FAU Institutional Repository This
Total Page：16
File Type：pdf, Size：1020Kb
FAU Institutional Repository http://purl.fcla.edu/fau/fauir This paper was submitted by the faculty of FAU’s Harbor Branch Oceanographic Institute. 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.