y. Nishimura, and N. Yamagishi 11977) Stellenbosch 381Ser. A.I:I05~153. Electron microscopic studies on spermiogenesis of Limax Eakin, RM., and J.L. Brandenburger 119751 Retinal differ­ [laous L. In KG. Adiyodi and R.G. Adiyodi leds): Ad­ ences between light-tolerant and light-avoiding slugs vances in Invertebrate Reproduction, Vol. I. Calicut, In­ (Mollusca: Pulmonatal, J. Ultrastruct. Res 53:382 ~394. dia: Mathrubhumi Press. Eddy, E .M. 119751 Germ plasm and the differentiation of the Takaichi, S. 11978) Spermiogenesis in the pulmonate snail, germ cell line. In G.H. Bourne and J.F. Danielli (eds): In­ Euhadra hickonis II. Structural changes of the nucleus. ternational Review of Cytology, Vol. 43. New York: Aca­ Dev. Growth Diff. 20(41:301~315. demic Press. Takaichi, S., and J.C. Dan 119771 Spermiogenesis in the pul­ Eyster, L.S. 11980) Distribution and reproduction of shell­ monate snail, Euhadra hickonis. I. Acrosome formation. less opisthobranchs from South Carolina. Bull. Mar. Sci. Dev. Growth Diff. 19: 1~ 14. II 30:580~599. Thomas, R.F. (19751 The reproductive system of Bursatella Favard, P., and J. Andre (1970) The mitochondria of sperma­ leachi plei (Opisthobranchia: Aplysiaceal with special tozoa. In B. Baccetti (ed): Comparative Spermatology. reference to its histology. Malacologia 15(11:113~121. New York: Academic Press. Thompson, T.E. 1196II The structure and mode of function­ Franzen, A. (1955) Comparative morphological investiga­ ing of the reproductive organs of Tritonia hombergi (Gas­ tions into the spermiogenesis among mollusca. Zool. Bid­ tropoda Opisthobranchia). Q. J. Microsc. Sci. 102:1 ~14. rag, Uppsala 30:399~456. Thompson, T.E. (19661Studies on the reproduction of Archi­ Garreau de Loubresse, N.119711 Sperrniogenese d'un gaster­ doris pseudoargus (Raon) (Gastropoda Opisthobranchia). opode prosobranchi: Nerita senegolensis; evolution du Phil. Trans. R. Soc. 250:343~375. canal intranucleaire. J. Microsc., Paris 12:425~440. Thompson, T.E. (1973) Euthyneuran and other molluscan Gilula, N.B. (1974) Junctions Between Cells. In Rody P. spermatozoa. Malacologia. 14:167~206. Cox (ed): Cell Communication. New York: John Wiley & Thompson, T.E., and A. Bebbington 11969) Structure and Sons. function of the reproductive organs of three species of Giusti, F. 119711 L'ultrastruttura della spermatozoa nella Aplysia IGastropoda: Opisthobranchial, Malacologia. filogenesi e nella sistematica dei molluschi gasteropodi. 7:347~380. Atti Soc. Ital. Sci. Nat. 112:381~402. Tilney, L.G., S. Hatano, H. Ishikawa, and M.S. Mooseker Henley, C. (1973) Chromatin condensation involving lamel­ 11973) The polymerization of actin: Its role in the genera­ lar strands in spermiogenesis of Goniobasis proxima. tion of the acrosomal process of certain echinoderm Chromosoma 42:163~174. sperm. J. Cell BioI. 59:109~126. Hill, R.S. 119771 Studies on the ovotestis of the slug Agrioli­ Walker, M., and H.C. MacGregor 11968) Spermatogenesis max reticulatus (Muller). I I. The epithelia. Cell Tissue Res. and the structure of the mature sperm in Nucella lapillus 183:131~141. L. J. Cell Sci. 3:95~104. Holman, J.A. 11972) The ultrastructure and activity of West, D.L. (1978) Reproductive biology of Colus stimpsoni sperm in Acanthodoris pi/osa, a nudibranchiate mollusk. IProsobranchia: Buccinidael, I I. Spermiogenesis. Veliger. Mar. BioI. 12:179~183. 21:1 ~9. Huebner, E., and E. Anderson 119761 Comparative spiralian Yasuzumi, G.119621 Spermatogenesis in animals as revealed oogenesis - Structural aspects: An overview. Am. Zool. 16: by electron microscopy. XI I. Light and electron micro­ 315~343. scope studies on spermiogenesis of Cipangopaludina mal­ Hyman, L.H. (19671The Invertebrates, Vol. VI., Mollusca I. leata Reeve. J. Ultrastruct. Res. 7:488~503. New York: McGraw-Hill. Yasuzumi, G., H. Tanaka, and O. Tezuka (1960) Spermato­ Karnovsky, M.J. 119651 A formaldehyde-glutaraldehyde fix­ genesis in animals as revealed by electron microscopy. ative of high osmolarity for use in electron microscopy. J VIII. Relation between the nutritive cells and the develop­ Cell BioI. 27:137 A. ing spermatids in a pond snail, Cipangopaludina malleata Kaye, J .S. 119581 Changes in the fine structure of mitochon­ Reeve. J. Biophys. Biochem. Cytology 7:499~519. dria during spermatogenesis. J. Morphol. 102:347~369. Yasuzumi, G., T. Sugioka, I. Tsubo, F. Yasuzumi, and Y. Luft, J.H. 11961) Improvements in epoxy resin embedding Matana (1970) Spermatogenesis in animals as revealed by methods. J. Biophys. Biochem. Cytology 9:409~414. electron microscopy. XX. Relationship between chroma­ Marcus, E. 119571 On opisthobranchia from Brazil. J. Linn. toid bodies and centriole adjunct in spermatids of grass­ Soc. Lond. 43:390~486. hopper, Acrida lata. Z. Zellforsch. 110:231~242. Miller, M.C. 119771 Aeolid nudibranchs (Gastropoda: Opis­ Yasuzumi, G., Y. Takahashi, Y. Nishimura, N. Yamagishi, thobranchial of the family tergipedidae from New Zealand H. Yamamoto, and K Yamamoto 11974)Spermatogenesis waters. J. Linn. Soc. Lond. 60:197~222. in animals as revealed by electron microscopy. XXVIII. Reger, J.F., M.A. Fain-Maurel, and P. Cassier 119771 The ori­ Development of flagella of spermatozoa of Limax flavus L. , gin, distribution, and fate of the chromatoid body (Germ Okajimas Fol. Anal. Jpn. 51:11~28.