survey of oviducal gland

7/28/2019 Survey of Oviducal Gland

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THE J OURNAL OF EXPERIMENTAL ZOOLOGY 282:399420 (1998)

1998 WILEY-LISS, INC.

Survey of Oviducal Gland Structure and

Function in ElasmobranchsW.C . H AMLE TT,1,5* D.P. KNI G HT,2 T.J . KOOB ,3 M. J EZIOR,4,5 T. LU ONG ,2T. R OZYCK I, 1 N. BRUNETTE,1AND M.K. HYSE LL1,51Department of Anatomy, South Bend Center for Medical Education, Indiana

University School of Medicine, Notre Dame, Indiana 465562Department of Biosciences, King Alfreds University College, Winchester,

SO22 4NR, United Kingdom3Skeletal Biology, Shriners Hospital for Children, Tampa, Florida 336124Uniformed Services University of the Health Sciences, Bethesda,

Maryland 20814-47995Department of Biological Sciences, University of Notre Dame, Notre Dame,

Indiana 46556

ABSTRACT We report the results of a comparative survey of the structure and function ofoviducal glands (OG) of selected elasmobranch fish with differing modes of reproduction usinglight a nd sca nnin g electr on microscopy, e.g., Scyliorhinus canicula, S. stellaris, Raja erinacea, R.eglanteria, R. clavata, Squalus acanthias, Mustelus canis, a nd Urolophus jamaicensis. Oviduca lgland s consistently display four funda menta l zones regardless of the type of reproduction of thepart icular species. The zones correspond to lamellae that extend full width across the glandlumen. Formerly, zones of OG were designated as albumen secret ing and shell secret ing. Thisoversimplified terminology does not accurately apply to all species. We have adopted the termi-nology recently introduced (Hamlett et al. [1998] Cybium, in press) that refers to the four basiczones on a morphological basis rather than on a purported function that may not be applicableacross species lines. This allows comparisons to be accurately made between species. Oviducalglands have a proximal club zone, papillary zone, baffle zone, and terminal zone. Variat ions inthe ma keup of each zone may show species varia bility , but t he fundam enta l orga nizat ion is ma in-

tained. The club and papillary zones replace the former designat ion of the albumen zone. Theclub zone is so named because of its shape when viewed in transverse sect ion with the lightmicroscope. Similarly, the papillary zone is cha ra cterized by a papillary or conical profile whenviewed in section. The club and papillary zones are responsible for producing the various jellycoa ts t ha t surround t he egg. The ba ffle zone produces the va rious types of egg investments seenin elasmobra nchs. In ovipa rous species, such a s th e sha rks S. canicula a nd S. stellaris and t heskates R. erinacea, R. eglanteria, a nd R. clavata, tubula r glan ds produce secretory componentstha t pass t o secretory ducts. Secretory ducts a re confluent wit h a spinneret tha t ha s paired baffleplates that manipulate the secretory material as it emerges from the secretory duct . Secretorymaterial from adjacent secretory ducts blends in transverse grooves that extend across the fullwidth of the gland, thus one transverse groove is responsible for one secreted layer. The yellowspotted st ingra y, U. jamaicensis, is unusual in that it does not produce an egg invest iture othertha n jelly coat s a nd t herefore lacks baffle plates. Despite varia t ions among species, the capsule-producing OG we studied seems to use the same basic assembly process to produce tough, flex-

ible, an d selectively permea ble egg capsules, cand les, or egg envelopes. This involves the extr usionof a capsule mat erial th rough dies, each of which opens between tw o baffle plat es and extrudes aflattened ribbon containing precisely and complexly orientated molecules. The dies dischargeinto transverse grooves in the main lumen of the gland, each groove thus secret ing a singlelamella of the egg covering. J. Exp. Zool. 282:399420, 1998. 1998 Wiley-Liss, In c.

Gr ant sponsor: India na University S chool of Medicine.*Correspondence to: William C. Hamlett, South Bend Center for

Medical Educat ion, Indiana Univers i ty School of Medicine, B-10Ha ggar Ha ll, Notre D ame, IN 46556. E-ma il: [email protected]


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400 W.C. HAMLETT ET AL.

The terms shell, nidamental, a nd oviducal glandha ve historica lly been used interchangea bly to re-fer to the region of the elasmobran ch oviduct t ha tproduces jelly coats and various egg coveringsaround the ovulated and fertilized eggs. The re-gion of the oviduct t ha t produces a tough egg casedeposited to the exterior in oviparous species isaccurately termed the shellgland because thisterm denotes its function. A shell is defined as arigid, outer covering, hence the designation. Theterm nidamentalgland is properly only used torefer to the thin egg coverings of viviparous spe-cies. The term nidamental is derived from nidusL. for nest. In many placental species, each em-bryo is surrounded by its own egg covering, andthe embryo and its covering develop and reside inits own uterine compar tment , hence the nest. Nei-th er of the previous t erms can be correctly a pplied

to th e region of th e oviduct in some rays wh ere noegg covering is produced because n o shell or nidusis formed. To esta blish a consist ent term inology,we have adopted the convention of Hamlett et al.(98) a nd h a ve chosen not t o use the t erms sh ell ornidam enta l but to use only the term oviducal gland(OG) to refer t o any of the aforementioned glan dsbecause they are all derived from the oviduct. Wehave also adopted the terminology of the trans-verse zones of all OGs going from proximal to dis-tal : c lub zone, papil lary zone, baff le zone, andterminal zone (Hamlett et al., 98).

Borcea ( 06), Widakowich (07), Filhol and

Garrault (38), Nalini (40) and Prasad (45, 48)provided early descriptions of OGs from variouselasmobran chs. The ma jority of the published de-scriptions of the structure and function of elas-mobranch OGs have centered on the oviparousdogfish, Scyliorhinus canicula. Metten (39) de-scribed the structure of the glan d, and several a u-th ors considered its h istochemical char a cteristics(Threadgold, 57; Rusaoun, 76). Additionally,Krishnan (59) provided information on the his-tochemistry of the OG in Chiloscyllium griseus.The formation and nature of the components ofth e egg case of S. canicula has been well studied

(Hobson, 30; Kn ight a nd H unt , 76, 86; Rusa ounet a l., 76; Ru sa oun-In nocent, 85, 90; Hu nt , 85;Feng and Knight, 92, 94a,b; Knight et al. , 93,96; Kn ight a nd F eng, 92, 94a ,b; Hepworth et a l. ,94; Thomason et al., 94). The composition, poly-meriza tion chemistr y, a nd physicochemica l prop-erties of little skate, Raja erinacea, egg capsuleshave been analyzed (Koob and Cox, 88, 90, 93).P ra tt (93) ha s discussed sperm st orage in t he OGof some elasmobra nchs.

The OG may perform the following functions: (1)produ ction of egg jellies , (2) egg ca se/cand le/enve-lope formation, (3) transport of fertilized eggs, and(4) sperm recepta cle an d/or sperm st ora ge. Wepresent a preliminary comparative account of thestructure and function of the OG in selected elas-mobranch fish. Selachian OGs merit study for sev-eral reasons. They demonstrate a complex designof novel extrusion dies that produce and assemblematerials that have a range of potentially impor-tant biological properties, many of which are spe-cies specific, including extreme toughness andstrength, flexibility, moderate extensibility, hightra nspar ency in some species, and a high, selectivepermeability to low molecular weight substancesand ions (Knight et al ., 94, 96). They have alsobeen reported to have antifoulant and antibacte-rial properties (Thomason et al., 94, 96). The chemi-

cal and morphological complexity of the capsulematerials raises questions about how animals as-semble such structures showing remarkable re-giona l differentia tion an d a high degree of order a tup to six hierar chical levels of orga nizat ion (Knightet a l., 96). A comparative study of OGs and the en-casements they secrete provides an opportunity tostudy a wide range of functional modifications tothe basic design. It may also provide informationabout the function and evolution of the female re-productive system in vertebrates. We describe how,despite variations from species to species, the samebasic functional design seems to be present in all

selachian fish examined in th is study.Sha rks, skat es , an d ray s consti tut e the elasmo-

bra nch fish. These cart ilaginous fish possess a di-verse ar ray of reproduct ive s tra tegies rangingfrom oviparit y t o severa l ty pes of vivipa rity, bothaplacental and placental (Hamlett, 87, 89, 93;Hamlett et al. , 93a; Hamlett and Koob, 99). Allemploy internal fer ti l ization and have a typicalrepertoire of vertebrate hormones that mediatesexual cycles and gonadal maturation (Callard eta l., 93; C a llar d a nd Koob, 93). Ovipar ous speciessuch as th e clearnose skate, R. eglanteria, the littleskate, R. erinacea, and the dogfish, S. canicula,

release egg ca ses conta ining fertilized eggs into thesea where they will develop at the expense of yolksequestered in their yolk sac. Viviparous specieshave a var iety of methods for nourishing theiryoung. Some sha rks, such a s the a pla cent al spinydogfish, S. acanthias, are completely dependent fornutrition on the yolk sequestered by the motherin t he egg (J ollie an d J ollie, 67). In t he smoothdogfish,Mustelus canis, initial development relieson yolk sa c content s. After t his yolk-relian t pha se,


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OVIDUCAL GLAND IN ELASMOBRANCHS 401

th e yolk sac becomes modified int o a y olk sa c pla -centa that provides for nutr ient and respiratoryexc hange d u r ing the r em a ind er o f ges t a t ion(Hamlett et al. , 85a,b,c, 93a,b,c; Hamlett, 87,89). Other modes of viviparity include uterinevil l i , known as trophonemata, in s tingrays thatsecrete histotroph, also known as uterine milk,that the developing embryos ingest (Hamlett etal. , 85d, 96a,b) and the practice of oophagy bysevera l large sha rks such a s th e porbeagle shark,Lamna nasus. The mother continues to ovulateeggs through early pregnancy that the young in-g e s t . I n a d d i t i o n t o o o p h a g y , t h e s a n d t i g e r ,Carcharias taurus, practices intra uterine embry-onic cannibalism in which more developmentallyadvanced embryos feed on more immature sib-lings (G ilmore, 93;Ha mlett et a l . , 93a).

MATERIALS AND METHODS

Female clearnose skates, R. eglanteria, spinydogfish, S. acanthias, and smooth dogfish, M.canis, were obtained via long line from shallowwaters in the Chesapeake Bay . Female yel lowspotted stingrays , Urolophus jamaicensis, wereobta ined via ha nd nets in sha llow wa ters off LongKey, FL. Raja erinacea w ere collected fr om Fren ch-man Ba y, a t The Mount Desert Is land B iologicalLaboratory, Maine. Females were anesthetizedwith MS 222, humanely sacrif iced by cervicaltra nsection an d pithing, an d opened by a longitu-dinal ventral incis ion, and the OG was isolated.

For light microscopy, glands were fixed in 10%neutral buffered formalin. Samples were dehy-drated through a graded series of alcohols andembedded in J B -4 glycol metha cry la t e (P oly-sciences, Wa rr ing t on, PA). Tw o-microm et er-t hicksections were cut w ith gla ss knives a nd a ffixed toglass s l ides . Sections were stained with eithertoluidine blue, periodic acid-Schiff, or methyleneblue-bas ic fuchsin. Sections w ere photogra phed ona Nikon (Melville, NY) Optiphot -2 light microscopeequipped with a Microflex AFX-DX photomicro-graphic at ta chment. For sca nning electron micros-copy (SEM), tissues were immersed in a primary

fixative consisting of 3%glut a ra ldehyde in 0.1 Mphosphat e buffer wit h 0.4 M sucrose a nd 10 dropsof 1%CaCl2 per 10 ml fixative. Fixation was car-ried out at room temperature for 6 to 8 hr. Tis-sues were then washed several t imes in freshbuffer. Samples were transferred to a secondaryfixat ive (1.0% osmium tet roxide in 0.1 M phos-pha te buffer) a nd postfixed for 1.5 hr a t room tem-perature. Specimens were subsequently washedwith d is t i l led water and dehydra ted through a

gra ded ser ies of t-buty l a lcohol from 30%t o 100%,frozen and dried in a refrigerated vacuum desic-cator. Specimens were mounted on stubs to whichsilver conductive paint had been added. Sampleswere coated in a Denton Desk II Sputter coaterwith a thin layer of gold and viewed on a J EOLJ SM -T300 SE M a t 10 to 25 kV.

Alternatively, for SEM, female S. canicula, R.clavata, a nd some sam ples of R. erinacea were per-fused wit h filtered sea w a ter, follow ed by 300 ml offixative (20 C) injected int o the dorsal a orta aft erdecapitation. The auricle was cut to facilitate es-cape of the fixative. Karnovsky (65) fixative wasprepared in 0.1 M cacodylate buffer (pH 7.4) andha d a f inal concentra tion of 1% glutara ldehyde,3%formaldehyde, 0.27 or 0.45 M sodium chloride,and 2 mM calcium chloride. After perfusion, OGswere removed and cut wit h a ra zor blade into lon-

gitudinal slices. Fixation was carried out by im-mersion in the same fixative for another 2 hr at4C followed by washing overnight in the samebuffer and postfixation in Daltons dichromate-buff-ered osmium tetroxide. Some fixed material wasinfiltrated with dimethyl sulfoxide, frozen in liq-uid nitrogen, fractured with a cold metal rod, orcut with a cold razor blade before thawing andwashing to remove the dimethyl sulfoxide. Afterdrying by the critical point method, material wassputt er coat ed w ith gold/palla dium. An S -8000SEM f i t ted wi th a n LBH gun was used .

RESULTSWe have chosen to survey OG structure in a

select group of elasmobranchs based on their di-versity in reproductive modes. It is hoped tha t a smore information is generated on the details ofOG from these species we will be able to makecomments on the evolution of reproductive modesas relat ed to the OG.

Oviparous species such as S. canicula, S. stel-laris, R. erinacea, R. eglanteria, a nd R. clavataproduce a hard, permanent egg case that is de-posited externally. In aplacental yolk sac speciessuch as S. acanthias, a tra nsient, flexible candle

case is produced an d reta ined in utero until it dis-appears . The yellow spotted stingray, U. jam-aicensis, is aplacental with trophonemata and isunusual in that i t does not form any type of eggcovering except for mucous and jelly and lacks abaffle zone. In placenta l species such a s M. canis,a plia ble egg envelope is formed tha t is ma inta inedthroughout gestat ion a nd is incorpora ted into th eplacenta . Species tha t produce an egg ca se, ca ndlecase, and egg envelope all share similar design


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featur es of the ba ffle zone or egg investment zone,e.g., a blown extrusion die complex that manipu-lat es th e combined secretions of gland tubules ina spinneret region of the ba ffle zone t o form com-plex la mellat ed structu res (Fig. 1). Secretory unit seject their products into transverse grooves thatextend across the width of the gland. Adjacentsecretory units thus form a part of each subse-quent layer of the covering. Species differences inOG architecture a re noted.

Scyliorhinus canicula

The functional anatomy of the OG of S. cani-cula i s the most s tud ied of se lachian f ish andp r ov id es a f r am ew or k fo r u nd er s t and ing theglan d in other species . Previous s tudies (Knigh t

et a l. , 96) ha ve ident ified several zones tha t h a vebeen designa ted zones AF 3 a long wi th severa lsubzones. According to the terminology used inthis investigation, their zone A corresponds toth e club/papillary zone; zones B , C, D , a nd E cor-respond t o the ba ffle zone; a nd zone F correspondsto the terminal zone. The gland consists of twoidentical halves surrounding a highly f la ttenedlumen. The gla nd is la rgely const ructed from nu-merous simple tubular glands about 100 m indiameter and wi th a length tha t var ies sys tem-a tically in the glan d, reaching approximat ely 1.5mm in t he termina l zone. The gland tubules arearranged in transverse zones distinguished byhist ochemist ry, tinctorial a ffinity, morphology, a ndultrastructure (Rusaoun 76; Rusaoun et a l . ,

Fig . 1. Composite l ine diagra m of a generic OG show-ing club, papillary, baffle, and terminal zones. Lower illus-tra tion is of a baffle zone unit composed of a secretory gland

tubule, secretory duct, spinneret with baffle plates, and trans-verse groove between plateau projections.


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76; Feng and Knight, 92; Knight et a l . , 96).Each zone adds a successive layer surroundingth e ovum. Thus, t he club and papil lary zones se-crete je l ly , which immedia te ly surrounds theovum. The ba ff le zone secretes t he la mellae th a tform the bulk of the thickness of the capsulewall . Zones discharge their secretion by way ofa series of transverse grooves, each transversegroove secreting a s ingle lamella of the capsulewall . The transverse grooves vary in depth indif ferent loca tions in the gla nd a pproxima tely inproportion to the thickness of the lamellae theysecrete and t he length of the g lan d tubules tha td ischa rge into them.

We ha ve concentr a ted m uch of our work on th eD zone w ith in th e baffle region. This zone secretesth e lamellae th a t form more tha n 80% of th ethickness of the capsule wall . The baff le zone

secretory tubules are approximately 10 mm inlength a nd a re constructed from a columnar epi-th elium conta ining t w o types of cells, ciliat ed cellsand secretory cel ls tha t s tore and secrete thesecretory product.

The secretion seems to undergo a series of liq-uid crysta l tra nsitions during concentra tion in theG olgi appara tus, stora ge in th e secretory droplets,merocrine secretion into the lumen of the secre-tory t ubule, and passage t hrough the extra cellu-lar secretory pathway. It is thought to be presentas an isotropic phase in the endoplasmic reticu-lum cisterna e but t o condense in t he G olgi cister-

nae into a lamellar liquid crystalline phase. Thel i q u i d c r y s t a l t r a n s i t i o n s d u r i n g s t o r a g e a r eth ought t o depend on the low pH a nd progressiveconcentr a tion of the secretion w ithin t he G olgi a p-para tus a nd secretory droplets. Wa ter is thoughtto flood back into the secretory granule when thelimiting membra ne of the secretory gra nule is loston merocrine secretion, allowing t he fibrilla r ma -terial to revert to a lamellar l iquid crystal l inephase. The secretory granules fuse in the lumento give a stra nd of liquid crysta lline ma terial. Thisis tra nsported t o the extrusion die a t a relativelylow pH t o prevent aggregat ion.

Each gland tubule contributes its secretion toits own extrusion die. The output opening of eachextrusion die is an oblique output slit measuringa pproximat ely 350 10 nm t ha t extrudes a highlyflat tened ribbon of the sa me dimensions. The rib-bon emerges between a pair of curved lips formedfrom hemispherica l ba ffle plat es covered on bothsides wit h ciliat ed epithelium. The extr usion diesare arranged in a geometric overlapping fashionin t he ba se of ea ch tra nsverse groove like tiles on

a roof. As the ribbons emerge from between thepairs of baffle plates they fuse to give rise to aunique plywood-like construction. The lamellaeth en fuse on rea ching the ma in lumen to producethe multilaminate construction of capsule wall,ma rginal r ib , a nd t endril. Thus, capsule wa ll andma rginal ribs are formed as blown, multilam inat e,cont inuous extrusions. Fina l a ssembly into fibrilsoccurs when the viscous material in the lamellaereaches the main lumen. Fibril assembly occurswith out a s ignif icant cha nge in the pat tern of mo-lecular orientations defined in the extrusion die.I t is thought to be init ia t ed by an increase in pHpossibly produced by the epithelial transport ofhydr ogen ions. The a ssembled fibrils show a highdegree of crysta l l inity an d conta in systema ticallykinked molecula r segment s.

Scyliorhinus stellaris

The egg capsule and associated tendrils in S.stellaris a re approxima tely tw ice the size of thoseof S. canicula but a re practically identical in sha peand construction. The capsule wall is approxi-mately twice as thick, this increase in thicknessarising from a doubling of the numbers of lamel-lae rat her tha n a n increase in lamellar thickness.This difference is reflected in t he OG , w hich ha sdouble th e number of rows of baffle zone t ubulesand baffle zone transverse grooves but is closelysimilar to tha t of S. canicula in a ll oth er respects.

Raja erinaceaa n d

Raja eglanteriaThe OG of R. erinacea is crescent shaped with

the lateral margins upturned (Fig. 2). The ante-rior oviduct leads to the OG, and the uterus exitsfrom it. The lateral margins of the gland expandgreatly prior to the arrival of an ovum. When theintern a l structure of th e gland is examined grossly,severa l zones are evident , including t he baffle an dterminal zones (Fig. 3).

The egg capsule is fundamentally similar to thatin the Scyliorhinus species described above butdiffers in the following features: (1) tendrils areabsent, (2) bundles consisting of numerous fine

hairs run the length of the lateral margin origi-na ting a t t he poster ior end and inserting into theanterior end of this structure, and (3) the outer-most surface of the dorsal capsule wall also car-ries numerous similar fine hairs. The outermostsurface of both dorsal and ventral capsule wallscarries deep pits that are roughly elliptical whenviewed from above, with the long axis of the el-lipse oriented la tera lly across the capsule.

The zones , ex t r u s ion d ies , and t r ans ver s e


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grooves of the OG in R. erinacea (Figs. 4 and 5)

are closely similar in appearance to their coun-terpar ts in Scyliorhinus spp. described above.Examination of natural casts of secreted mate-rial shows secretory product present in gland tu-bules and the spinneret region (Figs. 6 and 7).The fine hairs of the lateral margin (Fig. 8) aresecreted from compound tubular glands (Fig. 9)arranged in the anterolateral margins of the OG.Each tubule of the compound gland secretes asingle hair t ha t t hen passes down common ducts

to give rise to the hair bundles. All the lamellae

of the capsule wall have a more or less constantth ickness (Fig. 10).

The gland tubules of the baffle zone sweep pos-teriorly below th e termin a l zone (Fig. 11). E xam i-

Fi g. 2. Externa l vi ew of the OG of R. erinacea. The ovi-duct (o) is confluent with the OG. The lateral compartment(asterisk) of the OG expands greatly prior to the arrival of

an ovum. This expanded region merges with the uterus (u).F i g. 3. In te rna l v iew of the OG of R. erinacea showing

th e baffle zone (b), termin al zone (t), and ut erus (u).

Figs. 4 and 5. Scanning electron microscopy monta ge oflongitudina l slices cut from ha lf of the OG of R. erinacea show-ing glan d tubules (g), baffle plat es (a rrow), tra nsverse grooves(arrowheads) in the main lumen, and different zones of thegland: pa pillary (p), ba ffle (b) and termina l (t). 60.


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Figures 4 and 5.


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Figur es 610.


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nation of this region with SEM reveals secretoryproduct in the gland tubules as well as secretoryvesicles in the epithelial cells and cilia projectinginto the lumen (Figs. 12 and 13).

We have chosen to use the OG of R. eglanteriato i llustrat e the fundamenta l organ ization of OG.The oviduct in all species thus far examined isv ir tua l ly ident ica l . Longi tud ina l fo lds extendthrough the entire oviduct. Surface epithelium issimple columna r w ith cilia. The curve a t t he baseof the folds is char a cterized by t wo cell types: (1)pyriform sustentacular cells with cilia and nucleisituated near the lumen and (2) secretory cellswith basal nuclei (Fig. 14).

The club zone is cha ra cterized by r oughly T-sha ped lamella e. The surfa ce epithelium is simplecolumnar with cilia. A fairly dense lamina pro-pria is subjacent to the epithelium. Simple tubu-

lar gla nds connect to the lumen between a djacentclub lamellae (Fig. 15). E xam ina tion of a club zonetubule in cross sec t ion revea ls the secretoryvesicles to have a dense core and a lighter-stain-ing periphera l ha lo (Fig. 16).

In the papillary zone, the surface epitheliumcontinues as simple columnar with cilia. Simpletubular glan ds empty int o the lumen between ad-jacent lamellae (Fig. 17). Gland tubules have py-riform sustenta cular cells with cilia, a nd secretory

cells have secretory vesicles with homogeneouscont ent s (Fig. 18).

Secretory g land tubules are s imple and un-branched in the baffle zone. They are confluentwith the secretory duct that delivers secretoryproduct to the spinneret region where cilia-cov-ered baffle plates manipulate the secretory prod-uct (Fig. 19). From the spinneret region, secretedma terial enters t ran sverse grooves. Plat eau pro-jections of differing dimensions are situated be-tween ad jacent rows of tubules . The p la teauprojections are truncated at the base and havedram at ically extended a pical portions t ha t sweeptoward the terminal zone (Figs . 4, 5, and 19).Gland tubules most frequently contain secretoryproduct (Figs. 11, 12, 13, an d 20). P yriform sust e-ntacular cells have nuclei s i tuated near the lu-men, whereas secretory cells have basal nuclei.

Secretory cells are engorged with vesicles (Figs.13, 20, a nd 21). Nona ggrega ted, individua l sperma re occasionally encount ered in the glan d tubules(Fig. 21), but only in the region nearest the lu-men. They a re ra rely seen in t he deeper recessesof the gland.

The terminal zone has short , s imple tubularglands composed of two types of cells. Pyriformsustent a cula r cells have cilia, a nd t heir nuclei arelocated near the lumen, whereas secretory cellsare columnar and are f i l led with frothy, l ight-sta ining material tha t ha s the appeara nce of mu-cous (Fig. 22). Some of the glands are strictly

mucous (Fig. 22), whereas others have a mixtureof mucous a nd serous cells (Fig. 23).

Raja clavata

The egg capsule wall of R. clavata is broadlysimilar to that of R. erinacea but the layer se-creted by the baffle zone is split into two by anadditional layer of material. This is the alveolarlayer described by Faur-Frmiet (38). The zonethat secretes this layer stains differently from thea djacent baffle zone an d is secreted by tw o to threetransverse folds that protrude into the main lu-men of the gland sl ightly more tha n t he adjacent

baffle zone folds. The secretion t ha t gives rise tothe a lveolar layer ha s not yet been chara cter izedbiochemically or histochemically. It has the ap-pearance of a stabilized emulsion composed ofrat her large droplets .

Squalus acanthias

This is an aplacental yolk sac species, the em-bryo being reta ined wi thin the mother s bodywithin a thin, transparent, yellow f lexible egg

Fig. 6. Raja erinacea. Scan ning electron microscopy of al-dehyde-stabil ized natural internal cast of the extrusion diesstripped from the secretory pathway of part of the D-zoneafter prolonged fixation. Each die secretes a flattened rib-bon. These adh ere together on discha rge into th e tra nsversegroove to form the lamellae. The lamellae adhere on reach-ing the main lumen of the glan d. t = gland tu bule lumen, s =secretory duct, b = spaces left by ba ffle plates. 300.

Fig. 7. Raja erinacea. Pa rt of a cast of the secretory path-way as in Fig. 6 but showing where the transverse pits areformed in conta ct with t he termina l zone an d how th ey deepenprogressively as the thickening outermost surface moves pos-teriorly. 350.

Fig. 8. Raja erinacea. Scan ning electron microscopy show-ing transversely orientated pits and longitudinally orientedhairs on the outermost dorsal surface of the capsule wall .

These hairs are secreted from the openings of short tubularglands shown in Fig. 9. 75.

Fig. 9. Raja erinacea. The longitudinally orientated slit-sha ped openings of short tubular glands in t he terminal zoneare thought to be intermittently active to secrete the hairsseen on th e outer dorsa l surface of the capsule wa ll . 650.

Fig. 10. Raja erinacea. Sca nning electron microscopy of alongitudinal razor blade section of the full thickness of theegg capsule wall showing lamellar construction. The outerlayer is secreted by the papillary zone. The successive lamel-lae are secreted by the baffle zone and a thin inner layer issecreted by the t erminal zone. 900.


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Fig. 11. Raja erinacea. Scanning electron microscopy ofrazor blade section through the tubular glands of the bafflezone (b) and the terminal zone (t). Baffle zone tubules sweepposteriorly under the secretory tubules of the terminal zone.Solid rods of secretory ma terial (arrowh eads) can be seen inbaffle zone tubules. 65.

Fig. 12. Raja erinacea. Higher magnification of Fig. 11reveals rods of secretory ma terial (arrowh eads) and ba ffle zoneglands (ast erisk). 550.

Fig. 13. Raja erinacea. Sustentacular cells have cilia (c)and secretory cells of the baffle zone are dominated by secre-tory vesicles (circle). Arrowh ead = secreted ma teria l. 3,000.


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Figures 1417. Caption on next page.


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candle until it disappears prior to parturition. Thegland is proportionally smaller than in the egg-la ying species described above. The gla nd is sim pleand barrel shaped, with no lateral extensions asin oviparous species (Fig. 24). The structure of theOG and transverse zones are s imilar in generalorganizat ion to those in th e Scyliorhinus a nd Rajaspp. described above but there are fewer trans-verse grooves and rows of baffle zone gland tu-bules. The spinneret of the baffle zone has whatappear to be two sets of baffle plates. These are,in reality, the next or adjacent baffle plates. Thebaffle plates lie obliquely across the transversegroove so that the ribbons of egg case materialth a t t hey secrete overlap like tiles on a roof. P iecesof a djacent baffle pla tes a re, therefore, frequentlyseen in the same section (Fig. 25). The terminalzone extends several inches and is populated by

a homogeneous layer of simple columna r cells w ithfrothy contents as well as ci l ia ted sustentacularcells (Fig. 26).

Mustelus canis

This species of shark is viviparous with a yolksac placenta. The egg envelope is secreted in asimilar way to the egg case of egg-laying speciesbut w ith one significa nt difference. The w idth (150mm) is vast ly great er tha n tha t of the widest eggcapsule of a ny egg-lay ing species we h a ve studiedbut t he OG is proport iona lly sma ller. How is sucha wide envelope secreted by such a compact OG ?

The OG extrudes t he egg envelope into t he reser-voir, a modified part of the oviduct posterior tothe OG . The ma teria l of th e envelope is not ma rk-edly extensible but is neatly folded transverselyand pleated longitudinally within the reservoirlike a J a pan ese fan. The developing fetus pullsout t he folds and pleats as i t grows in t he uterus.The problem of forming such a wide envelope froma compact gland is solved by coiling the walls of

the OG through gyres on each side of the ante-rior oviduct to give a double helter-skelter struc-ture much like a rams horn (Fig. 27). This givesa n extremely w ide loom from w hich the egg enve-lope is extr uded. As in oth er OG s, th e lam ella e ofthe egg envelope of M. canis are secreted fromtra nsverse grooves tha t run up ar ound the helter-skelter. As in t he species described a bove, th e tr a ns-verse grooves cont a in extrusion dies equipped wit hbaff le plates . Sections of the baff le plates fre-quently include portions of the next set of baffleplates (Fig. 28). These a re t hought to extrud e rib-bons whose widt h an d arra ngement defines wherethe longitudinal pleats will form in the egg enve-lope. The terminal zone has columnar secretorycells in various stages of synthesis and secretion.The lumen of the gla nds frequent ly ha rbor bundlesof lat erally a ligned sperm (Fig. 29).

Urolophus jamaicensis

The external structure of the OG in the yellowspotted ray is grossly s imilar to that of S. acan-thias in that i t is small and barrel shaped (Fig.30). The internal organization is similar to theother OG s examined. A baffle zone wit h t he char-a cteristics found in other species is lacking. In U.jamaicensis we ha ve termed this zone the baff lezone equivalent. Glands are populated by twotypes of cells. Ciliated sustentacular cells havenuclei near the lumen, whereas the nuclei of thesecond cell type is basal. Columnar cells contain

enormous mem bra ne-limit ed rods (Fig. 31).

DISCUSSION

Early descriptions of the elasmobranch OG in-clude th ose of P erra vex (1884), Hen neguy (1893),B orcea (04, 06) a nd Wida kowich (07). All used lightmicroscopy to describe the fundamental organiza-tion of th e gla nd. R etzius (02) described the spermof elasmobranchs and commented on the spiral

Fig. 14. Raja eglanteria. Epith elium of crypt between lon-gitud ina l folds of the oviduct. Two cell types a re appa rent :pyriform sustentacular cells with cilia (c) and nuclei (open

arrows) near the lumen and secretory cells engorged witha pical vesicles (as terisk) and ba sal nu clei (da rk a rrows). 200.

Fig. 15. Raja eglanteria. Club zone folds (asterisks) haveciliated surface epithelium (e). Gland tubules (g) empty intothe OG lumen between a djacent club projections. 100.

Fig. 16. Raja eglanteria. Cross section of club zone glandtubule showing dense core secretory vesicles (arrows) with aperipheral ha lo. 600.

Fig. 17. Raja eglanteria. Papil lary zone folds (asterisks)have ciliated epithelium (e1) that continues onto projectionsof the a djacent ba ffle zone (e2). Gla nd t ubules (g) empty theircontents into the lumen betw een papillary folds. 100.

Fig. 18. Raja eglanteria. Cross section of papil lary zonegland tubule showing homogeneous vesicles (circle) in secre-tory cel ls a nd c i l ia (c) on the su stenta cular cel l s . 600.

Fig. 19. Raja eglanteria. G land tubules (aster isks) ofthe baffle zone deliver their secretory products to the spin-neret region (two-headed arrow) by way of the secretoryduct (arrow). Pa i red baf f le plat es (b) ma nipulate t he secre-tory product into f l a t r ibbons that are del ivered to the lu-men between adjacent plateau project ions (p) where theyas semble into lamel lae of the egg case . 100.

Fig. 20. Raja eglanteria. Nuclei of sustentacular cells(open arrows) wi th c i l i a (c) are located near the lumen.Nuclei of secretory cells ar e basa l (dark a rrows), whereasthe apical cy toplasm is dominated by secretory vesic les(circles). 600.


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Figures 1820.


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Figures 2123.


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shape of the head. Lo Bianco, (0809) introducedthe possibility t ha t t he OG ma y function as a spermstorage device when he noted tha t nearly a ll eggsof Raja asterias a nd R. undulata laid in tanks werefertilized. In 1922 Clark noticed that female R.brachyura kept isolated from males in tanks laida bout 25 egg cases all w ith fert ilized eggs in 5 to 8weeks an d concluded th a t sperm must be stored bythe female. Although he could not locate the sightof storage, he suggested that storage occurred inthe upper oviduct. Metten (39) demonstrated theoccurrence of sperm in the OG of S. canicula. H eidentified albumen-, mucous-, and shell-secretingzones and commented on mucous glands in whatw e have t ermed the t ermina l zone. Pr a sa d (45) pre-sented a description of the light microscopy of apla-cental yolk sac guitarfish, sawfish, and viviparousplacental sharks. He provided line drawings of the

glands and noted three constant zones: albumen,mucous, and shell secreting.Our terminology, based on Hamlett et al. (98),

identifies an a nt erior club zone followed by papil-lary, baffle, and terminal zones. Earlier descrip-tions a nd t erminologies generally refer to t he clubzone as the albumen zone; the papillary zone asth e mucous zone; and t he baffle zone va riously a sthe zottehan (Widakowich, 07), oblique plates(Mett en, 39), a nd tuft s (Pra sad , 45). B eca use nobiochemical studies have been performed on thesecretions of the va rious regions of th e glan ds inany species, we have chosen to follow terminol-

ogy based on morphology rather than terminol-ogy ba sed on composition of secretions, w hich ma ynot be const a nt a cross species.

Although the baffle zone occurs in all the OGsexamined here, except in U. jamaicensis, whicha pparent ly does not produce a n egg envelope, th esize, extent, a nd organ izat ion of this region w ithinthe gland is species specific. Baffle zone complex-ity reflects th e complexity of the ca psule produced.

Despite t he differences in egg ca psules producedby these species, which range from substantialstructures in oviparous species to thin diaphanousmembra nes in some vivipa rous species, the sa me

fundamental mechanism for assembly of capsuleproteins is employed. Epithelial cells secrete cap-sule precursors int o the t ubules, where th ey coa -lesce . This mater ia l i s then moved down thetubules presumably by t he ciliat ed cells lining t hetubule t o the extrusion dies. The ma teria l is spunout of the dies tha t open betw een the baffle pla tesa nd from there into th e tra nsverse grooves, whichthen a ssemble the laminat e s tructure of the cap-sule wall. The number of transverse grooves de-termines the n umber of layers in t he capsule wa ll.

Tw o aspects of th is process a re crucial t o thesuccess of the extrusion molding and assemblymechan isms, at least for th e oviparous species andlikely for th e vivipa rous species as well. First, th eproteins and enzymes that make up the capsulemust be maintained in a prepolymerized condi-tion within intracellular s torage compartments .

These mat erials a re segregat ed with in cells in dis-tinct secretory granules and in the gland withinfunctionally separate regions, thereby permittingactivation of the polymerization process only af-ter secretion and coalescence in the tubules. Thesecond aspect is th e timing of th e polymerizat ionreactions. Stabilization of the admixture occursafter organization of the components into the as-sembled matrix.

The morphological feat ures common t o all of th eOGs examined in this survey are the club, papil-lary, baffle or ba ffle equivalent, a nd t erminal zones.The club and papillary zones are present in both

oviparous and v iv iparous spec ies , even in U.jamaicensis, which lacks the ba ffle zone and seemsnot t o produce a n egg envelope. B oth of th ese zonesseem to secrete material that directly surroundsth e ovulat ed egg and fills th e lumen of the egg cap-sule not occupied by the egg itself. The function ofthis material is not known, including whether itfunctions identically in oviparous and viviparousspecies. Nonetheless, given that it seems to be aubiquitous feature in all elasmobranch OGs, thefunction of the ma teria l must be crucial for a t lea stth e early st a ges of embryonic development .

The na tur e of the secretory products a rising in

the club and papillary zones has been examinedwith histochemistry, and with that adequately inonly one spec ies , the oviparous S. canicula.Threa dgold (57) observed t ha t th e secretory prod-ucts in the club zone of S. canicula stained in-tensely with PAS and sulphated toluidine blue.Reactions with other histochemical s tains werew eak or a bsent, most n ota bly th ose specific to pro-tein stains. Threadgold (57) concluded that thesecretory product was a carbohydrate and possi-

Fig. 21. Raja eglanteria. Occasionally the luminal portionof baffle zone glan d t ubules contain individual , disa ggregatedsperm (s). 600.

Fig. 22. Raja eglanteria. The proximal portion of the ter-minal zone has cil iated (c) sustentacular cells (arrow) thatalterna te w ith secretory cells engorged with secretory prod-uct (asterisk). 600.

Fig. 23. Raja eglanteria. The distal portion of the termi-nal zone has both mucous (asterisk) and serous (s) secretorycells in the sam e crypt. 600.


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Fig. 24. Squalus acanthias. The OG (double arrows) issi tua ted betw een the proximal (p) an d dista l (d) oviducts.

Fig. 25. Squalus acanthias. Simple ciliated (c) columnarepithelium covers baffle plates (1), adjacent baffle plates (2),a nd pla tea u projections (p) of the baffle zone. 600.

Fig. 26. Squalus acanthias. The terminal zone extends

out into the posterior oviduct several cm. The distal oviductis occupied by a prolongation of the terminal zone before itscomposition changes to ciliated, nonsecretory columnar epi-thelium. The posterior oviduct is separated from the uterusby a prominent sm ooth mu scle sphincter. Ciliat ed sustenta cu-lar cells (c) alt erna te w ith secretory cells (s). 600.


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bly a neutral mucopolysaccharide and not an al-bumen as Filhol and Garrault (38) had earliersurmised. Rusaoun (76) applied s imilar his-tochemical methods together w ith other s t ains tothis same region of the gland. Although the in-tense periodic acid-Schiff staining was again ob-served in this region, secretory materials alsostained with Alcian blue at acid pH, indicatingth e presence of str ong polya nions. A strong meta -chromatic reaction was obtained with toluidineblue, leading to the speculation that these prod-ucts are sulphated polysaccharides. Her conclu-s ion was tha t th is region elabora ted abundantneutral mucopolysaccharides and acidic polysac-char ides . Given the apparent absence of his-tochemically reactive proteins, she assumed thatth ese polysa ccha rides were not bound t o protein.H er r esults a lso supported Threa dgolds (57) con-

tention that the secreted material wa s not relat edto a lbumen. Feng a nd K night (92) more recentlyreported simila r observa tions.

B ased on these few observat ions, it a ppears t ha tth e principal product of the club zone in th e ovipa-rous S. canicula is a combina tion of predomina nt lyneutral mucopolysaccharide and lesser amountsof acidic polysaccharides. Unfortunately, the ex-act chemical composition of these carbohydrateshas yet to be determined. Moreover, there is noreason to assume that the products of this zoneare s imi lar in ov iparous sharks vs . ska tes , inoviparous vs. viviparous sha rks, or in th e vivipa -

rous rays. However, it is clear that this region ofthe gland produces the more or less fluid com-part ment sur rounding eggs in ca psules of all elas-mobranchs that encapsulate eggs and l ikely inthose that lack an encapsulating membran e.

The composition and function of the secretoryproducts of the papillary zone are equally enig-matic . Based on staining character is tics , Nalini(40) believed the product to be a type of mucin inChiloscyllium griseum, sepa ra ting the fluid origi-na ting in the club zone from the capsule and func-tioning as a lubricant during encapsulation so asto reduce friction between the fluid and forthcom-

ing capsule. Threadgold (57) found that this thinregion in S. canicula sta ined metachromat icallya nd concluded th a t t he secretory product conta inedcarbohydr a te. In contra st, Rusa oun (76) report edthat the product was a s trongly sulfated muco-polysaccharide. Feng and Knight (92), based onthe observation that Alcian blue stains this mate-rial, concluded tha t t his mat erial is a sulfated gly-cosaminoglycan. Final conclusions about the exactnature of this material await chemical analyses .

As with the club zone secretions, it would be pre-sumptuous to speculat e on t he na ture of the prod-uct of this zone in other elasmobran chs.

In only t wo species, S. canicula a nd R. erinacea,have capsule composition, assembly, and stabili-za tion been examin ed in any det a il (Koob a nd Cox,93; Knight et al., 96). The process of capsule for-ma tion is simila r in both s pecies. The OG secretesa l iquid crystal l ine material that is then moldedinto sheets tha t a re layered together by the t ran s-verse grooves in t he gla nd. The liquid crysta llin-ity of the material enables it to be oriented byflow through th e gland tubules w ith subsequentcomplex molecular orientation produced by theextrusion dies . The result is a highly orderedfibrillar component in each layer. The liquid crys-tallinity of the coalesced capsule materials, to-gether with the ordered arrangement achieved

during a ssembly, also provides for the syst emat icintroduction of polymerizing molecular interac-tions but only af ter the f ibr i l lar or ientation hasbeen a ccomplished.

Regional specialization in the gland is respon-sible for the distinct morphological organizationof the ca psule wa ll in S. canicula. The ba ffle zonecan be subdivided on the basis of the chemistryand morphology of the products each secretes(Knight et al. , 96). The thin inner layer of thecapsule arises from the baffles neighboring thepapillary zone. The product of this zone forms aphenolic varnish stabilized by quinones and pos-

sibly peroxidative crosslinking. The bulk of thebaffle zone produces the fibrillar lamellae com-pr is ing the major i ty o f the capsule wal l . Thefollow ing zone secretes hyd rophobic gra nules con-ta ining t yrosine-rich protein. The next zone a ddsa thin layer of phenolic varnish to the outer as-pect of the capsule wa ll.

Based on available data , the chemistry of thec ap s u le c ons t i tu en ts and the p o lym er i za t ionmechanisms in S. canicula a nd R. erinacea seemto differ in some import a nt respects. The predomi-na nt fibrillar component in S. canicula capsule isa protein with certa in structura l affinities to base-

ment membrane collagens. According to Knightet a l. (96), it is a sheet-forming m ini-collagen w itha molecular length of about 40 nm with a promi-nent nonhelical doma in at one end and a smallernonhelical domain at the other end. Other pro-teins that are feasibly incorporated into the dog-fish ca psule ha ve not been identified. In t he littleskate, R. erinacea, there are six major proteinscont a ined in th e assembled capsule mat rix (Kooband Cox, 93). None of these proteins gave an


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Figures 2729.


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a mino a cid composition similar to int erstitia l col-lagens, an d none mat ch the molecular w eight ofthe putative dogfish capsule collagen. However,one or more of the skate capsule proteins mayha ve collag enous doma ins. Tw o of t he low m olecu-lar weight ska te proteins contain relat ively hightyrosine levels and may be analogous to the ty-rosine proteins in the h ydrophobic gran ules in th edogfish ca psule. The ma jor protein component s ofth e egg envelope in vivipa rous species ha s yet tobe examined, so compa risons between th e capsulesof oviparous species an d live-bearing sha rks a ndra ys is not possible.

The molecular events responsible for polymer-izing the a ssembled capsule mat r ix may also dif-fer in the dogfish and ska te examined here. Ba sedon histochemical tests on dogfish OGs and eggcapsules , a quinone tanning process involving

phenoloxidase seems to operat e aft er a ssembly ofth e capsule precursors (Rusaoun, 76; Rusa ounet al. , 76; Feng and Knight, 92). The nature ofthe enzym es and r eac tan ts has no t b een u n-equivoca lly delineat ed. However, Feng an d K night(92) identified free 3,4-dihydroxyphenylalanine inextracts from w hole dogfish OG s a nd speculat edthat i t was the catechol involved in quinone sta-bilizat ion. P eroxidase a ctivity wa s a lso observedhistochemically , leading to the hypothesis thatperoxidase-catalyzed dityrosine crosslinking is apivotal mecha nism for capsule s ta bilizat ion.

Polymerization of the assembled capsule ma-

trix in the little skate relies on two postsecretoryevents (Koob and Cox, 90). The first is disulfidebond formation between two of the capsule pre-cursor proteins. The second involves th e int roduc-tion of catechols after assembly of the capsulematr ix in the gland and their subsequent oxida-tion t o quinones in the ut erus. Tw o enzym es ar einvolved in the quinone tanning mechanism. Atyrosine hydroxylase activity hydroxylates pep-

tid e-bound t yrosine residu es forming 3,4-dihy dro-xyphenylala nine. Evidently, in contra st to t he dog-fish, 3,4-dihydroxyphenylalanine is absent fromthe secreted and assembled matr ix and appearsonly in ut ero as a result of th is enzyme. The sec-ond enzyme is a cat echol oxidase (Koob a nd C ox,88). This enzyme oxidizes a portion of the newlyformed 3,4-dihydroxyphenylalanine to the corre-sponding quinone. This quinone then goes on topolymerize the matrix. Although peroxidase ac-tivity is a lso found in t he l i t t le skate OG , i ts rolein capsule format ion is unclea r.

The OG of selachia n fish is a complex str uctureused in most species to extrud e th e successive la y-ers of jelly a nd egg ca se, ca ndle, or envelope th a tsurround t he fertilized ovum. The sa me ba sic or-ganization is found repeatedly in both egg-layingand live-bearing species.

Such funda menta l informa tion as the actua l siteof fertilization and the site of sperm storage inelasmobranchs remains unsettled. Lo Bianco (0809) suggested th a t t he OG performs t he functionof sperm storage but did not present any histo-logical observat ions. C lar k (22) suggested th a t fer-tiliza tion occurred in th e coelom, an d Mett en (39)believed that fertilization occurred simultaneouslywith egg case secretion and that fertilization oc-curred in t he OG . H e described bundles of spermin the lumen of the baffle plate zone and isolatedsperm in the tubules. He never found sperm inth e club or papillar y zones. He d escribed mucous

glan ds in the t ermina l zone but did not see spermthere. It was not indicated if he took serial sec-tions of the entire gla nd or simply only a few sec-tions. He may not, therefore, have identified thefull expanse of the terminal zone tubules . Hethought tha t sperm st ora ge occurred in th e baff lezone and that these tubules secreted a nutr i t ivematerial for sperm in the shell substance.

P ra tt (93) described sperm stora ge in n ine spe-cies of elasmobranchs. He sampled the posteriorone-third of the OGs and examined them withlight microscopy. This corresponds to the baffleand terminal zones. His published micrographs

were transverse sections, and he noted sperm inthin-walled tubules of the lower OG around thecurves of t he S -sha ped lumen. We interpret th isto be equivalent to the terminal zone. He alsonoted sperm in tubules that course posteriorly/anteriorly along the peripheral edge of the shell-secreting zone in Sphyrna lewini. We also inter-pret this as the termina l zone. In a l l spec iesexamined by us, the terminal zone swings backto the periphery of the baffle zone to envelop a

Fig. 27. Mustelus canis. The OG (g) ha s coiled lat era l ex-tensions (aster isk) much l ike a rams horn. An egg (e)

tra verses the oviduct (o) to pass t hrough the OG , where i t ispacked in its individual egg envelope (ee) and thence to theuteru s (u).

Fig. 28. Mustelus canis. The baffle zone is characterizedby paired baffle plates (1) and small segments of adjacentbaffle plat es (2) tha t ma nipulate secretory mat erial (arr ows)as i t passes through the spinneret to emerge between adja-cent plat eau projections (p). 200.

Fig. 29. Mustelus canis. Termina l zone tubules a re deepand populated by large cells (asterisk) in various stages ofsecretion an d ciliat ed (c) sustent acula r cells. Lat erally a lignedsperm aggrega tes (arrow) occur in t he gland lumen. 600.


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Fig. 30. Urolophus jamaicensis. The ovidu ct (o) is confluen twith the sma ll, bar rel-sha ped OG (g), which leads to th e uterus(u) with luminal t rophonemat a (ar row). Scale is cm.

Fig. 31. Urolophus jamaicensis. The baffle zone equiva-lent does not produce an egg capsule but is characterized by

cells with enormous membrane-limited, rod-shaped bodies(curved a rrow); basa l nuclei (da rk a rrow); an d ciliat ed (c) sus-tentacular cells whose nuclei (open arrow) are located nearerthe lumen. 600.


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portion of it. The egg coverings of elasmobranchslack a micropyle and cannot, therefore, be fertil-ized in the posterior oviduct a fter format ion of theegg covering. The sequence of events during en-capsulation of the ovum in S. canicula (Knighta nd Feng, 92) suggests th a t fertiliza tion must oc-cur before passage of the ovum through the pap-illary zone.

We ha ve observed sperm in bundles in th e deep-est recesses of the t ermina l zone in M. canis a ndin an equivalent zone in other species studied. Wehave also seen individual sperm in tubules nearth e lumen in ba ffle plates. It seems plausible th a tsperm bundles in the terminal zone are releasedunder the stimulation of hormones associated withovulation and encapsulat ion a nd then migra te tothe OG lumen. In the skate, the region of the up-per uterus imbibes water and dramatically ex-

pan ds to receive the egg case a nd ovum (Koob an dHa mlett , 98). This migh t a lso contribut e to spermrelease. These phenomena would explain indi-vidual sperm being present near the openings ofthe baffle plates and the observation of sperm inthe upper reaches of the oviduct as reported inChimaera (Dea n, 1895). Fert ilization migh t th enoccur in the oviduct or upper OG. The fertilizedovum would them be coa ted w ith jelly a nd descendinto t he pa rt ially formed egg capsule. We presentthis as a working hypothesis. We are in the pro-cess of mapping the full extent of all tubules inthe OG of our study species so that we can pre-

cisely identify sperm storage areas. What havebeen previously described as sperm storage inth e baffle plat es may merely represent incidencesof sperm occurrence as they move anteriorly tofertilize freshly ovulat ed eggs.

ACKNOWLEDGMENTS

We thank Steve Hunt, Larry Gathercole, DianFeng , X iao Wen H u , Mu r r ay S tew ar t , Dav idHepwort h, J ulian Vincent, Allen B a iley, MaggieI n n o c e n t , R o b e r t G a r o n n e , R i c h a r d N e w t o n ,Ma rt a Cippolone, and Ma e Wa n H o for helpful col-laboration. This work was supported in part byfunds provided to W.C.H. by I ndia na U niversitySchool of Medicine.

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