the ultrastructure of spermatid development during spermiogenesis within the rosebelly lizard,...

The Ultrastructure of Spermatid Development DuringSpermiogenesis Within the Rosebelly Lizard, Sceloporusvariabilis (Reptilia, Squamata, Phrynosomatidae)

Kevin M. Gribbins,1* Caroline L. Matchett,1 Kathryn A. DelBello,1 Justin Rheubert,2

Maricela Villagr�an-SantaCruz,3 Gisela Granados-Gonz�alez,4 and Oswaldo Hern�andez-Gallegos4

1Department of Biology, Wittenberg University, Springfield, Ohio 455012Department of Biology, St. Louis University, St. Louis, Missouri 631033Laboratorio de Biolog�ıa de la Reproducci�on Animal, Departamento de Biolog�ıa Comparada, Facultad de Ciencias,Universidad Nacional Aut�onoma de M�exico, Distrito Federal, M�exico 045104Facultad de Ciencias, UAEM�ex, Instituto Literario # 100 Centro, Toluca, Estado de M�exico, M�exico 50000

ABSTRACT Several recent studies have mapped out thecharacters of spermiogenesis within several species ofsquamates. Many of these data have shown both conservedand possibly apomorphic morphological traits that couldbe important in future phylogenetic analysis within Repti-lia. There, however, has not been a recent study that com-pares spermiogenesis and its similarities or differencesbetween two species of reptile that reside in the samegenus. Thus, the present analysis details the changes tospermiogenesis in Sceloporus variabilis and then comparesspermatid morphologies to that of Sceloporus bicanthalis.Many of the morphological changes that the spermatidsundergo in these two species are similar or conserved,which is similar to what has been reported in other squa-mates. There are six main character differences that canbe observed during the development of the spermatidsbetween these two sceloporid lizards. They include thepresence (S. variabilis) or absence (S. bicanthalis) of amitochondrial/endoplasmic reticulum complex near theGolgi apparatus during acrosome development, a shallow(S. variabilis) or deep (S. bicanthalis) nuclear indentationthat accommodates the acrosomal vesicle, filamentous(S. variabilis) or granular (S. bicanthalis) chromatin con-densation, no spiraling (S. variabilis) or spiraling(S. bicanthalis) of chromatin during condensation, absence(S. variabilis) or presence (S. bicanthalis) of the longitudi-nal manchette microtubules, and the lack of (S. variabilis)or presence (S. bicanthalis) of nuclear lacunae. This is thefirst study that compares spermiogenic ultrastructuralcharacters between species within the same genus. Thesignificance of the six character differences between twodistantly related species within Sceloporus is stillunknown, but these data do suggest that spermiogenesismight be a good model to study the hypothesis that sper-matid ontogeny is species specific. J. Morphol. 275:258–268, 2014. VC 2013 Wiley Periodicals, Inc.

KEY WORDS: lizard; spermatids; Phrynosomatidae;spermiogenesis; ultrastructure; Sceloporus

INTRODUCTION

Much has been published in the last 5 yearsregarding the histology or the process of spermato-genesis within the genus Sceloporus (Villagr�an-

Santa Cruz et al., 2009; Gribbins et al., 2011;Goldberg, 2012; M�endez-de la Cruz et al., 2013;Rheubert et al., in press). These histological stud-ies utilize the light microscope level exclusively.The Sceloporus genus, like most taxa within Squa-mata, is underrepresented as far as testiculararchitecture and ultrastructural descriptions ofgerm cells during spermatogenesis.

Reproductive morphological data such as theontogenic changes germ cells undergo during sper-miogenesis and observations on the structure ofthe testis not only offer important reproductivemarkers as proposed by Guillette and Casas-Andreu (1987), but also have implications on ourcomprehension of the evolution of reproductivemorphology in amniotes. Spermatozoal ultrastruc-ture has been a major focus of reproductive biologyin reptiles for well over 30 years. The ultrastruc-ture of the mature spermatozoa provides a charac-ter matrix that displays both variability(synapomorphies) and conserved structures (sym-pleisomorphies) across chordate taxa (Jamiesonet al., 1995; Jamieson, 1999). Thus, there has beena number of studies recently that concentrate onthe microscopic anatomy of the spermatozoa in rep-tiles and its relevance as a phylogenetic tool for

Contract grant sponsors: Wittenberg University, UAM�ex andPROMEP (Financial support for lizard collections and research toGG-G and OH-G); Contract Grant numbers: 9003/2013CAFF,PROMEP FE022/2012.

*Correspondence to: Kevin Gribbins, Biology Department, Witten-berg University, PO Box 720, Springfield, OH 45501.E-mail: [email protected]

Received 11 August 2013; Revised 6 September 2013;Accepted 10 September 2013.

Published online 4 November 2013 inWiley Online Library (wileyonlinelibrary.com).DOI 10.1002/jmor.20212

VC 2013 WILEY PERIODICALS, INC.

JOURNAL OF MORPHOLOGY 275:258–268 (2014)

amniotic systematics (Oliver et al., 1996; Jamieson,1999; Vieira et al., 2005; Tourmente et al., 2006;Tavares-Bastos et al., 2008; Rheubert et al.,2010a,b; Gribbins and Rheubert, 2011).

The ultrastructural characteristics of the maturespermatozoa are generated during spermatogenesisand the formation of these mature structures spe-cifically can be observed during the phases of sper-miogenesis (Rheubert et al., 2011a,b). Previousstudies have concluded that all vertebrates followthe same overall three-step process of acrosomedevelopment, nuclear elongation/DNA condensa-tion, and flagellar development during spermiogen-esis (see Russell et al., 1990). Thus, spermiogenesishas the potential to reveal ontogenically howmature structures of the spermatozoa originateduring germ cell development. There also may bediversity in the developmental pathway that leadsto the formation of various structures seen withinthe spermatozoa between closely related taxa. Forexample, some species of lizard do not display themicrotubules of the manchette during spermiogene-sis (Rheubert et al., 2010c), which results in widerspermatozoal nuclear diameters than sperm fromother species that have well-developed microtu-bules of the manchette throughout late spermatiddevelopment (Gribbins, 2011).

The reptilian, specifically Squamata, spermato-zoon has more variation across species (apomor-phies) than many other vertebrate taxa (Jamiesonet al., 1995). Most of this variation is based on thelevel of compartmentalization and protein stratifi-cation found within the acrosome (Healy and Jamie-son, 1992; Jamieson, 1999; Jamieson, 2007). Whilespermatozoal studies have increased in numberrecently, most of the 9,4001 species of Squamatastill lack ultrastructural data for spermatid devel-opment. Also, multiple data sets on spermiogeniccharacters within a single monophyletic group donot currently exist in the literature. Thus, eachstudy that provides ultrastructural descriptions ofthe spermatozoa or spermatids increases our under-standing of the development involved in producingthe highly differentiated male gametes.

Although there are many studies that outlineincomplete ultrastructural descriptions (Clark,1967; Butler and Gabri, 1984; Hondo et al., 1994;Al-Dokhi, 2004; Mubarak, 2006) and comprehen-sive details (Courtens and Depieges, 1985; Carcu-pino et al., 1989; Vieira et al., 2001; Ferreira andDolder, 2002; Gribbins et al., 2007; Rheubertet al., 2011a,b; Rheubert et al., 2012; Gribbinset al., 2013) regarding the developmental steps ofspermiogenesis within squamates, no study todate offers a comparison between the spermiogenicevents within two or more species within the samegenus. Rheubert et al. (2012) provided the firstultrastructural account of spermiogenesis for Sce-loporus bicanthalis (within the scalaris group;Leach�e, 2010). The present study describes the

spermiogenic events in the testis of Sceloporusvariabilis (within the variabilis group; Leach�e,2010) and then compares spermatid developmentbetween these two species within Sceloporus andto other reptilian taxa. These two species of lizardwithin the Sceloporus genus have very differentmodes of parity. S. variabilis practices oviparity,while S. bicanthalis is exclusively viviparous(M�endez-de la Cruz et al., 1998). This will be thefirst attempt to compare spermiogenic morphologybetween two lizard species within the same genusand thus will facilitate the further testing of thehypothesis provided by Jamieson (1999) that sug-gests spermatid development is species specific.

MATERIAL AND METHODSTissue Preparation

Four mature spermiogenic male S. variabilis (Wiegmann,1834) were collected (scientific collector permit: FAUT 0186,SEMARNAT) from an open riverbed outside of Veracruz, M�exico,within the Ejido Adolfo L�opez Mateos, Selva del Marinero, LosTuxtlas (18.44100�N, 094.96440�W) in April 2012. This entirestudy was conducted in accordance with the ethical principles forthe laboratory use of animals; all work met the relevant legal reg-ulations as well as institutional procedures for animal researchof the Facultad de Ciencias, UAEM�ex. Lizards were sacrificedusing a 0.1% injection of sodium pentobarbital, testes were thenremoved, cut into small pieces (2–3 mm blocks), and submergedin Trumps fixative (0.2 mol formalin and glutaraldehyde in0.2 mol cacodylate buffer, pH 7.4) and stored under refrigeration.The specimens were deposited in the Centro de Investigaci�on enRecursos Bi�oticos, Facultad de Ciencias, UAEM�ex.

After refrigeration for over 48 h, the tissues were washedthree times with cacodylate buffer (pH 7.4) for 20 min. Testicu-lar tissues were then postfixed in 2% osmium tetroxide for 2 h,washed again in cacodylate buffer (pH 7.4) three times for 20min, dehydrated in ascending concentrations of ethanol (70%,85%, 95% 3 2, 100% 3 2), and cleared twice with 10 min incu-bations in propylene oxide. The testicular pieces then wereslowly introduced to a graded series of epoxy resin (Embed 812,Electron Microscopy Sciences, Hatfield, PA; 2:1 and 1:1 solutionsof propylene oxide: epoxy resin). The final step in the epoxyinfiltration was a 24 h incubation in pure 100% Epon 812. Freshresin was prepared and testicular tissues were embedded insmall beam capsules and cured for 48 h at 70�C in a Fisher iso-temperature vacuum oven (Fisher Scientific, Pittsburgh, PA).Once hardened, sections (90 nm) were cut from the blocks via aDelaware diamond knife (Wilmington, DE) on a Leica UC7ultramiucrotome (Leica Microsystems Inc. Buffalo Grove, IL).Sections were then placed on copper grids and stained for 18min with uranyl acetate and for 5 min with lead citrate.

Ultrastructural Analysis

The samples of testis were viewed using a JEOL JEM-1200EX II transmission electron microscope. Spermatids andorganelles associated with spermiogenesis were identified andmicrographs were taken with a Gatan 785 Erlangshen digitalcamera (Gatan, Warrendale, PA). Micrographs were analyzedand composite plates were constructed using Adobe PhotoshopCS software (Adobe Systems, San Jose, CA).

RESULTS

The beginning phase of spermiogenesis includesacrosome development within the round spermatidcytoplasm. The acrosomal vesicle (Fig. 1A; Ac)

259SPERMIOGENESIS IN SCELOPORUS VARIABILIS

Journal of Morphology

enlarges juxatpositioned to the apical nucleus astransport vesicles (Fig. 1B; white arrowhead) bud-ding from the Golgi apparatus (Fig. 1B; blackarrow) fuse with the developing acrosomal vesicle.The acrosomal vesicle at first stands alone in thecytoplasm making little contact with the nuclearmembrane surface (Fig. 1A, Ac). The acrosomalvesicle undergoes substantial growth beforeactually making significant contact with thenucleus; once contact is made at the apex of thenucleus a very shallow wide nuclear indentationoccurs (Fig. 1B; Ac). There are few observablemyelin figures within the acrosomal vesicle duringits development and the only structure typicallypresent in the acrosome vesicle is a basally locatedacrosomal granule (Fig. 1D, white arrowhead).The deepest penetration of the acrosomal vesicleoccurs during the flattening of the apical nucleusat the end of the round spermatid stage (Fig. 2D,inset). Also noteworthy, is the association of mito-chondria (Fig. 1B, black arrowhead) and roughendoplasmic reticula (Fig. 1B; white arrow) with

the Golgi apparatus (Fig. 1B, black arrow) duringacrosome vesicle formation. The ER ring surround-ing the mitochondria that is consistently situatedbetween the Golgi and the acrosomal vesicle iswhat is different than that reported for othersquamates. This new association of these twoorganelles with the Golgi apparatus during acro-some development is here termed the mitochon-drial/ER complex. As the acrosomal vesicleincreases in size, the associated nuclear indenta-tion remains shallow and the vesicle begins to col-lapse onto the surface of the nucleus early inround spermatid development (Fig. 1B,D; Ac).During the round spermatid stage, the surround-ing cytoplasm has abundant cellular machinerysuch as the Golgi apparatus (Fig. 1B; black arrow),rough ER (Fig. 1B; white arrow), mitochondria(Fig. 1B,D; black arrowheads), and lipid inclusions(Fig. 1A; *). The centrioles migrate to the oppositeside of the nucleus away from the developing acro-some complex and start the formation of the flagel-lum (Fig. 1C). By the midround spermatid stage,

Fig. 1. S. variabilis, round spermatids in the early stages of spermiogenesis. A: Early acrosomal vesicle formation. The acrosomalvesicle (Ac) has made little contact with the nuclear surface (Nu). Lipid inclusion; *. Bar 5 1 mm. B: The acrosomal vesicle (Ac)increases in size at the apex of the nucleus (Nu) and a prominent Golgi apparatus (black arrow) can be seen close to the developingacrosome. However, rough ER (white arrow) and a mitochondrium (black arrowhead) are between the Golgi and the developing vesi-cle, this structure has been termed the Mitochondrian/ER complex. Transport vesicles, white arrowhead; Bar 5 0.5 mm. C: Earlydevelopment of the proximal centriole (Pc), which becomes situated at the basal portion of the nucleus (Nu), and the distal centriole(Dc) radiates from the proximal centriole at a 90� angle. Pericentriolar material (P) accumulates near the proximal centriole. Mito-chondrium, black arrowhead; endoplasmic reticula, ER. Bar 5 1 mm. D: The midacrosomal vesicle formation stage with a visible acro-somal granule (white arrowhead) within a large acrosomal vesicle (Ac) that has flattened most of the apex of the nucleus (Nu). Notethe large shallow nuclear indentation between the acrosome and the nucleus. Mitochondria, black arrowhead. Bar 5 1 mm.

260 K.M. GRIBBINS ET AL.


the proximal centriole (Fig. 1C; Pc) is enclosedwithin a nuclear fossa and associated with the dis-tal centriole (Fig. 1C; Dc), which forms the begin-ning of the principal/mid piece. Pericentriolarmaterial also begins to accumulate around thedeveloping centrioles (Fig. 1C; P).

Once the acrosomal vesicle attains its maximumsize, it completely collapses covering the apical sur-face of the nucleus (Fig. 2A–C; Ac). The subacroso-mal space, a conspicuous area separating the innermembrane of the acrosomal vesicle from thenuclear membrane, develops and accumulates agranular protein layer (Fig. 2B,C; Sa). During thelate round spermatid stage, the nuclear materialbegins to condense into filaments (Fig. 2C,D; Nu).As the acrosomal vesicle (Fig. 2B,C; Ac) envelopsthe apex of the nucleus, a compact dark stainingaccumulation, the subacrosomal granule (Fig. 2B;black arrow), forms in the middle of the subacroso-mal space just below the basally positioned acro-some granule (Fig. 2A,B; white arrowheads). Theacrosomal shoulders, or leading edges of the acro-some, start to round off the apical nucleus toward

the end of the round spermatid stage (Fig.2C; As).Caudally, the flagellum continues to develop as themid/principal piece (Fig. 2D; Pp) and elongatesaway from the distal centriole (Fig. 2D; Dc). Thetermination of the round spermatid stage includesthe initiation of nuclear elongation, which can beseen in Figure 2C,D.

During elongation, the spermatid nucleus ismore cylindrical in shape (Fig. 3A, inset). In earlyelongation, the spermatid nucleus migrates to theapical cytoplasm causing most of the germ cell cyto-plasm shifts posteriorly (Fig. 3A, Inset). The endresult of this shift is that no cytoplasm is locatedbetween the outer acrosomal membrane and thecellular membrane of the elongating spermatid(Fig. 3A; black arrow). The acrosomal lucent ridge(Fig. 3A; Ar) is visible within the subacrosomalspace and multiple Sertoli cell membrane processes(Fig. 3C, Ca) border the developing germ cell’s acro-some complex and this membrane cap helps anchorthe spermatid within the seminiferous epithelium.The acrosomal vesicle shoulders extend laterallyalong either side of the apical nucleus, which starts

Fig. 2. S. variabilis, late stages of the round spermatids. A and B: The acrosomal vesicle (Ac) has collapsed and rests on the flat-ten nuclear apex (Nu). The acrosomal granule (white arrowhead) sits in a basal depression with the acrosomal vesicle. The subacro-somal granule (Bp) forms juxtapositioned to the acrosomal granule; however, it is located in the subacrosomal space (Sa).Mitochondria, black arrowhead; endoplasmic reticula, ER. Bars 51 mm. C and D: The start of chromatin condensation occurs aselongation begins at the termination of the round spermatid stage. The acrosomal vesicle (Ac) with its acrosomal granule (whitearrowhead), and the acrosomal shoulders (As) round off the nucleus (Nu) at the end of early nuclear elongation. During the initialelongation and condensation phases, the acrosomal vesicle finally penetrates deeply into the nucleus proper (inset, Bar 5 200 nm).The centrioles (Pc, Dc) are anchored in the nuclear fossa (Nf) and the principal piece (Pp) is elongating away from the Dc. Mitochon-dria, black arrowhead; ER, white arrows. Bars in C 5 2 mm and in D 5 2 mm.



thinning the apical nucleus into what will be therostrum in later elongating spermatids (Fig. 3C;Ac, As). The nuclear contents (Fig. 3B,C) becomemore electron dense during this phase of spermio-genesis as the chromatin continues to condenseinto thicker and longer filaments (Fig. 3B; Nu).During early elongation, circum-cylindrical (Fig.3D, Ma) microtubules of the manchette are presentand the condensing chromatin filaments (Fig. 3D;Nu) in cross section show little to no spiraling. Theproximal (Fig. 3B, inset; white arrow) and distal(Fig. 3B, inset; white arrowhead) centrioles stilloccupy a well-developed nuclear fossa (Fig. 3B,inset; black arrowhead) on the caudal end of thenucleus, which anchors the elongating flagellum tothe nuclear body.

The chromatin of the nucleus (Fig. 4A, Nu) fin-ishes condensing during late elongation and thenucleus becomes homogeneous in electron density.Toward the end of condensation, the longitudinalmicrotubules of the manchette develop (Fig. 4A;

black arrowhead), however in cross section they arestill often absent or underdeveloped. At the climaxof elongation, the nucleus develops a slight curva-ture and the germ cell cytoplasm is moved into aninner radial area within the concavity of thenucleus. Caudally, the distal centriole (Fig. 4B; Dc)continues to elongate and mitochondria (Fig. 4C,D;Mi) and dense bodies (Fig 4C,D; Db) now surroundthe distal centriole and the beginning of the fibroussheath (Fig. 4C,D; Fs) in the developing midpiece. Aprominent annulus (Fig. 4B–D; An) separates thenow developing midpiece from the distal principalpiece (Pp) of the flagellum. The acrosomal complex(Fig. 4A) is completely enveloped by multiple Sertolicell processes (Fig. 4E; black arrowhead). The acro-somal vesicle (Fig. 4E; Ac) surrounds the subacroso-mal space (Fig. 4E; Sa) that has an epinuclearlucent zone (Fig. 4E; black arrow) and a very dis-tinct subacrosomal lucent ridge (Fig. 4E; Ar). Theepinuclear lucent zone is capped with the darkstaining remains of the subacrosomal granule

Fig. 3. S. variabilis, chromosome condensation continues in elongating spermatids. A: The acrosomal vesicle (Ac) and shoulders(As) have enveloped the rounded nuclear apex (Nu). The acrosome has been pushed against the cell membrane (black arrow) andcytoplasm of the spermatid has been shifted posteriorly (Inset, Bar 5 1 mm). Mitochondria (black arrowhead, inset) and other organ-elles are relocated closer the developing flagellum (white arrowhead, inset). Subacrosomal space, Sa; Subacrosomal lucent ridge, Ar;acrosomal granule, white arrowhead. Bar 5 1 mm. B: The chromatin of the nucleus (Nu) condenses in a filamentous manner and themanchette microtubules (Ma) are arranged in an organized fashion around the nucleus. Inset: Bar 5 200 nm. The proximal (whitearrow) and distal (white arrowhead) centrioles reside in a deep nuclear fossa (black arrowhead) caudally. Subacrosomal space, Sa;acrosomal vesicle, Ac; Acrosomal shoulders, As; mitochondria, black arrowhead. Bar 5 1 mm. C: The acrosomal shoulders (As) of theacrosomal vesicle (Ac) continue to envelope the apex of the nucleus (Nu) and multiple Sertoli cell membrane layers (Ca) cap the acro-some complex. Subacrosomal space, Sa; Manchette, Ma; cell membrane, Cm. Bar 5 0.5 mm. D: The filamentous chromatin in thenucleus (Nu) shows no spiraling in cross section and the circum-cylindrical microtubules (Ma) of the manchette are well-developedduring this stage. Bar 5 0.5 mm.



(Fig. 4E; *). The acrosomal vesicle shoulders havenarrowed the nuclear apex into a rostrum (Fig. 4E;Nr) that is surrounded by paracrystalline subacro-somal proteins (Fig. 4E; Sa). The basal plate of theperforatorium is located just proximal to the suba-crosomal granule and is within the acrosomal vesi-cle (Fig. 4E; white arrowhead).

Upon completing spermiogenesis, the matureelongating spermatid (Fig. 5A) is anchored withinthe seminiferous epithelium and awaits spermia-tion. The acrosomal complex, which is surroundedby two Sertoli cells’ cytoplasmic processes (Fig. 5B;1 and 2), has an acrosomal vesicle (Fig. 5B–E; Ac)resting on top of a subacrosomal space (Fig. 5E;Sa) that surrounds a narrow nuclear rostrum (Fig.5E; Nr). A basal plate (Fig. 5A; white arrow) sepa-rates the perforatorium (Fig. 5C, black arrowhead)

at the base of the acrosomal vesicle from the epi-nucelar lucent zone (Fig. 5D; black arrow) withinthe subacrosomal space. The subacrosomal para-crystalline proteins are stratified or separated intoan inner and outer layer of proteins via a veryprominent subacrosomal lucent ridge (Fig. 5D,E;Ar). The nuclear body is surrounded by a conspicu-ous manchette (Fig. 5F, black arrow) that isalmost completely made up of circum-cylindricalmicrotubules. The microtubule triplets (Fig. 5G;white arrow) of the distal centriole are each sur-rounded by an enlarged peripheral fiber (Fig. 5G;white arrowhead). The peripheral fibers surround-ing microtubule doublets 3 and 8 are the onlyfibers to continue into the principal (Fig. 5I; whitearrowhead) and endpiece (Fig. 5J; white arrow-head). Dense bodies (Fig. 5G,H; Db) and

Fig. 4. S. variabilis, late elongating spermatid which has reached its maximum nuclear length. A: Sagittal section of the nucleus(Nu) and acrosome (Ar) showing the manchette (black arrowhead) and the nuclear fossa (Nf). Bar 5 2 mm. B: Caudal nucleus (Nu)with Proximal (Pc) and Distal (Dc) centrioles and visible annulus (An) of the very early midpiece. Bar 5 1 mm. C: Oblique section ofthe midpiece later in development with mitochondria (Mi) and dense bodies (Db) associated with the disal centriole (Dc) and thegrowing principal piece (Pp). The annulus (An) marks the termination site of the midpiece. Nuclear fossa, white arrowhead; proximalcentriole, Pc; cell membrane, black arrowhead. Bar 5 1 mm. D: Midpiece in sagittal section late in development. The oblong mitochon-dria (Mi) and dense bodies (Db) are associated with the fibrous sheath of the midpiece (Fs), which begins at mitochondria tier 2. Theannulus (An) appears as a perfect ring in Sagittal section. Nucleus, Nu. Bar 5 2 mm. E: High magnification of the layers of the acro-some complex. Two Sertoli cells (1,2) form a cellular membrane cap (black arrowhead) that covers the acrosome vesicle (Ac), whichsits on a subacrosome cone (Sa) that is divided in layers by the acrosomal lucent ridge (Ar). The nuclear rostrum (Nr) penetrates thesubacrosome cone and the epinuclear lucent zone (black arrow) sits just cephalic to the rostrum and is capped by the remnants ofthe subacrosome granule (*). Basal perforatorial plate, white arrowhead. Bar 5 0.5 mm.



mitochondria (Fig. 5H; Mi) surround the distalcentriole and beginning part of the axoneme cov-ered by the fibrous sheath within the midpiece ofthe developing elongating spermatid (Fig. 5G,H).The fibrous sheath in S. variabilis starts at mito-chondria tier two within the midpiece (Fig. 4D).Typically there are 3–5 dense bodies and 4–6 mito-chondria per tier within the midpiece. The princi-pal piece begins right after the annulus of themidpiece and its axoneme (Fig. 5H; Ax) is sur-rounded by a thick fibrous sheath (Fig. 5H,I; Fs).The mitochondria are fusiform in proper sagittalsection (Fig. 4D) and circular (Fig. 5G,H) in crosssection and the midpiece contains between 4 and 6tiers of mitochondria in sagittal section (Fig. 4C,D)during development. The principal piece (Fig. 5I)continues past the annulus for 40–50 mm and theflagellum terminates in the endpiece (Fig. 5J),which lacks the fibrous sheath (Fig. 5I; Fs) but

retains the 9 1 2 microtubule arrangement of theaxoneme (Fig. 5J; white arrow).

DISCUSSION

The early round spermatid stages of spermio-genesis of S. variabilis are similar to what hasbeen described in all squamates to date except fortwo notable ultrastructural differences. First,there is a ring of ER surrounding a mitochondriaconsistently located between the Golgi apparatusand the developing acrosomal vesicle during thefirst two steps of early spermiogenesis; we termthis new complex the mitochondrial/ER complex.Though other species seem to have either mito-chondria or ER near the Golgi (e.g., Ferreira andDolder, 2002), none have described the ER sur-rounding a mitochondria and the specific, consist-ent location of these two organelles between the

Fig. 5. S. variabilis, the final step of elongation and condensation during spermiogenesis. A: Sagittal section of the mature sperma-tid showing the acrosome, basal perforatorial plate (white arrow), nucleus, and flagellum. Lettered perpendicular lines correspond tocross sections B-J. Bar 5 1 mm. B: Cross section through the apical acrosome vesicle (Ac) and its Sertoli membrane (1,2) cap. C: Crosssection through the acrosome vesicle (Ac) and the perforatorium (black arrowhead). D: Cross section through the epinuclear lucentzone (black arrow) of the subacrosomal cone (Sa). Subacrosomal lucent ridge, Ar; Acrosomal vesicle, Ac. E: Cross section through thenuclear rostrum (Nr). Subacrosomal space, (Sa); Subacrosomal lucent ridge, Ar; Acrosomal vesicle, Ac. F: Cross section through thenucleus proper (Nu). Manchette microtubules, black arrow. G: Cross section through the midpiece and distal centriole. Mitochondria,black arrowhead; dense body, Db; microtubule triplet, white arrow; peripheral fiber, white arrowhead. H: Cross section through thedistal midpiece. Mitochondria, Mi; dense body, Db; axoneme, Ax; fibrous sheath, Fs. I: Cross section through principal piece. Fibroussheath, Fs; axoneme, white arrow; cell membrane, Cm; enlarged peripheral fiber 8, white arrowhead. J: Cross section through theendpiece. Cell membrane, Cm; peripheral fiber number 3, white arrowhead; axoneme, white arrow. Bars 5 1 mm.



Golgi and the developing acrosomal vesicle. Thelocation and arrangement of these two organellesis not observed in S. bicanthalis (Rheubert et al.,2012), and thus may be a synapomorphy for thevariabilis group (Leach�e, 2010) within the Scelopo-rus genus or more unlikely an autopomorphy forS. variabilis. The function of this complex isunknown; however, we propose that it simplyexpedites delivery of processed ER proteins andenergy (Adenosine triphosphate) delivery to theGolgi during this highly active time of acrosomedevelopment. Ferreira and Dolder (2002), duringtheir study of Iguana iguana, note that the roughER produces transport vesicles that play a directrole in acrosomal vesicle formation. In S. variabi-lis, even with the ER’s close association no vesicu-lar transport directly to the acrosomal vesicle wasobserved. The only transport vesicles in this spinylizard that were involved with acrosomal vesicledevelopment originated from Golgi cisternae.

The second major difference in S. variabilisobserved during acrosome development was thevery large and shallow apical nuclear indentationthat houses the acrosomal vesicle during earlyspermiogenesis. Typically as the acrosomal vesiclegrows, it produces a much deeper nuclear indenta-tion in early to mid acrosomal development (seeGribbins, 2011) within Reptilia, as is true for S.bicanthalis. The main reason for this differenceseems to be related to the longer duration of timethat the acrosomal vesicle grows in size before itmakes substantial contact with the S. variabilisspermatid nucleus. Thus, the apical nuclear inden-tation appears much larger once contact is madein order to accommodate the larger-sized vesicle.

At the climax of acrosomal vesicle growth, theacrosomal granule is located in a basal position inS. variabilis, in S. bicanthalis, and in most othersquamates studied to date (Gribbins, 2011). Paral-lel to the formation of the acrosomal vesicle, a sub-acrosomal granule originates juxtapositioned tothe acrosomal granule; however, the subacrosomalgranule is located outside of the acrosomal vesiclewithin the subacrosomal space within S. variabi-lis. There has been much confusion on what thesetwo granules give rise to in the mature spermato-zoa. In early studies of spermiogenesis withinbirds and lizards, the perforatorium was thoughtto arise from the subacrosomal granule (Hum-phreys, 1975; Del Conte, 1976) or the acrosomalgranule (Adelina et al., 2006). More recently inBarisia imbricata (Gribbins et al., 2013) and in S.bicanthalis (Rheubert et al., 2012) the same gran-ule appears to develop either during the lateround or early elongation phases of spermiogene-sis. However, these two studies call the granulewithin the subascrosomal space the developingbasal plate of the perforatorium. In both previousstudies, it seemed hard to discern whether thisgranule arose from the acrosomal granule or was

a new protein accumulation within the subacroso-mal space (see Fig. 2C in B. imbricata, Gribbinset al., 2013). At least in S. variabilis it is apparentthat this granule arises independently of the acro-somal granule and most likely gives rise to theepinuclear lucent zone based on its close associa-tion with this zone during midelongation (see Fig.4E,*). Thus, the authors agree with Ferreira et al.(2006) in that the perforatorium most likely origi-nates from the acrosomal granule and the epinu-clear lucent zone is hypothesized to arise from thesubascrosomal granule based on location and spa-tial organization within the acrosome complex.

During nuclear elongation and condensation inS. variabilis, chromatin condenses into filamentsalmost immediately. This is similar to B. imbricata(Gribbins et al., 2013), but different from manyother lizards, including S. bicanthalis (Rheubertet al., 2012) that typically have granular and thenlater filamentous condensation (Clark, 1967; Fer-reira and Dolder, 2002; Vieira et al., 2004; Vieiraet al., 2005; Gribbins, 2011; Rheubert et al.,2011a). Also, S. variabilis differs from S. bicantha-lis and many other lizards (see Gribbins, 2011) inthat there is little spiraling of the chromatin dur-ing nuclear condensation. Historically, elongationof the nucleus has been thought to be aided by themicrotubules of the manchette during late sper-miogenesis, which run the length of the developingspermatid. A manchette has been observed inevery squamate studied to date, except Anolis line-atopus (Rheubert et al., 2010c). Interestingly, S.variabilis has well-developed circum-cylindricalmicrotubules and under developed longitudinalmicrotubules within its manchette. This is directlyopposite of what is seen in S. bicanthalis, whichhas a very well developed longitudinal array ofmicrotubules (Rheubert et al., 2012). H€ofling andLandim (1978) and Adelina et al. (2006) suggestthat non- histonic nuclear proteins, nuclear waterloss, and/or Sertoli cell mediation may also playkeys roles in nuclear elongation, which theauthors agree with as components of or the wholemanchette is absent in some lizards but nuclearelongation still appears normal.

The mature S. variabilis spermatid prior tospermiation resembles the Phrynosomatidaemature spermatozoa described by Scheltinga et al.(2000). The only notable ultrastructural variationsare the absence of a nuclear lacuna and a moreround acrosomal complex in S. variabilis. S. bican-thalis (Rheubert et al., 2012), and Sceloporus con-sobrinus (Rheubert, personal communication) bothhave nuclear lacuna during late elongation of sper-miogenesis similar to that described for the phry-nosomatid spermatozoa, but S. variabilis lackslacunae during late elongation. The acrosomalcomplex in S. variabilis is similar to thatdescribed for other lizards. The acrosome vesicleoverlies the subacrosomal space or cone, which



Fig. 6. Comparison of ultrastructural character differences observed during spermiogenesis between S. variabilis and S. bicantha-lis. There are six main character differences seen during spermiogenesis between these two species within the same genus, Scelopo-rus. These include: 1) presence (white arrow)/absence (*) of a mitochondrial/ER complex near Golgi complex (white arrowhead), 2)deep or shallow nuclear depression accommodating the acrosome, 3) filamentous versus granular condensation of chromatin, 4) spira-ling (*) or lack of spiraling during condensation of chromatin, 5) presence (black arrow) or absence of the longitudinal manchettemicrotubules, and 6) presence or absence of nuclear lacunae. Bar 5 1 mm. [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]



has been described as a pleisomorphic condition inreptiles (Oliver et al., 1996; Teixeira et al., 1999;Scheltinga et al., 2000). The paracrystalline pro-tein that makes up of the subacrosome space orcone is considered a synapomorphy for squamates,as may be the subacrosomal stratification causedby the subacrosomal lucent ridge. There is also aprominent basal plate, extranuclear perforatorium,and epinuclear lucent zone within the S. variabilismature spermatids. The epinuclear lucent zone isconsidered by most to be another synapomorphy ofSquamata (Jamieson et al., 1995; Oliver et al.,1996; Teixeira et al., 1999).

The observed nuclear body, nuclear rostrum,nuclear shoulders, basal nuclear fossa, proximaland distal centriole structure and location withinS. variabilis spermatids all appear to be consistentwith what has been described for most of Squa-mata. The principal source of variation in the flag-ellum of Squamata typically occurs in themidpiece (Jamieson et al., 1995; Scheltinga et al.,2000). The mitochondrial numbers and dense bodyinterspersed in the incomplete rings or tiers ofmitochondria found in the midpiece of S. variabilismature spermatids is similar to that described forPhyrnosomatid (Scheltinga et al., 2000) and Igua-nid lizard (Furieri, 1970; Oliver et al., 1996) sper-matozoa, and the mature spermatid of S.bicanthalis (Rheubert et al., 2012). The S. variabi-lis midpiece also has the origin of the fibroussheath starting at mitochondrial tier two, which isconsistent among all iguanids studied to date (Fer-reira and Dolder, 2003; Scheltinga et al., 2000;Vieiria et al., 2004; Rheubert et al., 2012). Theaxoneme of the flagellum with its borderingfibrous sheath continues past the midpiece in S.variabilis as the principal piece. The peripheralfibers 3 and 8 do continue into the principal piecebut are not as conspicuous as some squamates(Jamieson et al., 1995; Oliver et al., 1996; Teixeiraet al., 1999). The termination of the developingelongating spermatid is the endpiece, like all lepi-dosaurians studied to date (Jamieson et al., 1995;Jamieson, 1999), which has no associated fibroussheath but retains the 9 1 2 microtubule arrange-ment of the flagellar axoneme.

Largely, spermiogenesis in S. variabilis is com-parable to that of other squamates and mostamniotes. However, there are a few distinctiveultrastructural features to their spermatids thatmay either be autapomorphies for this species orpossibly synapomorphies within the Sceloporusclade. These include the mitochondrial/ER complexduring acrosome development and the large shal-low nuclear indentation accommodating the acro-some vesicle. Sceloporus is a diverse taxon oflizards, which contains over 901 species (Leach�e,2010). There has been controversy in the past onthe monophyly of this clade (Frost and Etheridge,1989). However, recent data support the phyloge-

netics of Sceloporus as a monophyletic clade andrecent nuclear and mitochondrial DNA studies(Leach�e, 2010) suggest that S. variabilis andS. bicanthalis are distintly related within the Sce-loporus clade. This study is the first to directlycompare spermiogenesis within two lizards fromthe same genus. The six spermiogenic characterdistinctions (see Fig. 6) between S. variabilis andS. bicanthalis spermatids preliminarily supportsthe distant kinship between these two spiny lizardproposed by Leach�e (2010). Although the sperma-tid data presented here suggests that spermiogen-esis may be a good ultrastructural model tocompare character differences phylogenetically,further research within clades such as Sceloporusmust be completed between sister and more dis-tantly related species to test the robustness of thehypothesis that spermiogenic morphology is spe-cies specific.

ACKNOWLEDGMENT

We thank the community of the Ejido AdolfoL�opez Mateos for facilities during fieldwork.

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