Reproductive morphology of the male Tuatara, …Reproductive morphology of the male Tuatara, Sphenodon punctatus Justin L. Rheubert,1 Alison Cree,2 Matthew Downes2 and David M. Sever3

Reproductive morphology of themale Tuatara, Sphenodon

punctatus

Justin L. Rheubert,1 Alison Cree,2 Matthew Downes2 and DavidM. Sever3

1Department of Biology, Saint Louis Uni-

versity, 3507 Laclede Ave., St. Louis,

MO, 63103, USA; 2Department of

Zoology, University of Otago, 340 Great

King St., Dunedin, 9016, New Zealand;3Department of Biological Sciences, South-

eastern Louisiana University, SLU 10736,

Hammond, LA, 70401, USA

Keywords:

Tuatara, reproduction, histology, testicular

ducts, sperm

Accepted for publication:

31May 2012

Abstract

Rheubert, J.L., Cree, A., Downes, M. and Sever, D.M. 2013. Reproductive

morphology of the male Tuatara, Sphenodon punctatus. —Acta Zoologica (Stock-

holm) 94: 454–461.

Over the past decade, studies on reproductive morphology in the Squamata

(snakes and lizards) have expanded tremendously. With the accumulation of

these studies and revisions of the terminology based on structural similarities

and differences, it is imperative to review the work on tuataras to determine

whether the structural organization fits the revised terminology of vertebrates.

We investigated the morphology of the male reproductive system in the Tuatara,

Sphenodon punctatus (Rhynchocephalia), the sister taxon to the Squamata. Previ-

ous studies on the Tuatara used a nomenclature for the testicular ducts different

from the current terminology for amniotes. The reproductive system in the Tua-

tara is consistent with reports in the Squamata. Two rete testis tubules exit the

testis within a connective tissue sheath similar to that shown in other squamate

species and the protherian Echidna. Each rete testis divides into multiple ductuli

efferentes that fuse with the epididymis. The epididymis transitions into the duc-

tus deferens where the sperm become more concentrated into spherical bundles.

The ductus deferens enters the cloacal urodeum separately from the ureter. An

ampulla ureter or ampulla urogenital papilla was not observed, which differs

from previous studies of lepidosaurians. Furthermore, a sexual segment of the

kidney (SSK) was not observed, consistent with previous studies on the Tuatara.

Justin L. Rheubert, Department of Biology, Saint Louis University, 3507 Lacl-

ede Ave., St. Louis, MO 63103, USA. E-mail: [email protected]

Introduction

The Tuatara, Sphenodon punctatus (Gray), is the only extant

member of the reptilian order Sphenodonta. This species is

limited to a few islands off the coast of New Zealand, although

attempts are being made to re-establish populations on the

mainland (Besson et al. 2012). Morphological (including

some reproductive characters) and molecular data indicate a

sister relationship between Sphenodon and Squamata, the liz-

ards and snakes (Healy and Jamieson 1994; Evans 2003; Rest

et al. 2003). The minimum divergence time between Rhyn-

chocephalia and Squamata is estimated at 230 million years

(Rest et al. 2003).

The first description regarding morphology of the repro-

ductive tract of male S. punctatus was given by Gunther

(1867). However, the observations made by Gunther (1867)

were based solely on gross morphology. The first histological

study of the testis and its ducts, based upon a single specimen,

was conducted by Osawa (1897). Subsequently, Hogben

(1921) described spermatogenesis, Gabe and Saint Girons

(1964) more thoroughly described the histology of the uro-

genital system, and Saint Girons and Newman (1987) and

Cree et al. (1992) described the histology of the spermato-

genic cycle of S. punctatus. These studies were limited in the

number of specimens examined because of the highly pro-

tected status of this species.

Gabe and Saint Girons (1964) used the nomenclature

developed by Volsøe (1944) for snakes, in which the seminif-

erous tubules pass sperm sequentially into ‘ductuli efferentes’

and ‘ductuli epididymides’ prior to the ductus epididymis.

Sever (2010) demonstrated that in snakes, the ductuli efferen-

tes of Volsøe (1944) should be named the rete testis and that

his ductuli epididymides represent the ductuli efferentes as

recognized in other amniotes, that is, lizards (Akbarsha et al.

© 2012 The Authors

Acta Zoologica© 2012 The Royal Swedish Academy of Sciences454

Acta Zoologica (Stockholm) 94: 454–461 (October 2013) doi: 10.1111/j.1463-6395.2012.00574.x

2007), turtles (Holmes and Gist 2004), crocodilians (Guerre-

ro et al. 2004), birds (Aire 2007), and mammals (Jones

2001). This synonymizing of the nomenclature was developed

using histological techniques and noting that the various ducts

were similar across vertebrate taxa at the cellular level. The

rete testis is made up of a simple squamous epithelium that is

typically non-ciliated (Rheubert et al. 2010; Sever and Free-

born 2012). The ductuli efferentes are the smallest of the tes-

ticular ducts (Sever 2010) and have a heavily ciliated simple

cuboidal epithelium. The epididymis is the largest duct adja-

cent to the testis and has a pseudostratified secretory colum-

nar epithelium (Jones 2001). The epididymis transitions into

the ductus deferens, which also possesses a pseudostratified

columnar epithelium that is less secretory. In snakes, the duc-

tus deferens may have a distal ampulla ductus deferentis and

join with the ureter to form an ampulla–ureter complex

(Trauth and Sever 2011). An ampulla ductus deferentis is also

known in lizards (Akbarsha and Meeran 1995), but the anat-

omy of sperm duct junctions with the ureter has not been

studied in lizards.

The first objective of this study is to examine the testicular

ducts of male S. punctatus to determine whether they are

unique among amniotes and deserving of a different nomen-

clature or whether they appear structurally and functionally

homologous to those ducts in other amniotes. The second

objective is to examine the kidneys of S. punctatus for the pres-

ence of a sexual segment of the kidney (SSK), a secondary sex

structure otherwise unique to male Squamata (Aldridge et al.

2011; Rheubert et al. 2011). Gabe and Saint Girons (1964)

could find no evidence for a SSK, but indicated this may be

due to the inactive reproductive condition of their specimens.

Saint Girons and Newman (1987) revisited this with five

specimens and once again found no SSK.

Materials andMethods

Left reproductive tracts were taken from three adult male

Tuatara at the Otago Museum, Dunedin, New Zealand. The

specimens had been fixed in 10% formalin and stored in 70%

ethanol. No collection data were available for the specimens

utilized in this study. Tissue samples were dehydrated with a

graded series of ethanol solutions (95%, 100% 9 2) and

cleared with two changes of xylene for 20 min each. Tissues

were then placed in melted paraffin at 60 °C under vacuum

overnight before being embedded in fresh paraffin using a

Leica EG 1120 wax embedding station (Leica Microsystems,

Wetzlar, Germany). Tissue blocks were sectioned to 10 lmon a Leica RM 2125RT microtome (Leica Microsystems),

placed on slides flooded with deionized water, and dried on a

slide warmer for 1 h. Alternate slides were stained with Gills

II hematoxylin and eosin, Coomassie brilliant blue R-250,

and periodic acid Schiff’s reagent with an alcian blue at pH

2.5 counterstain for general cytology, proteins, and carbohy-

drates and carboxylated glycosaminoglycans, respectively

(Hayat 1993). Slides were viewed using an Olympus BX 51

(Olympus Global, Center Valley, PA, USA) compound

microscope and photographed using an Olympus DP 25 digi-

tal camera (Olympus Global). Select photographs were com-

piled into composite micrographs using Adobe Photoshop

CS5 (Adobe Systems, San Jose, CA, USA). Select serial sec-

tions were photographed using a Canon EOS 50D digital

camera (Canon USA, Lake Success, NY, USA) with a 100-

mm macro lens for complete overviews. Slides were placed in

the OtagoMuseum where the specimens were obtained.

Results

The testes are suspended by the mesorchium into the pleuro-

peritoneal coelom posterior to the liver. Each testis is elon-

gated along its anteroposterior axis and flattened along its

dorsoventral axis, although overall shapes are highly variable

(A. Cree, pers. obs.). The testes are composed of seminiferous

tubules (Fig. 1A,B, St) that consist of a germinal epithelium

where germ cells develop; however, the germ cell development

strategy was unable to be determined owing to the inactive

state of the testes in the specimens used for this study. The

superficial covering of the testes is the visceral pleuroperito-

neum but posteriorly the ductus deferens, kidney, and ureter

are retroperitoneal. Deep to the serous covering of the testis is

a connective tissue sheath (Fig. 1A, Ct) homologous to the

tunica albuginea.

The seminiferous tubules, which react positively to Coo-

massie blue for proteins and react weakly to alcian blue at pH

2.5 for carboxylated glycosaminoglycans, merge into the rete

testis (Fig. 1, Rt) tubules intratesticularly prior to the rete tes-

tis exiting the testis proper at both the anterior and posterior

axes (results not shown), resulting in two main rete testis

tubules. The rete testis tubules are contained within a connec-

tive tissue sheath that is continuous with the tunica albuginea

(Fig. 1A,C,D, Ct) that Siegel et al. (2009) termed the epidid-

ymal sheath. The rete testis is composed of a simple squa-

mous epithelium (Fig. 1C, Rt) with basophilic nuclei,

eosinophilic cytoplasm, and positive reactions to protein stain-

ing. These tubules (Fig. 1D, Rt) divide before merging with

the ductuli efferentes (Fig. 1D, De).

The transition from the rete testis (Fig. 2A, Rt) to the

ductuli efferentes (Fig. 2A, De) is distinct as the epithelium

changes from simple squamous to simple cuboidal. The

nuclei are basophilic, but the cytoplasm is eosinophilic, which

reacts positively to protein staining, and multiple cilia

(Fig. 2A, Ci) can be seen protruding into the lumina of the

ductuli efferentes. The ductuli efferentes remain within the

connective tissue sheath (Fig. 2B, Ct) and extend posterior

alongside the epididymis (Fig. 2C, Ep). The ductuli efferen-

tes which can even be seen at the level of the kidney (Fig. 2D,

K), and ductus deferens (Fig. 2D, Dd), makes them the

longest (relatively) ductuli efferentes of any vertebrate studied

to date.

Although the ductuli efferentes can be seen at the level of

the ductus deferens, they appear to only merge with the

© 2012 The Authors

Acta Zoologica© 2012 The Royal Swedish Academy of Sciences 455

Acta Zoologica (Stockholm) 94: 454–461 (October 2013) Rheubert et al. � Reproductive morphology of S. punctatus

epididymis (Fig. 3A, Ep). The epididymis is encased in a thin

layer of smooth muscle (Fig. 3B,C, Sm) and consists of what

we believe is a pseudostratified epithelium, although the

condition of the specimens did not allow the observation of

basal cells. The epithelium reacts positively to Coomassie blue

for proteins but negatively to alcian blue for carboxylated

A B

C D

Fig. 1—Overview of the testis and rete testis tube.—A. The seminiferous tubule (St) of the testis merges with a rete testis tubule (Rt). The testis

and rete tubule are encapsulated within a connective tissue (Ct) sheath.—B.The rete testis (Rt) extends laterally from the testis. Seminiferous

tubule (St).—C. The rete testis (Rt) is composed of simple squamous epithelium and encapsulated in the connective tissue (Ct) sheath.—D.

The rete testis (Rt) divides into multiple ducts before merging with the ductuli efferentes (De). Connective tissue (Ct).

A B

C D

Fig. 2—Overview of the ductuli efferentes.—A. The rete testis tubule (Rt) merges with the ductuli efferentes (De), and the epithelium changes

from simple squamous to simple cuboidal with multiple cilia (Ci) extending into the lumen of the ductuli efferentes.—B. The ductuli efferentes

(De) remain within the connective tissue (Ct) sheath.—C. The ductuli efferentes (De) extend posterior in juxtaposition to the epididymis (Ep).

—D. The ductuli efferentes (De) extend posterior to the level of the kidney (K) near the ductus deferens (Dd).

© 2012 The Authors


Reproductive morphology of S. punctatus � Rheubert et al. Acta Zoologica (Stockholm) 94: 454–461 (October 2013)

glycosaminoglycans. Superficial to the smooth muscle is a thin

layer of connective tissue (Fig. 3B,C, Ct) that appears to be

continuous with the visceral pleuroperitoneum. Sperm can be

found within the epididymis, although the testes are inactive

and no sperm are found within the rete testis and ductuli effer-

entes, suggesting the epididymis as a site for sperm storage. As

sperm progress posteriorly within the epididymis, they

become concentrated into spherical bundles (Fig. 3C, Sp)

with their heads oriented toward the center of the bundle

(Fig. 3D, Spz).

At the level of the kidney (Fig. 4A,K), the epididymis tran-

sitions into the ductus deferens (Fig. 2A, Dd). Within the

ductus deferens, sperm are tightly packed, and minimal free-

floating sperm are noticed. The ureter (Fig. 4A,B, Ur) of the

kidney is located lateral to the kidney opposite of the ductus

deferens (Fig. 4A, Dd), which runs along the medial side of

the kidney. Like in other vertebrates, the visceral layer of Bow-

man’s capsule (Fig. 4C, Bc) consists of flattened cells (podo-

cytes) investing the glomerulus (Fig. 4C, Gl). The proximal

convoluted tubule (Fig. 4C,D, Pct) extends from Bowman’s

capsule and consists of a simple cuboidal epithelium with cen-

tralized nuclei. The presence of a brush border was difficult to

ascertain owing to the poor fixation of the specimens. The

proximal convoluted tubule reacts positively to alcian blue at

pH 2.5 for carboxylated glycosaminoglycans. The proximal

convoluted tubule transitions into the distal convoluted tubule

(Fig. 4C, Dct), which consists of a simple columnar epithe-

lium with basal nuclei. The distal convoluted tubule drains

into the collecting duct (Fig. 4B, Cd), which consists of a

cuboidal to low columnar epithelium and contains debris

within the lumen. A sexual segment of the kidney is not

evident, lending support to the hypothesis of the absence of

the SSK in tuataras (see Discussion). All kidney tubules stain

positively with Coomassie blue for proteins and periodic acid

for neutral carbohydrates. But only the proximal convoluted

tubules (Fig. 4D, Pct) show a positive reaction to alcian blue

for carboxylated glycosaminoglycans (Fig. 4D).

The collecting duct (Fig. 5A, Cd) extends posteriorly

(Fig. 5A,B,K) to transition into the ureter outside of the kid-

ney (Fig. 5B, Ur). The ureter (Fig. 5B, Ur) opens into the

urodeum (Fig. 5C,D,E, Ud), and the ductus deferens

(Fig. 5C,D,E, Dd) remains dorsal to the urodeum. The kid-

ney (Fig. 5E,K) extends posteriorly to lie lateral to the urode-

um (Fig. 5E, Ud) as the ductus deferens (Fig. 5F, Dd) opens

into the urodeum (Fig. 5F, Ud) via an orifice in the roof of

the urodeum (Fig. 5F, Or). The presence of an ampulla duc-

tus deferentis, ampulla ureter, or ampulla urogenital papilla

(sensuTrauth and Sever 2011) was not observed.

Discussion

The epithelial structure of the testicular ducts resembles that

of other vertebrates studied to date and follows the synonymi-

zation of the nomenclature proposed by Sever (2010). The

rete testis exits the testis proper at two locations: anterior and

posterior. This overall structure resembles that of the echidna

A B

C D

Fig. 3—Overview of the epididymis.—A. The ductuli efferentes (De) merge with the epididymis (Ep), and all ducts remain within the connective

tissue (Ct) sheath.—B. The epididymis (Ep) is composed of a tall columnar epithelium, which appears pseudostratified. Connective tissue (Ct).

—C. Sperm bundles (Sp) can be found within the epididymis (Ep). Connective tissue (Ct), ductuli efferentes (De), smooth muscle (Sm).—D.

The sperm aggregate in a spherical fashion within the epididymis (Ep) with the heads facing the interior of the sphere (Spz).

© 2012 The Authors



(Djakiew and Jones 1981). The gross morphology shows that

two connective tissue sheaths, which the authors termed

gonadal ligaments, extend from the testis similar to that seen

in S. punctatus. Recent data suggest that in squamates, the

number of rete tubules exiting the testis is highly variable, and

no correlation has been observed between the number of rete

tubules and testicular size (Sever and Freeborn 2012). The

rete tubules originate intratesticularly, contrasting with the

observations made by Rheubert et al. (2010), in the gecko,

Hemidactylus turcicus. However, it appears that most squa-

mates have an intratesticular origin of the rete testis (J. L.

Rheubert, unpublished data).

The rete testis tubules are contained within an extension

of the connective tissue sheath covering the testis that (Siegel

et al. 2009) referred to as the epididymal sheath. Within the

sheath, the rete tubules divide into multiple ductuli efferentes,

which consist of a low cuboidal epithelium that is highly cili-

ated. This structure is consistent with observations made in

other amniotes (mammals, Ilio and Hess 1994; birds, Aire

2007; reptiles, Guerrero et al. 2004; Akbarsha et al. 2007;

and Rheubert et al. 2010). However, in the Tuatara, the duct-

uli efferentes extend posteriorly to the level of the kidney.

Thus, the ductuli efferentes are greatly elongated, which Gabe

and Saint Girons (1964) noted in their review of the testicular

ducts.

The ductuli efferentes merge with the epididymis along its

length. This differs from other vertebrates in which the ductuli

efferentes only merge with the anterior portion of the epididy-

mis (Rheubert et al. 2010). The epididymis is highly convo-

luted and possesses a secretory columnar epithelium

consistent with reports in other squamates (Dufaure and Saint

Girons 1984; Akbarsha et al. 2006; Sever and Freeborn

2012). The condition of the specimens did not allow observa-

tion of a pseudostratified epithelium (by presence of basal

cells), a feature that has been noted in all vertebrate species

studied to date (Hermo and Robaire 2001). Large amounts of

spermatozoa are observed within the epididymis, although the

testes were spermatogenically inactive, which suggests male

Tuatara are capable of sperm storage, at least for a short per-

iod of time. Fox (1977) suggests the epididymis as the sperm

storage site for lizards, which differ from snakes which store

sperm in the ductus deferens. The specimens observed in this

study could have recently ended their reproductive cycle, or

sperm could be stored within both the epididymis and ductus

deferens.

In contrast to squamates, the spermatozoa are arranged in

spherical bundles with their heads pointing toward the center

of the circle. This differs from previous descriptions of

sperm bundle organization in salamanders in which the heads

are all aligned (spermatophores) (Sperone et al. 2009) or

B

A

C D

Fig. 4—Overview of the kidney.—A. The kidney (K) is lobed, and the ureter (Ur) and ductus deferens (Dd) lie juxtapositioned to the kidney

proper contralateral to one another.—B. The collecting duct (Cd) is situated between the two lobes of the kidney proper.—C. Bowman’s capsule

(Bc) surrounds the glomerulus (Gl), and the proximal convoluted tubule (Pct) extends from Bowman’s capsule. The distal convoluted tubule

(Dct) extends from the proximal convoluted tubule (Pct).—D. The proximal convoluted tubule (Pct) is demarcated by staining positively with

alcian blue. Glomerulus (Gl).

© 2012 The Authors



chondrichthyans in which the sperm are aligned on a single

plane with the heads directly facing one another (spermato-

zeugmata) (Moura et al. 2011), but resembles sperm bundle

organization in the Echidna, Tachyglossus aculeatus (Djakiew

and Jones 1981). Although Djakiew and Jones (1981) did not

describe the organization of the sperm bundles in the

Echidna, our examination of their plates indicates that they

resemble those of the Tuatara. This organizational scheme

may aid in the transfer of spermatozoa in the absence of an

intromittent organ.

The epididymis transitions into the ductus deferens, which

extends lateral to the kidney. No morphological differences

between the epididymis and ductus deferens were observed in

these specimens (potentially owing to poor fixation) except

that the spermatozoa seem to be more densely packed with

fewer free-floating spermatozoa in the ductus deferens. Kid-

ney nephrons follow the usual vertebrate pattern of a glomeru-

lus surrounded by Bowman’s capsule, a proximal convoluted

tubule, distal convoluted tubule, and collecting ducts. The

morphology of the nephron resembles that of other verte-

brates with the exception of the presence of a brush border in

the proximal convoluted tubule. The brush border may have

been present, as light microscopy shows a conspicuous stain-

ing material at the luminal edge of the epithelium, but the fix-

ation quality limits the certainty of this interpretation. Gabe

and Saint Girons (1964) noted the presence of a brush border

A B

C D

E F

Fig. 5—Serial sagittal sections through the posterior region of the urogenital system.—A. The collecting duct (Cd) within the posterior end of the

kidney (K).—B. The ureter (Ur) exits the kidney (K) posteriorly.—C. The ductus deferens (Dd) lies anterior to the urodeum (Ud). Collecting

duct (Cd).—D. The ductus deferens (Dd) containing sperm (Sp) widens slightly but remains anterior to the urodeum (Ud).—E. The ductus

deferens (Dd) continues to widen slightly remaining anterior to the urodeum (Ud), and the kidney (K) extends lateral to the urodeum (Ud).—F.

The ductus deferens (Dd) containing mature sperm (Sp) opens into the urodeum (Ud) through a dorsal orifice (Or).

© 2012 The Authors



that was less definitive than in other reptilian species. The

presence of a sexual segment, which has been described in

every squamate species studied to date, was not observed.

Gabe and Saint Girons (1964) noted the absence of the sexual

segment but proposed this was because of the inactivity of

their specimens. The testes were inactive in the specimens

investigated in this study, but sperm were present in the testic-

ular ducts. Previous studies on squamates suggest that if

sperm are present in the testicular ducts, the SSK is hypertro-

phied. Rheubert et al. (2011) suggested this as the explanation

for the difference in hypertrophy duration between lizards and

snakes. As snakes exhibit long-term sperm storage, sperm are

found in the ductus deferens throughout the entire year, and

thus, the SSK is hypertrophied throughout the entire year. As

sperm are present in the testicular ducts and an SSK was

not observed in this or previous studies (Saint Girons and

Newman 1987), we reject the hypothesis of the presence of an

SSK in Sphenodon, which further suggests the SSK is a syna-

pomorphy for the Squamata.

The collecting duct of the kidney extends into the ureter,

which opens into the urodeum of the cloaca. The ductus def-

erens does not fuse with the ureter and empties into the urod-

eum separately. In snakes, considerable variation occurs in the

morphology of the distal ends of the ureters and ductus defer-

entia, but invariably a common urogenital papilla opens into

the cloaca (Siegel et al. 2012; Trauth and Sever 2011). A uro-

genital papilla was not observed in our specimens but was

reported by Osawa (1897) and (Robb 1977). A comparative

study of the distal ends of these ducts has not been made in

lizards.

Overall, the reproductive system of S. punctatus resembles

that of other vertebrates (especially squamates with the lack of

an anastomizing intratesticular rete testis), and the nomencla-

ture used for other vertebrates (Sever 2010) is deemed appro-

priate. Only two rete testis tubules exit the testis, which

preliminarily appears to reject the hypothesis suggested by

Volsøe (1944) who stated snakes contain the ancestral condi-

tion with multiple rete testis tubules exiting the testis; how-

ever, more data on a variety of taxa are needed. The ductuli

efferentes in Sphenodon are relatively longer than in any squa-

mate studied to date, extending posteriorly to the level of the

kidney. Sperm are condensed in the epididymis into packets

that are organized differently than other organizational

schemes observed in vertebrates except the Echidna. Distal

ends of the ureters and the ductus deferens enter the cloaca

separately, unlike the situation in snakes. The absence of the

SSK further suggests the SSK as a synapomorphy for the

Squamata.

Acknowledgements

The authors would like to thank the Otago Museum for

access to preserved specimens, especially Lucy Rowe and

Cody Fraser for the endless help during the development of

this study. Sincere appreciation goes to KenMiller at the Uni-

versity of Otago for his help with photographic techniques.

We would also like to thank Amanda Fisher and the Histology

Unit, Department of Pathology, University of Otago, for help

with slide preparation as well as Anthony Wilmes, Stanley

Trauth, and an anonymous reviewer for their comments on

earlier drafts of the manuscript. This study was undertaken

following consultation with the Ngai Tahu Consultation

Committee of the University of Otago. Funding was provided

by the National Science Foundation Grant no. DEB-

0809831 to DMS. This work is dedicated to the memory of

Manfred Gabe and Hubert Saint Girons whose work on the

histology of the tuatara inspired ours.

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Reproductive morphology of the male Tuatara, …Reproductive morphology of the male Tuatara, Sphenodon punctatus Justin L. Rheubert,1 Alison Cree,2 Matthew Downes2 and David M. Sever3