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Review Article
Sex Dev 2008;2:115–127
DOI: 10.1159/000143429
Reproductive Biology in Egg-Laying
Mammals
F. Gr ü tz n er a B. Nixon b R.C. Jones b
a School of Molecular & Biomedical Science, The University of Adelaide,
b Discipline of Biological Sciences,
School of Environmental and Life Sciences, The University of Newcastle, Callaghan , Australia
[Flannery and Groves, 1998]. The 2 genera split 25–100
million years ago, have evolved a very different general
appearance, and adapted to completely different habitats.
Although the fossil record shows a wider distribution of
monotremes more than 100 million years ago [Rowe et
al., 2008], platypus are now only found in Australia and
Zaglossus are only found in Papua New Guinea, while
Tac hygl os su s are widely distributed in both Australia and
Papua New Guinea. As very little is known about the bi-
ology of Zaglossus, and its survival is highly endangered,
this review focuses on Tachyglossus and the platypus.
The general importance of research on monotreme
mammals lies not only in the highly unusual biology of
these animals. Monotremes are the most distant living
mammalian relatives of humans and other eutherians.
Divergence time estimates range from 210 million years
ago (mya) to a more recent redating of only 166 mya
[Woodburne et al., 2003; Bininda-Emonds et al., 2007].
There has been some lingering discussion about the pos-
sibility that monotremes have diverged from the marsu-
pial lineage (marsupionta theory, reviewed in Grützner
and Graves [2004]), but this has been refuted by recent
analysis of individual nuclear genes [van Rheede et al.,
2006] as well as a systematic analysis as part of the platy-
pus genome project [Warren et al., 2008] and the recent
discovery of much older monotreme fossils [Rowe et al.,
2008]. The general importance of monotremes in com-
parative genomics is reinforced by the whole-genome se-
quencing project of the platypus which was published re-
cently [Warren et al., 2008].
Key Words
Development ⴢ Echidna ⴢ Fertilization ⴢ Lactation ⴢ Ova ⴢ
Platypus ⴢ Reproduction ⴢ Sex chromosomes ⴢ Sperm
Abstract
The duck-billed platypus and short-beaked echidna are
iconic species in Australia. Their morphology and physiolo-
gy have puzzled scientists all over the world for more than
200 years. Recent genetic studies, particularly the platypus
whole-genome sequencing project, have revealed the mo-
lecular basis of some of the extraordinary characteristics of
monotremes. This and other works demonstrate the great
value of research on our most distantly related mammalian
relatives for comparative genomics and developmental biol-
ogy. In this review we focus on the reproductive biology of
monotremes and discuss works that unravel genes involved
in l acta tion, testicular de scent , gamete biology and fer tiliz a-
tion, and early development. In addition we discuss works
on the evolution of the complex sex chromosome system in
platypus and echidna, which has also significant impact on
our general understanding of mammalian sex chromosomes
and sex determination.
Copyright © 20 08 S. Karger AG, Base l
Monotremes, the mammalian subclass Prototheria,
comprise one living species of platypus (Ornithorhynchus
anatinus), and 2 genera of echidnas, the long-beaked
echidna (3 species of Zaglossus ) and one species of short-
beaked echidnas (4 subspecies of Tachyglossus aculeatus )
Received: May 07, 2008
Accepted : June 09, 2008
Frank Grützner
Discipline of Genetics
School of Molecul ar & Biomedical S cience
The University of Adelaide, 5005 SA (Australia)
Tel. +61 8 8303 4812, Fa x +61 8 8303 4362, E-Mai l frank.g rutzner@adelaide.edu .au
© 200 8 S. Karger AG, Basel
1661–5425/08/0023– 0115$24.50/0
Accessible online at:
www.karger.com/sxd
Grützner /Nixon /Jones
Sex Dev 2008;2:115–127
116
Monotremes are in many ways extraordinary mam-
mals, but clearly the reproductive biology is by far the
most bizarre aspect of their biology. After their discovery
by Europeans, even their possession of mammary glands
was controversial for a long time. The absence of nipples
led the earliest workers to consider for 25 years that mam-
mary glands were absent until they were described by
Johann Meckel [Meckelio, 1826]. In fact, the size of the
lactating glands is enormous considering the size of the
animal [Griffiths, 1978]. The question of whether mono-
tremes lay eggs was settled much later. Despite earlier re-
ports by aborigines and settlers, it was not until 1884
when on the same day William Caldwell (for platypus)
and Wilhelm Haake (for echidna) reported separately to
the scientific community that monotremes lay eggs.
These findings came at a high price as thousands of ani-
mals were killed and preserved during expeditions lead
by William Caldwell in the 1880s. In addition to these 2
obvious aspects of monotremes, more recent histological
and molecular studies of their reproductive biology con-
tinues to reveal an intriguing amalgam of reptile-like,
mammal-like, and monotreme-specific characteristics.
All the extant monotremes are seasonal breeders. The
little data available on Zaglossus indicates that they can
mate between late June and September [Griffiths, 1968].
Ta chyglossu s may mate during the second half of July to
22nd October showing little variation in this period with
latitude and occurring even in alpine areas where mating
occurs after the final arousal from hibernation [Griffiths,
1968, 1978; Beard et al., 1992; Beard and Grigg, 2000; Ris-
miller a nd McKelvey, 2000]. The mating pattern of Tachy-
glossus is reminiscent of induced ovulators: courtship
lasts 7 to 37 days and intercourse lasts 30 to 180 min [Ris-
miller and McKelvey, 2000; Augee et al., 2006]. Presum-
ably during oestrus, females have been seen followed by
a ‘train’ of 1 to 10 males [Griffiths, 1968, 1978; Rismiller
and McKelvey, 2000] probably attracted by a female pher-
omone [Rismiller, 1992, 1993]. However, females have
only been observed to mate once and with only one male
during the mating period on Kangaroo Island [Rismiller
and McKelvey, 2000]. On the other hand, mainland fe-
males have been seen to mate with a second male during
an oestrus (Tony Corrigan, personal communication)
and to have conceived a second time during a season, pre-
sumably when the first conception failed [Beard and
Grigg, 2000]. It is generally considered that females only
breed every second year although there are at least 2 re-
ports of some females breeding in consecutive years
[Beard and Grigg, 2000; Rismiller and McKelvey, 2000].
Male and Female Reproductive Tracts
The reproductive tracts of monotremes are structur-
ally similar to one another [Griffiths, 1978, 1999; Jones et
al., 1992] and are considered to be ancestral compared to
other mammals. They are unique among mammals in
having a single external orifice, the cloaca, for excretory
and reproductive functions [Griffiths, 1978, 1999]. The
female tract ( fig. 1 ) is quite similar to the reptilian system
and also the marsupial system except that a median va-
gina forms in marsupials. The ovaries are ovoid and are
suspended below the kidneys by a fold of the peritoneum,
and each is completely enclosed by an infundibulum.
Both ovaries are functional in the echidna, but only the
left ovary is functional in the platypus as in many avian
and some reptilian species. The right ovary of the platy-
pus is embryonic retaining numerous primordial follicles
20 mm
Fig. 1. Female reproductive tract of the platypus showing both
uteri are well developed, but only its left ovary is developed. The
inserts at the top of the f igure show the ovaries dissected from the
tract.
Monotreme Reproduction Sex Dev 2008;2:115–127
117
( fig. 2 ). The platypus usually lays 2 (and sometimes 3)
eggs whereas Tachyglossus usua lly only lays 1 egg [Burrell,
1927; Griffiths, 1968, 1978]. The female reproductive
ducts are separate from one another and connect sepa-
rately to the urogenital sinus. Each duct is differentiated
into an oviduct, uterus and cervix. As in other mammals,
there is a junctional region between the oviduct and uter-
us which contains numerous tubular glands.
The male reproductive system retains some reptilian
characteristics but has some characteristics which are
unique to mamma ls [Griff iths, 1978, 1999; Djakiew, 1982;
Djakiew and Jones, 1982, 1983; Jones et al., 1992]. The
testes of monotremes are relatively large compared to
marsupials and eutherians [Jones et al., 2004]. According
to values published by Griffiths [1978], the combined
mass of the testes of Zaglossus, Tachyglossus and the
platypus are 1.2, 1.3 and 1.4% of their body mass, respec-
tively. However, the size of the testes must increase with
age (and possibly vary between location) because our es-
timates for animals with sperm throughout the epididy-
mis are only half of the above for the platypus and Tachy-
glossus except for one Tachyglossus which was 1.3%. The
testes are situated intra-abdominal as in reptiles and
birds and some other mammals such as the elephant, du-
gong, elephant shrews, rock hyrax, and marsupial mole.
However, it is unlikely that they ever exceed their body
temperature (32
° C) while they are reproductively active
as the platypus’s environment is below body temperature
and the echidna avoid high environmental temperatures
[Brice et al., 2002a, b; Jones et al., 2004]. The testes are
suspended in the abdominal cavity medial and just distal
to the kidneys. They are supported by anterior and pos-
terior gonadal ligaments and a peritoneal fold, the mesor-
chium, which also encloses the epididymis ( fig. 3 ). How-
ever, the gubernaculum, which is involved in transab-
dominal testicular descent in scrotal eutherians and
marsupials, is absent in monotremes and non-mamma-
lian vertebrates [Griffiths et al., 1993; van der Schoot,
1996; Werdelin and Nilsonne, 1999; Coveney et al., 2002;
Mackay et al., 2004; Wilhelm and Koopman, 2006]. Con-
sistent with this finding is that the specificity of INSL3
(Insulin like factor 3) for LGR8 (orphan leucin-rich repeat
containing G-protein coupled receptors) is absent in
200 µm
20 mm
Fig. 2. Light micrograph of a paraffin section of the right ovary of
a platypus showing it mainly consists of primordial follicles em-
bedded in connective tissue.
Fig. 3. Testis and epididymis of the echidna showing it undissect-
ed (top) and dissected from the supporting peritoneal fold. From
the testis distally the arrows delineate the extent of the ductuli ef-
ferentes and initial and terminal segments of the ductus epididy-
midis.
Grützner /Nixon /Jones
Sex Dev 2008;2:115–127
118
monotremes and has evolved in therian mammals by a
si ng le poi nt mut at ion [Pa rk et al., 20 08]. Th is is of inter es t
as, in the mouse, Insl3 acting through Lgr8 is essential for
the mediation of testicular descent via its effect on growth
of the gubernaculum’s primordia and the caudal genitor-
inguinal ligament [Nef and Parada, 1999; Zimmermann
et al., 1999; Bogatcheva et al., 2003; Kamat et al., 2004;
Wilhelm and Koopman, 2006].
Sperm are carried from the testis via an extratesticular
rete. It is restricted to the dorsal surface of the testis and
does not run the length of the testis as in reptiles and
birds. About 7 ductuli efferentes leave the rete in Ta chy-
glossus and some join together before joining the ductus
epididymidis at right angles [Djakiew and Jones, 1981].
As in other mammals, the cord formed by the ductuli is
structurally differentiated into 2 zones and the ductuli
reabsorb most of the fluid and solute (including proteins)
leaving the testis.
Most of the epididymis lies on the testis ( fig. 3 ), and it
forms an isthmus where the cord is folded through 180°
as it leaves the surface of the testis and narrows. The duc-
tus epididymidis is, relative to body mass, as long as in
other mammals. However, it is only differentiated into 2
segments whereas in other mammals it is differentiated
into about 6 structurally distinct segments [Jones, 1998,
2002]. The proximal region of the monotreme epididy-
mis, the initial segment, has characteristics unique to the
mammalian epididymis [Jones et al., 1992]. In mono-
tremes it comprises 96% the length of the duct and is sep-
arated by the isthmus from the terminal segment. The
latter is wider and the epithelium lower than more prox-
imally, and the epithelium is thrown into folds forming
villi as in the elephant epididymis [Jones and Brosnan,
1981]. The diameter and muscular coat of the terminal
segment increase distally and no distinct ductus deferens
is present nor is one necessary as the terminal segment is
adjacent to the urogenital sinus which it joins at its ante-
rior end. Djakiew [1982] estimated that it takes about 14.1
days for sperm to pass through the extragonadal ducts:
29 min to pass through the ductuli efferentes, and 9.9 and
4.2 days to pass through the initial and terminal seg-
ments of the epididymis, respectively.
A number of authors have described disseminate ure-
thral glands in the platypus and Tachyglossus which are
branched and tubuloalveolar like the mammalian pros-
tate gland. However, the glands are not sufficiently large
in the monotremes to be obvious macroscopically as in
eutherians and marsupials, and they would not contrib-
ute a significant volume to an ejaculate. Temple-Smith
[1973] found no seasonal variation in secretory activity of
the glands that were associated with changes in testis ac-
tivity in the platypus, whereas Djakiew [1978] and Jones
et al. [1992] provided evidence that the glands were an-
drogen dependent in Tachyglossus. Both the platypus and
Ta chyglossu s have a pair of androgen dependent Cowp-
er’s (bulbourethral) glands which empty into the urethra
[Griffiths, 1968, 1999; Temple-Smith, 1973; Jones et al.,
1992]. However, they are also small and would contribute
little to the volume of an ejaculate.
The platypus and Ta ch yg lo ss us each have a well-devel-
oped penis ( fig. 4 ) which is normally retracted into a pre-
putial sac in the ventral cloaca. In contrast to other mam-
mals, except the marsupial bandicoot Perameles nasuta
[Griffiths, 1978], only sperm pass through t he penis while
urine is excreted through the cloaca. When erect, the pe-
nis protrudes through the cloacal opening [Griffiths,
1968, 1999; Temple-Smith, 1973]. The glans penis is bifid,
as in marsupials [Biggers, 1966], with each part contain-
ing a branch of the urethra. In the platypus the surface of
the distal third of the penis is spined and the end of each
bifid has an eversible papilla containing 4 spines. The dis-
tal end of the penis of Tachy gl os su s is different. It bears a
pair of eversible papillas containing another urethral
branch [Griffiths, 1968; Carrick and Hughes, 1978]. A re-
cent study reports that successive ejaculates are delivered
alternatively from each pair of papilla [Johnson et al.,
2007]. Temple-Smith and Grant [2001] suggest that in-
Fig. 4. Penises of the echidna (top) and platypus (bottom) show-
ing the differences in structure and urethral opening at the base
where urine normally is excreted. The insert at the left side of the
platypus penis shows that the end of each bifid has an eversible
papilla containing 4 spines.
Monotreme Reproduction Sex Dev 2008;2:115–127
119
semination in monotremes is intrauterine. The mono-
treme penis shares some characteristics with the single
penis of turtles and crocodilians in that both have cor-
pora cavernosa which become engorged with blood to ef-
fect erection. However, the reptilian penis is different in
that sperm pass along a groove whereas in the mono-
tremes and other mammals they pass through a duct.
Sperm Production
The structure of the seminiferous tubules of the platy-
pus and Tachyglossus is similar to other amniotes in that
clones of at least 4 generations of germ cells are supported
by a group of Sertoli cells forming cellular associations
[Benda, 1906; Djakiew, 1982; Lin and Jones, 2000]. The
area of tubular wall covered by an association is small so
that numerous cellular associations (stages of spermato-
genesis) are present in a cross-section of a seminiferous
tubule as in man, some other primates, and sauropsids
[Setchell, 1978; Jones and Lin, 1993]. This is in contrast to
most mammals in which the associations are large and
extend several millimeters along a seminiferous tubule.
Most features of spermiogenesis in the platypus are sim-
ilar to sauropsids including the absence of acrosomal
granule development [Carrick and Hughes, 1982; Lin and
Jones, 1993]. However, some fe atures a re unique to mam-
mals. For example, as in other mammals, the acrosome
develops a thin, lateral margin which expands over the
nucleus more than in sauropsids. A tubulobulbar com-
plex develops around the spermatid head, a feature which
appears to be unique to mammals. Also during spermia-
tion the residual body is released from the caudal end of
the nucleus leaving a cytoplasmic droplet located at the
proximal end of the middle piece as in marsupial and eu-
therian mammals, but different to the avian model. Oth-
er features of spermiogenesis appear to be unique to
monotremes. Nuclear condensation involves the forma-
tion of a layer of chromatin granules under the nucleo-
lemma, and development of the fibrous sheath of the
principal piece starts much later in the platypus than in
birds or eutherian mammals.
The rate of sperm production has been estimated in
Tachyglossus [Djakiew, 1982], and values fall within the
range estimated for other mammals [Setchell, 1978].
Djakiew [1982] identified 6 stages of spermatogenesis in
Tachyglossus and estimated the duration of a cycle of the
seminiferous tubule to be 13.8 8 0.12 days and the dura-
tion of the whole spermatogenic process (4.5 cycles) to be
62 days. He worked with animals with a mean body mass
of 2.9 8 0.28 kg and mean testis mass of 9.190 8 0.94 g
and estimated that the daily sperm production was 6.0 8
0.27 million sperm per gram testis and 55.4 8 7. 3 7 m il-
lion sperm per testis.
Tachy gl os sus sperm are longer (120 m) t han plat ypu s
sperm (100 m), but there is little difference in their
structure [Carrick and Hughes, 1982]. They differ from
other mammalian sperm in that their nuclei are vermi-
form and cylindrical like sauropsids and that they are ar-
ranged in a helix. The acrosome is small and mainly lim-
ited to the rostral surface of the nucleus; there is no post-
acrosomal sheath over the nucleus as in eutherian sperm
[Jo ne s, 19 71; Fawc et t , 19 75] , a nd th ey ha ve no st ri at ed co l-
umns in the neck region or coarse fibers supporting the
principal piece. Also, the fibrous sheath supporting the
principal piece of monotreme sperm is spiraled whereas
in eutherian sperm it has longitudinally oriented connec-
tions between adjacent rings. Further, their nuclei and
tails are less rigid than those of eutherians and marsupi-
als owing to the absence of disulphide formation during
epididymal maturation which occurs in the higher mam-
mals, but not in lower vertebrates [Bedford and Calvin,
1974a, b; Bedford and Rif kin, 1979]. The lack of disul-
phide bonding in the nucleus is because monotremes
have no cysteine residues in their protamine-1 sequences
[Retief et al., 1993]. Orthologs of many of the eutherian
sperm membrane proteins related to fertilization are
present in platypus (and marsupial) genomes [Warren et
al., 2008]. Among these are the genes for putative zona
pellucida receptors and proteins implicated in sperm-
oolemmal fusion including: zona pellucida binding pro-
tein (Zpbp, Sp38) , zona pellucida binding protein 2
(Zpbp2) , zona pellucida sperm-binding protein 3 receptor
( Zp3r; Sp56) , hyaluronoglucosaminidase 5 (Hyal5; Spam1;
Ph20) , A disintegrin and metallopeptidase domain 1a
(Fertilin alpha) , sperm autoantigenic protein 17 (Spa17;
Sp17) , milk fat globule-EGF factor 8 protein (Mfge8; Sed1),
UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase
(B4galt1) , fucosyltransferase 5 (alpha (1,3) fucosyltrans-
ferase (Fut5) , mannosidase 2, alpha B2 (Man2b2) and izu-
mo sperm-egg fusion 1 (Izumo1) . The conservation of
such genes argues for their functional importance in
sperm and attests to the evolutionary conservation of the
mechanisms underpinning gamete interaction. Further,
as the zona pellucida of monotremes is much thinner
than the zona of eutherians [Bedford, 1991], it is of inter-
est that testis-specific proteases, which are considered to
play important functions in degrading the zona pellucida
of eutherians during fertilization, are all absent from the
platypus genome [Warren et al., 2008].
Grützner /Nixon /Jones
Sex Dev 2008;2:115–127
120
Epididymal sperm maturation in the platypus and
Tachyglossus is similar to descriptions of sauropsids and
the maturational changes, which are both unique and es-
sential for other mammalian sperm to be able to fertilize
an ovum, have not been identified [Bedford and Rifkin,
1979; Djakiew and Jones, 1983]. During passage through
the epididymis the cytoplasmic droplet migrates along
the middle-piece of monotreme spermatozoa as in other
mammals, and there is an increase in the proportion of
sperm that are motile. However, changes in the form of
mo ti lit y o r st at us o f the a cro some hav e no t be en de tecte d.
The terminal segment of the monotreme epididymis is
small and stores relatively fewer sperm (about 28% of ex-
tragonadal sperm in Tachyglossus and probably a smaller
proportion in the platypus) than most other mammals
[Jones and Djakiew, 1978; RC Jones, personal observa-
tion]. Consistent with these findings is the absence of
platypus genes for the epididymal specific proteins that
have been implicated in sperm maturation and storage in
other mammals [Warren et al., 20 08]. The most abundant
secreted protein in the platypus epididymis is lipocalin
whose homologs are the most secreted proteins in the
reptilian epididymis [Morel et al., 2000]. Interestingly,
Adam7, which is secreted in the epididymis of eutherians,
has an ortholog in the platypus. This is a bona fide pro-
tease with a characteristic Zn-coordinating sequence
HExxH in the platypus, in the opossum, and the tree
shrew (Tupaia belangeri), yet it has lost its proteolytic ac-
tivity in eutherians due to a single point mutation within
its active site (E 1 Q).
A unique feature of Tachy gl os su s and the platypus is
that their sperm form bundles of about 100 individuals
( fig. 5 ) as they pass through the distal part of the initial
segment of the epididymis [Djakiew and Jones, 1981,
1983; Jones, unpublished data]. The bundle formation is
correlated with the synthesis and secretion of 1 or 2 spe-
cific proteins by the epididymal epithelium [Jones et al.,
2007]. The motility of the bundles is about 3 times great-
er than individual sperm and it has been suggested that
the adaptation probably evolved due to sperm competi-
tion, the competition between males to achieve paternity
[Jones et al., 2004, 2007]. The bundles initially remain
intac t in v itro on dilution of epid idy mal f lu id or e jaculate
of Tachyglossus. Indeed, viable sperm remain in bundles
unless they are incubated for nearly 3 h in a specific me-
dium required for capacitation of eutherian sperm. Con-
sequently, it is suggested that monotremes provide an
ea rly example of capacitat ion [Jone s et al., 2 007]. Fur ther,
as the formation of bundles and their dispersal seems to
mainly involve an interaction of sperm with protein, it
seems that the process is quite different to the process in
eutherian mammals which mainly involves loss of cho-
lesterol from the sperm membrane [Davis, 1981].
The ancestral nature of monotreme sperm provides an
opportunity to obtain new insights into the evolution of
genome organization and the positioning of sex chromo-
somes in sperm. In this respect, since sperm development
in monotremes and birds is similar [Lin and Jones, 2000],
it is questionable whether there is a random distribution
of chromosomes in monotreme sperm as in the chicken
[Solovei et al., 1998], or whether there is a non-random
organization of chromosomes as in mammals [Haaf and
Ward, 1995; Zalensky et al., 1995; Meyer-Ficca et al., 1998;
Greaves et al., 2003; Foster et al., 2005; Zalensky and Za-
lenskaya, 2007]. Several studies on genome organization
in platypus provide clear evidence for a non-random or-
ganization of the genome in the sperm nucleus [Watson
et al., 1996; Greaves et al., 2003; Tsend-Ayush et al., sub-
mitted]. Both monotremes feature a complex sex chro-
mosome system (i.e., 10 sex chromosomes in platypus
and 9 in echidna). The sex chromosomes form a chain at
meiosis and segregate into X and Y bearing sperm
[Grützner et al., 2004]. Recently, localization of X and Y
specific BAC (bacterial artificial chromosome) clones in
platypus sperm showed that the multiple sex chromo-
somes cluster in the middle and caudal part of the sperm
nucleus, suggesting that the sex chromosomes remain as-
sociated after t hey have segregated from the meiotic chain
Fig. 5. Sca nning elec tron microgr aph of bundles of ec hidna sper m
in the terminal segment of the epididymis. Individual sperm are
packed tightly in the bundles and are held together by electron
dense material. Picture from Djakiew and Jones (1983) © Society
for Reproduction and Fertility (1983). Reproduced with permis-
sion.
Monotreme Reproduction Sex Dev 2008;2:115–127
121
in anaphase I [Tsend-Ayush et al., submitted]. The auto-
somes that share homology with the human X chromo-
some in chicken (GGA1 and GGA4) and platypus (OAN6)
are found in similar positions in the middle of the sperm
head. This shows that the original position of the X chro-
mosome, in the middle of the sperm head, has been con-
ser ved in chicken and platypus as well as in some marsu-
pials and eutherians [Tsend-Ayush et al., submitted]. In
other species like humans the X chromosome has moved
rostrally in the sperm head [Hazzouri et al., 2000].
Oogenesis and Fertilization
The early work on oogenesis in monotremes is sum-
marized by Griffiths [1968, 1978]. In monotremes it pro-
ceeds much the same as in other amniotes except for the
different amount of yolk that develops. Yolk consists of
large glycolipoproteins, and monotreme eggs contain
much less than oviparous amniotes whilst yolk is absent
in eggs of eutherians and marsupials. This variation is
consistent with the occurrence of genes for one of the
most commonly found egg-yolk precursor proteins, the
vitellogenins. These proteins are found in a wide range of
oviparous vertebrate species but are absent in marsupials
and eutherian mammals. On the other hand, at least 3
vitellogenin genes have been identified in the platypus
genome so far [Babin, 2008; Warren et al., 2008] indicat-
ing that these proteins may also contribute to the yolk-
rich ovum in monotremes.
Although Griffiths [1968] interpreted from earlier re-
ports that the ovarian follicles develop an antrum filled
with follicular f luid like other mammals and fundamen-
tally different to that of reptiles which lack follicular flu-
id, we have not observed an antrum in tertiary follicles
of the platypus ( fig. 6 ). At ovulation the ovum is small
(4 mm diameter) in monotremes relative to comparably
sized reptiles and birds. It is bounded by the vitelline
membrane within a very thin zona pellucida (ZP) and a
tunic of 2 layers of columnar, follicular cells. The first
meiotic division is complete when the first polar body
and the second maturation spindle have formed. The in-
fundibulum is lined by ciliated and no-ciliated secretory
cells, the latter secreting a clear viscous fluid just prior to
ovulation. The oviductal secretions form 2 layers of albu-
men around the ovum, the upper two-thirds secreting
the densest layer. A basal layer of the shell membrane is
formed in the distal third of the oviduct and a second,
denser layer is formed in the proximal uterus. Uterine
glands in the body of the uterus secrete a nutritive f luid
which is absorbed by the egg which grows to its full size
of 14–15 mm diameter. A third uterine secretion is the
precursor of the protective layer of the shell.
Fertilization occurs prior to or during the formation
of the albumen layer and the second meiotic division is
complete when the egg enters the uterus. The ZP proteins
are noteworthy as they are considered to be involved in
sperm-egg recognition in mammals. The 4 proteins of
the human ZP, Zp1, Zp2 (Zpa), Zp3 (Zpc), and ZP4/B [Jo-
vine et al., 2007] possess single orthologs encoded in the
platypus with a high level of homology (approximately
40–50% identity on the amino acid level). They also dis-
play a high level of homology with proteins in the chick-
en inner perivitelline membrane [Stewart et al., 2004]
which is analogous to the mammalian ZP. The avian
membrane consists of 3 major components, homologs of
the Zp1 and Zp3 families and a unique, phylogenetically
distinct protein, ZPD. The latter is absent in the platypus
and eutherian mammals which may explain why the avi-
an membrane does not appear to form a species-specific
barrier as stringent to fertilization in birds as the ZP does
in mammals [Stewart et al., 2004].
Early Development
A recent estimate of the gestation period, from mating
to egg laying, was 23 8 3 days for 10 Tachy gl os sus indi-
viduals [Rismiller and McKelvey, 2000] and is in agree-
500 µm
Fig. 6. Light micrograph of a tertiary follicle in a paraffin section
of the left ovary of a platypus.
Grützner /Nixon /Jones
Sex Dev 2008;2:115–127
122
ment with other estimates [Griffiths, 1999; Beard and
Grigg, 2000]. Estimates for the platypus vary from at least
9 days [Griffiths, 1999] to 15–20 days [Holland and Jack-
son, 2002].
Griffiths [1968, 1978] and Hughes and Hall [1998]
summarized structural work on early development in
monotremes. In the uterus the egg undergoes conjuga-
tion of the nuclei and incomplete meroblastic cleavage as
in lower amniotes and unlike the holoblastic process in
higher mammals. Like marsupials, which also become
dependent on lactation from a very early stage of develop-
ment, monotremes only develop a simple placenta. In
contrast to eutherian mammals, which form a chorioal-
lantoic placenta, marsupials form an invasive or non-in-
vasive yolk-sac placenta [Freyer et al., 2003] whilst the
platypus develops an allantoic vitelline placenta from vi-
tellocytes, trophectoderm-like cells [Griffiths, 1978; Sel-
wood and Johnson, 2006]. Two genes important for pla-
cental development and trophectoderm (TE) differentia-
tion have recently been studied in monotremes, the Peg10
and Pou5f1 gene. Peg10 is an imprinted gene essential for
placental development, and in the mouse model absence
of Peg10 causes severe placental defects which are lethal
during early embryogenesis [Ono et al., 2006]. However,
although Peg10 is present and imprinted in marsupials,
there is no ortholog in monotremes [Suzuki et al., 2007]
indicating that it is not required for their placental devel-
opment. Two other genes important for TE differentia-
tion are the POU-family transcription factor Oct3/4 en-
coded by Pou5f1 and the caudal-related homeobox tran-
scription factor Cdx2. During TE differentiation in mice,
Pou5f1 is repressed and Cdx2 is activated [Niwa et al.,
2005]. In contrast to Peg10, Pou5f1 and Cdx2 are present
in monotremes as in other mammals. There is also evi-
dence that the platypus Pou5f1 gene can be fully func-
tional in eutherian cells: the evidence is based on studies
of gene complementation in mouse Pou5f1 knock-out
mouse ES cell lines and promoter analyses. However, the
platypus Pou5f1 gene lacks part of a conserved regulatory
element which is essential for positive autoregulation of
Pou5f1 and the interaction with Cdx2 which is important
for TE differentiation in mice. It is suggested that the
change in Pou5f1 and Cdx2 regulation may represent a
step towards a more complex placental development in
therian mammals [Niwa et al., 2008].
Monotreme eggs are laid when the embryo has 19–20
pairs of somites and after some regression of the corpus
luteum formed from t he ruptured ova rian follicle at ovu-
lation. In Tac hy gl os su s, but not the platypus, the egg is
retained by hairs plastered across a ‘pouch’ formed as a
shallow depression in the belly. Griffiths concluded that
the egg is incubated for about 10.5 days until hatching in
Ta chyglossu s and at least 10 days in the platypus. The
young is about 15 mm long and ‘probably tears the shell
by the combined action of caruncle and egg tooth’
[Griffiths, 1968]. The forelimbs are well developed, the
hindlimbs are still at the bud stage, the snout region is
compressed and, like marsupials, they exhibit modifica-
tions associated with living in a pouch on a diet of milk.
Peggy Rismiller [Rismiller, 1999; Rismiller and Mc-
Kelvey, 2000] found that echidna young are carried in the
pouch for 45–55 days (mass of 180–260 g) before they are
ejected and placed in a burrow where they reside until
they are weaned 180–205 days after hatching (mass of
800–1300 g). A study of 2 platypuses in captivity found
that they emerged from their burrows at 131 and 136 days
after they were laid as eggs [Holland and Jackson, 2002].
L a c t a t i o n
Ma le and female monotremes posses mammar y glands
but, as in other mammals, only the females produce milk.
The glands are located between the dermis and abdomi-
nal muscle on either side of the abdomen and secrete di-
rectly onto the skin as there are no nipples. It has been
suggested that mammary glands evolved from an ances-
tral apocrine-like gland that was associated with hair fol-
licles to provide moisture, and possibly other constitu-
ents to protect porous eggs during incubation [Oftedal,
2002]. Like other mammals, the milk of monotremes is a
rich secretion containing sugars, lipids, and milk pro-
teins. It is controversial whether there are changes in milk
composition similar in magnitude to those reported in
marsupials. However, it is reported that Ta ch yg lo ss us ini-
tially secretes diluted milk, composed of 1.3% fat, 7.9%
protein, and 2.9% carbohydrate and minerals, whereas
mature milk is very concentrated, containing 31% fat,
12.4% protein, and 2.8% carbohydrate and other compo-
nents [Augee et al., 2006]. The fat content is similar to
other mammals, but echidna milk contains little and
platypus milk contains considerable polyunsaturated fat-
ty acid. The carbohydrate content differs from other
mammals [Messer and Kerry, 1973; Messer et al., 1983].
Echidna milk mainly contains sialyl- and fucosyllactose
and no lactose, and about half the carbohydrate in platy-
pus milk is difucosyllactose. Green et al. [1985] found
that the milk intake and weight gain of Tachyglossus is
similar to other fast growing species, such as rabbits, with
weight gains of 0.4 g per ml milk intake.
Monotreme Reproduction Sex Dev 2008;2:115–127
123
Only a few milk peptides have been studied in mono-
treme milk, including the whey proteins alpha-lactalbu-
min, lysozyme [Teahan et al., 1991; Acharya et al., 1994;
Messer et al., 1997; Kikuchi et al., 1998], and whey acidic
pr ot ei n WAP [S ha rp et al ., 20 07]. Ge ne ex pre ss io n of mi l k
proteins in the platypus is 50%  -lactoglobulin and 30%
calcium-binding caseins [Warren et al., 2008]. Echidna
milk contains a higher content of whey proteins and with
different domain architecture and more rapid amino acid
substitutions, without altering the cysteine residues, than
the whey acidic protein in platypus [Sharp et al., 2007].
Casein genes are tightly clustered together in the platy pus
genome as in other mammals, and they contain an addi-
tional and recently duplicated  -casein gene [Warren et
al., 2008]. The casein genes are of interest as they are
thought to have evolved by duplication of enamel matrix
protein genes, either enamelin or ameloblastin [Kawa-
saki and Weiss, 2003], which is consistent with the pres-
ence of teeth and enamel in the juvenile and fossil mono-
tremes [Lester et al., 1987]. Warren et al. [2008] interpret
that ‘the platypus genome is unique among sequenced
mammalian genomes in containing approximately 11
paralogs of xa nthine dehydrogenase/oxidase ( XDH gene).
Of these paralogs, 4 have been placed on platypus chro-
mosome X1, in conserved synteny with human XDH
(HSA2) . In addition to its housekeeping role in purine
metabolism, eutherian XDH appears to have roles in the
secretion of milk lipids. As its protein level correlates with
the maturation of mouse mammary tissue in pregnancy,
the platypus XDH paralogs may assist in regulating milk
lipid content during lactation.’
Sex Chromosome Organization and Evolution
In terms of sex chromosomes and sex determination,
monotremes are remarkably different from all other
mammals. They share a XY ma le, XX female sex chromo-
some system with other mammals as opposed to the ZZ
male ZW female system in birds. However, the sex chro-
mosome system in platypus contains 5 pairs of X chro-
mosomes in females and 5 X and 5 Y chromosomes in
males [Murtagh, 1977; Grützner et al., 2004; Rens et al.,
2004]. In order to ensure that the 5 X chromosomes and
the 5 Y chromosomes are segregated into sperm contain-
ing only X or Y chromosomes, the 10 sex chromosomes
assemble as a chain at the first meiotic division in males
via 9 pseudoautosomal regions. Chromosome painting
showed how the sex chromosomes adopt an X1-Y1-X2-
Y2-X3-Y3-X4-Y4-X5-Y5 alternating pattern in metaphase
I and segregate reliably into 5 Y bearing and 5 X bearing
sperm [Grützner et al., 2004; Rens et al., 2004]. In addi-
tion to having this complex sex chromosome system, the
genes residing on the sex chromosomes differ from other
mammals. Platypus sex chromosomes show extensive
homology with the chicken Z as well as a number of au-
tosomes [Grützner et al., 2004; Rens et al., 2007; Veyrunes
et al., 2008]. This includes genes of the major histocom-
patibility complex (MHC) which is split apart on the
pseudoautosomal regions of 2 pairs of sex chromosomes
[Dohm et al., 2007]. There is no homology between platy-
pus sex chromosomes and the eutherian X, which is
largely homologous to platypus autosome 6 [Waters et al.,
2005; Veyrunes et al., 2008].
A l t h o u g h Tachyglossus exhibits a meiotic chain of 9
chromosomes [Bick, 1992; Watson et al., 1992], the basic
features of its sex chromosome system are similar to the
platypus and chromosome painting has shown that most
chromosomes of the 2 species are homologous. The only
exceptions are platypus sex chromosomes Y3-X4 and
parts of Y4 which show homology with Tachyglossus
chromosome 27. In addition, platypus Y5 has been trans-
located to Y3 in Ta ch yg lo ss us , which explains why the
echidna chain contains one chromosome less than the
platypus [Rens et al., 2007]. Comparative mapping of
MHC genes between platypus and echidna also showed
that as a result of these differences the order of chromo-
somes in the meiotic chain is different between platypus
and Ta ch yg lo ss us [Dohm et al., 2007]. This means that
there have been ongoing changes to the meiotic chain af-
ter platypus and Tach yg lo ss us diverged. For our under-
standing of the evolution of mammalian sex chromo-
somes this means a much more recent origin of the X
chromosomes in therian mammals [Veyrunes et al.,
2008].
Several models have been developed to evaluate how
the sex chromosome system of monotremes evolved
[Gruetzner et al., 2006; Dohm et al., 2007; Rens et al.,
2007]. Interestingly, one of the models (model in Gruetz-
ner et al. [2006]) suggests that sequential translocations
would transfer one arm of the original sex chromosome
onto the latest autosome that gets included in the system:
in platypus this would be X1. It has therefore been hy-
pothesized that X1 would also contain some genes found
on the chicken Z. Although a large number of genes on
X1 are found on chicken chromosomes 3, 13 and 12, the
mapping of the Z linked gene PDE6A on X1p supports
this idea [Rens et al., 2007].
In terms of understanding sex determination in mono-
tremes, the total lack of homology to the eutherian X
Grützner /Nixon /Jones
Sex Dev 2008;2:115–127
124
chromosomes is important as it shows that a Y chromo-
some similar to the eutherian Y never existed in mono-
tremes. This explains why the mammalian male deter-
mining gene SRY was never identified in platy pus [Grütz-
ner et al., 2004]. Its closest relative, the SOX3 gene, has
been mapped to platypus chromosome 6 which shares
extensive homology with the human X chromosome
[Wallis et al., 2007]. Other genes involved in sex determi-
nation in eutherian mammals have been mapped to au-
tosomes in platypus [Grafodatskaya et al., 2007]. The
only gene involved in the male sex differentiation path-
way is GATA4 which has been mapped to the Y1X2 shared
region [Grafodatskaya et al., 2007]. Therefore, there is no
dosage difference between males and females. The chick-
en candidate sex determining gene DMRT1 could be a
candidate as it is located on the X specific part of X5, and
males would only have one copy [Grützner et al., 2004].
Paradoxically, in other mammals and birds 2 copies are
required for male development [Raymond et al., 1999].
Clearly more sequence information is needed from the Y
specific parts of platypus Y chromosomes. Since the ge-
nome of a female platypus has been sequenced, we only
have sequence information of the X chromosomes and
some XY share regions [Veyrunes et al., 2008].
C o n c l u s i o n
The reproductive biology of monotremes can offer
unique insights into the evolution of many reproductive
traits in mammals. Similarities with reptiles are likely to
represent ancestral mammalian characteristics. Egg-lay-
ing, some aspects of gamete production, sex chromo-
somes, and meroblastic cleavage are a few examples. In
terms of mammalian characteristics, monotremes may
represent important early steps in the development, for
example, of the placenta, mammary glands, and more
rigid spermatozoa that require post-testicular matura-
tion and a period of capacitation before they can fertilize
an ovum. In addition, monotremes have been separated
from the mammalian line for more than 160 million
years, and this has led to the establishment of unique fea-
tures. This may be the case for parts of the sex chromo-
some system and possibly includes the formation of
sperm bundles. On the other hand, the arrangement and
subsequent release of sperm from bundles may be an ear-
ly form of the processes involved in the capacitation of
sperm in higher mammals. We have begun to unravel the
molecular mechanisms and genes involved in some of t he
specific traits of reptiles, monotremes, and higher mam-
mals. The lack of entire genes like Peg10, changes of regu-
latory mechanisms as in Pou5f1 , or even point mutations
as in INSL3 are examples of individual changes being
linked to the lack of a sophisticated placenta or testicular
decent. The recently completed annotation and analysis
of the entire platypus genome takes this to another level.
With more that 18,000 predicted genes and annotated ge-
nomic and cDNA sequences whole pathways can be in-
vestigated and searches for the occurrence of genes in-
volved in fertilization and lactation are examples of how
this can be linked to the biology of monotremes. Other
developmental pathways like sex determination are still
lacking promising candidate genes, although a much bet-
ter picture of the gene content of X and XY shared regions
of the multiple sex chromosomes is an important step to-
wards finding regions that could harbor potential sex de-
termining genes.
Acknowledgement
We greatly appreciate the help and advice of Eileen McLaugh-
lin, Discipline of Biological Sciences, University of Newcastle, in
preparing the images of the ovarian follicles. FG is an Australian
Research Council Research Fellow.
References
Acharya KR, Stuart DI, Phillips DC, McKenzie
HA, Teahan CG : Models of the th ree-dimen-
sional structures of echidna, horse, and pi-
geon lysozymes: calcium-binding lysozymes
and their relationship with alpha-lactalbu-
mins. J Protein Chem 13:
569–584 (1994).
Augee M, Gooden B, Musser AM: Echidna: Ex-
traordinary Egg-laying Mammal. (CSIRO
Publishing, Collingwood 2006).
Babin PJ: Conservation of a vitellogenin gene
cluster in ov iparous vertebr ates and identifi-
cation of its traces in t he platypus genome.
Gene 413:
76–82 (20 08).
Beard LA, Grigg GC: Reproduction in the short-
beaked Echidna, Tachyglossus aculeatus:
f i e ld o b se r v at i on s a t a n el e v at e d s i te i n s o ut h -
east Queensland. Proc Linn Soc NSW 122:
89–99 (2000).
Beard LA, Grigg GC, Augee ML: Reproduction
by Echidnas in a cold climate, in Augee ML
(ed): Platypus a nd Echidnas, pp 93–100
(Royal Zoological Society of New South
Wales, Sydney 1992).
Bedford JM: The coevolution of mammalian
gametes; in Dunbar BS, O’Rand MG (eds): A
Comparative Overview of Mammalian Fer-
tilization, pp 3–35 (Plenum Press, New York
1991).
Monotreme Reproduction Sex Dev 2008;2:115–127
125
Be dford JM, Calv in HI: The occ urrence and pos -
sible functional significance of -S-S cross-
link s in sperm heads. J E xp Zool188:
137–156
(19 74a).
Bedford JM, Calvin HI: Changes in -S-S- linked
structures of the sperm tail during epididy-
mal maturation. J Exp Zool 187:
181–204
(1974b).
Bedford JM, Rif kin JM: An evolutionary view of
the male re productive trac t and sperm matu-
ration in a monot reme mammal – the echid-
na, Tachyglossus aculeatus . Am J Anat 156:
207–230 (1979).
Benda C: Die Spermiogenese der Monotremen.
Jen Z Med Natur wiss 6:
413–438 (1906).
Bick Y: The Meiotic Chain of Chromosomes of
Monotremata (Royal Zoological Society of
New South Wales, Sydney 1992).
Biggers JD: Reproduction in male marsupials.
Symp Zool Soc Lond 15:
251–310 (1966).
Bininda-Emonds OR, Cardillo M, Jones K E,
MacPhee RD, Beck RM, et al: The delayed
rise of present-day mammals. Nature 446:
507–512 (20 07).
Bo gatch eva N V, Truong A, Feng S, Engel W, Ad-
ham IM, Agoulnik AI: GREAT/LGR8 is the
only receptor for insulin-like 3 peptide. Mol
Endocrinol 17:
2639–2646 (2003).
Brice PH, Grigg GC, Beard LA, Donovan JA:
Heat tolerance of short-beaked echidnas
(Tachyglossus aculeatus) in the f ield. J Therm
Biol 27:
449–457 (2002a).
Br ice PH, Grigg GC, B eard LA, Donova n JA: Pat-
terns of inactivity in echidnas (Tachyglossus
aculeatus) free-ranging in a hot dry climate:
correlate s with ambient temper ature, time of
day and season. Aust J Zool 50:
461–475
(2002b).
Burrell H: The Platypus. Its Discovery, Zoologi-
cal Posit ion, Form and Charac teristics, H ab-
its, Life History, etc. (Angus and Robertson
Ltd, Sydney 1927).
Carrick FN, Hughes RL: Reproduction in male
monotremes. Austr Zool 20:
211–231 (1978).
Carrick FN, Hughes RL: Aspects of the structure
and development of monotreme spermato-
zoa and their relevance to the evolution of
mammalian sperm morpholog y. Cell Tissue
Res 222:
127–141 (1982).
Coveney D, Shaw D, Hutson JM, Renfree MB:
The development of the gubernaculum and
inguinal closure in the marsupial Macropus
eugenii . J Anat 201:
239–256 (2002).
Davis BK: Timing of fertilization in mammals:
cholesterol/phospholipid ratio as a determi-
nant of the capacitation interval. Proc Nat l
Acad Sci USA 78:
7560–7564 (1981).
Djakiew D: Studies of the male reproductive
tract of t he echidna (Tachyglossus aculeatus)
with particular emphasis on the epididymis.
Honours thesis, Department of Biological
Sciences, University of Newcastle, NSW,
Australia (1978).
Djakiew D: Reproduction in the Male Echidna
(Tachyglossus aculateus) with Particular
Emphasis on the Epididymis, pp 158 (De-
partment of Biological Sciences, University
of Newcastle, Newcastle 1982).
Djakiew D, Jones RC: Structural differentiation
of the male genital ducts of the echidna
(Tachyglossus aculeatus) . J A nat 132:
187–20 2
(19 81).
Djakiew D, Jones RC: Ultrastructure of the duc-
tus epididymidis of the echidna, Ta chy glo s-
sus aculeatus . J Anat 135:
625–634 (1982).
Djakiew D, Jones RC: Sperm maturation, f luid
transport, and secretion and absorption of
protein in the epididymis of the echidna,
Tac hyg los s us a cul e atu s . J Reprod Fertil 68:
445–456 (1983).
Dohm JC, Tsend-Ayush E , Rein hardt R, Gr utzner
F, Himmelbauer H: Disruption and pseudo-
autosomal localization of the major histo-
compatibility complex in monotremes. Ge-
nome Biol 8:R175 (2007).
Fawcett DW: The mammalian spermatozoon.
Dev Biol 44:
395–436 (1975).
Flannery TF, Groves CP: A revision of the genus
Zaglossus (Monotremata, Tachyglossidae)
with description of new species and subspe-
cies. Mammalia 62:
367–396 (1998).
Foster HA, Abeydeera LR, Griffin DK, Bridger
JM: Non-random chromosome positioning
in mam malian sper m nuclei, with migr ation
of the sex chromosomes during late sper-
matogenesis. J Cell Sci 118:
1811–1820
(2005).
Freyer C, Zeller U, Renfree MB: The marsupial
placenta: a phylogenetic analysis . J Exp Zoo-
log A Comp Exp Biol 299:
59–77 (2003).
Grafodatskaya D, Rens W, Wallis MC, Trifonov
V, O’Brien P C, et al: Search for the sex-de-
termining switch in monotremes: mapping
WT1, SF1, LHX1, LHX2, FGF9, WNT4,
RSPO1 and GATA4 in platypus. Chromo-
some Res 15:
777–785 (2007).
Greaves IK, Rens W, Ferguson-Smit h MA, Grif-
fin D, Marshall Graves JA: Conservation of
chromosome arrangement and position of
the X in mammalian sperm suggests func-
tional significance. Chromosome Res 11:
503–512 (2003).
Green B, Griffiths M, Newgrain K: Intake of
milk by suckling echidnas (Tachyglossus
aculeatus) . Comp Biochem Physiol A 81:
441–444 (1985).
Griff iths AL, Renf ree MB, Shaw G, Watts LM,
Hutson JM: The tammar wallaby (Macrop us
eugenii) and the Sprague-Dawley rat: com-
parati ve anatomy and physiolog y of ingui no-
scrotal testicular descent. J Anat 183:
441–
450 (1993).
Gr iffith s M: Echidnas (Per magon Press, Ox ford,
London, New York 1968).
Griffiths M: The Biology of the Monotremes
(Academic Press, New York, San Fransisco,
London 1978).
Gr iffit hs M: Monotremes, in Ne ill EKaJ (ed): En-
cyclopedia of Reproduction, pp 295–302
(Academic Press, New York 1999).
Gruetzner F, Ashley T, Rowell DM, Marsha ll
Graves JA: How did t he platypus get its sex
chromosome chain? A comparison of mei-
otic multiples and sex chromosomes in
plants and animals. Chromosoma 115:
75–88
(2006).
Grützner F, Graves JAM: A plat ypus’ eye view of
the mammalian genome. Curr Opin Genet
Dev 14:
642–649 (2004).
G r üt z ne r F, Re ns W, Ts en d- Ay u sh E , E l- M og h ar -
bel N, O’Brien PC, et al: In the platy pus a
meiotic cha in of ten sex chromosomes s hares
genes with the bird Z and mammal X chro-
mosomes. Nature 432:
913–917 (2004).
Haaf T, Ward DC: Higher order nuclear struc-
ture in m ammalia n sperm reveale d by in situ
hybridization and extended chromatin f i-
bers. Exp Cell Res 219:
604–611 (1995).
Hazzouri M, Rousseaux S, Mongelard F, Usson
Y, Pelletier R, et a l: Genome organization in
the human sperm nucleus studied by FISH
and confocal microscopy. Mol Reprod Dev
55:
307–315 (2000).
Hol land N, Jackson SM: R eproductive behav iour
and food consumption associated wit h the
captive breeding of platypus (Ornith orhyn-
chus anatinus) . J Zool (Lond) 256:
279–288
(2002).
Home E: A des cription of the anatomy of t he Or-
nithorhyncus paradoxus. Phil Trans R Soc
1802:67–84 (1802a).
Home E: A description of the anatomy of Orni-
thorhynchus hystrix. Phil Trans R Soc 1802:
348–364 (1802b).
Hughes RL, Ha ll LS: Early development and em-
bryology of the platypus. Phil Trans R Soc
Lond B Biol Sci 353:
1101–1114 (1998).
Johnson SD, Smith D, Pyne M, Stenzel D, Holt
WV: One-sided ejac ulation of ech idna sperm
bundles. Am Nat 170:E162–164 (2007).
Jones RC: Studies of the structure of the head of
boar sper matozoa from the e pididymis. J Re -
prod Ferti l Suppl 13:
51–64 (1971).
Jones RC: Evolution of t he vertebrate epididy-
mis. J Reprod Fertil Suppl 53:
163–182
(19 98).
Jones RC: Evolution of t he epididymis, in Ro-
baire B, Hinton BT (eds): The Epididymis.
From Molecules to Clinic al Practice: A com-
prehensive Survey of the Ef ferent Ducts, the
Epididymis and the Vas Deferens, pp 11–33
(Kluwer Academic/Plenum Publishers, New
York, London 2002).
Jones RC, Brosnan MF: Studies of the deferent
ducts from the testis of the Af rican elephant
Loxodonta africana . I. Structural differenti-
ation. J Anat 132:
371–386 (1981).
Jones RC, Lin M: Spermatogenic cycle in birds
and mammals, in Milligan SR (ed): The Ox-
ford Reviews of Reproductive Biology, pp
233–264 (Oxford University Press, Oxford,
New York, Tokyo 1993).
Jones RC, Stone GM, Zupp J: Reproduction in
the ma le echidna, in Augee ML (ed): Platy-
pus and Ec hidnas, pp 115–126 (Royal S ociety
of New South Wales, Sydney 1992).
Grützner /Nixon /Jones
Sex Dev 2008;2:115–127
126
Jones RC, Djakiew D, Dacheux JL: Adaptations
of the echidna (Tachyglossus aculateus) for
sperm production, particularly in an arid en-
vironment. Austr Mammal 26:
199–204
(20 04).
Jones RC, Dacheux JL, Nixon B, Ecroyd HW:
The role of the epid idymis in sp erm competi-
tion. Asian J Androl 9:
493–499 (2007).
Jo vi ne L, Qi H, Wi ll iam s Z, Lit sche r ES , Was sar-
man PM: Features that affect secretion and
assembly of zona pellucida glycoproteins
during mammalian oogenesis. Soc Reprod
Fertil Suppl 63:
187–201 (2007).
Kamat AA, Feng S, Bogatcheva NV, Truong A,
Bishop CE, Agoulnik AI: Genetic targeting
of relaxin and insulin-like factor 3 receptors
in mice. Endocrinology 145:
4712–4720
(20 04).
Kawasaki K, Weiss KM: Mineralized tissue and
vertebrate evolution: the secretory calcium-
binding phosphoprotein gene cluster. Proc
Natl Acad Sci USA 100:
4060–4065 (2003).
Kikuchi M, Kawano K, Nitta K: Calcium-bind-
ing and structural stability of echidna and
canine milk lysozymes. Protein Sci 7:
2150–
2155 (1998).
Le ster KS, Boyde A, Gil keson C, Archer M: M ar-
supial and monotreme enamel structure.
Scanning Microsc 1:
401–420 (1987).
Lin M, Jones RC: Spermiogenesis and spermia-
tion in t he Japanese quail (Cot urn ix co tur nix
japonica) . J Anat 183:
525–535 (1993).
Lin M, Jones RC: Spermiogenesis and spermia-
tion in a monotreme mammal, the platypus,
Ornithorhynchus anatinus . J Anat 196:
217–
232 (2000).
Mackay S, Xie Q, Ullmann SL, Gilmore DP,
Payne AP: Postnatal development of the re-
productive system in the grey short-tailed
opossum, Monodel phis domestic a . A nat Em-
bryol (Berl) 208:
121–133 (2004).
Meckelio IF: Ornithorhy nchi paradoxi Descrip-
tio Anatomica. Gerhardum Fleischerum
Lipsiae (1826).
Messer M, Kerry KR: Milk carbohydrates of the
echidna a nd the platypus. Science 180:
201–
203 (1973).
Messer M, Gadiel PA, Ralston G, Griff iths M:
Carbohydrates of the milk of the platypus.
Aust J Biol Sci 36:
129–137 (1983).
Messer M, Griffiths M, Rismiller PD, Shaw DC:
Lactos e synthesis i n a monotreme, the echid-
na (Tachyglossus aculeatus) : isolation and
amino acid sequence of echidna alpha-lact-
albumin. Comp Biochem Physiol B Biochem
Mol Biol 118:
403–410 (1997).
Meyer-Ficca M, Muller-Navia J, Scherthan H:
Clustering of pericentromeres initiates in
step 9 of spermiogenesis of the rat (Rattus
norvegicus) and contributes to a wel l defined
genome architecture in the sperm nucleus. J
Cell Sci 111:
1363–1370 (1998).
Morel L, Dufaure JP, Depeiges A: The lipocalin
sperm coating lizard epididymal secretory
protein family: mRNA structural analysis
and sequential expression during the annual
cycle of the lizard, Lacerta vivipara. J Mol
Endocrinol 24:
127–133 (2000).
Murtagh C: A unique cy togenetics system
in monotremes. Chromosoma 65:
37–57
(19 77).
Nef S, Parada LF: Cryptorchidism in mice mu-
tant for Insl3 . Nat Genet 22:
295–299 (1999).
Niwa H, Toyooka Y, Shimosato D, Strumpf D,
Takahashi K, Yagi R, Rossant J: Interaction
between Oct3/4 and Cdx2 determines troph-
ectoderm differentiation. Cell 123:
917–929
(2005).
Niwa H, Sekita Y, Tsend-Ayush E, Grützner F:
Platypus Pou5f1 revea ls the first steps in the
evolution of trophectoderm differentiation
and pluripotency in mammals. Evol Dev, in
press (2008).
Oftedal OT: The mammary gland and its origin
during synapsid evolution. J Mammary
Gland Biol Neoplasia 7:
225–252 (2002).
Ono R , Nakamura K, I noue K, Naruse M, Usa mi
T, et al: Deletion of Peg10 , a n imprinted gene
ac qu i re d f ro m a r et r ot ra ns po so n, ca us es ea r-
ly embryonic le thality. Nat Gene t 38:
101–106
(2006).
Park JL , Semyonov J, Chang CL, Yi W, Warren
W, Hsu SYT: Origin of INSL3 - med iated tes-
ticular descent in therian mammals. Ge-
nome Res 18:
974–985 (2008).
Raymond CS, Kettlewel l JR, Hirsch B, Bardwell
VJ, Zarkower D: Expression of Dmrt1 in the
genital ridge of mouse and chicken embr yos
suggests a role in vertebrate sexual develop-
ment. Dev Biol 215:
208–220 (1999).
Rens W, Grützner F, O’Brien P C, Fairclough H,
Graves JA, Ferguson-Smit h MA: Resolution
and evolution of t he duck-billed platypus
karyotype with an X1Y1X2Y2X3Y3X4Y4
X5Y5 male sex chromosome constitution.
Proc Natl Acad Sci USA 101:
16257–16261
(20 04).
Rens W, O’Brien PC, Grützner F, Clarke O,
Graphodatskaya D, et al: The multiple sex
chromosomes of platypus and echidna are
not completely identical and several share
homology wit h the avian Z. Genome Biol 8:
R243 (2007).
Retief JD, Winkfein RJ, Dixon GH: Evolution of
the monotremes. The sequences of the prot-
amine P1 gene s of platypus and ech idna. Eur
J Biochem 218:
457–461 (1993).
Rismiller PD: Field observations on Kangaroo
Island echidnas (Tachyglossus aculateus
multiaculateus) during the breeding season,
in Augee ML (ed): Plat ypus and Ech idnas, pp
101–105 (Royal Zoological Society of New
South Wales, Sydney 1992).
Rismiller PD: Overcoming a prickly problem.
Aust Nat Hist 4:
22–29 (1993).
R ismille r PD: The Echidna – Au stralia’s Enig ma.
(Hugh Lauter Levin, Southport 1999).
Rismiller PD, McKelvey MW: Frequency of
breeding and recruitment in the short-
beaked echidna, Tachyglossus aculeatus. J
Mammal 81:
1–17 (2000).
Rowe T, Rich TH, Vickers-Rich P, Springer M,
Woodburne MO: The oldest platy pus and its
beari ng on divergence ti ming of the platy pus
and echidna clades. Proc Natl Acad Sci USA
105:
1238–1242 (2008).
Selwo od L, Johnson MH: Trophoblas t and hypo-
blast in the monotreme, marsupial and eu-
theria n mammal: evolution and origins.
Bioessays 28:
128–145 (2006).
Se tchell BP: The Mamm alian Testis (Elek B ooks,
London Cornell University Press, London
1978).
Sharp JA, Lefèvre C, Nicholas KR: Molecu lar
evolution of monotre me and marsupia l whey
acidic protein genes. Evol Dev 9:
378–392
(2007).
Solovei IV, Joffe BI, Hori T, Thomson P, Mizuno
S, Macgregor HC: Unordered arrangement
of chromosomes in the nuclei of chicken
spermatozoa. Chromosoma 107:
184 –188
(19 98).
Stewart S, Bausek N, Wohlrab F, Schneider WJ,
Horrocks A, Wishart G: Species specificity
in avian sperm:perivitelline interaction.
Comp Biochem Physiol A Mol I ntegr Physiol
137:
657–663 (2004).
S uz uk i S, Ono R, N ar ita T, Pa sk A J, S haw G, e t a l:
Retrotr ansposon silenc ing by DNA methyla-
tion can drive mammalian genomic im-
printing. PLoS Genet 3:e55 (2007).
Teahan CG, McKenzie HA, Shaw DC, Griffiths
M: The isolation and amino acid sequences
of echidna (Tachyglossus aculeatus) milk ly-
sozyme I and II. Biochem Int 24:
85–95
(19 91).
Temple-Smith PD: Seasonal breeding biology of
the platy pus, Ornithorhynchus anatinus
(Shaw, 1799). PhD thesis submit ted to Aust
Nat Univ (1973).
Temple-Smith P, Grant T: Uncertain breeding:
a short history of reproduction in mono-
tremes. Reprod Fertil Dev 13:
487–497
(2001).
van der Schoot P: Foetal genital development in
Hyrax cape nsis , a species with primary testi-
condia: proposal for the evolution of Hunt-
er’s gubernaculum. Anat Rec 244:
386–401
(19 96).
van Rheede T, Bastiaans T, Boone DN, Hedges
SB, de Jong WW, Madsen O: The platypus is
in its place : nuclear genes and inde ls confirm
the sister group relation of monotremes and
Therians. Mol Biol Evol 23:
587–597 (2006).
Veyr unes F, Waters PD, Miet hke P, Rens W, Mc-
Millan D, et al: Bird-like sex chromosomes
of platypus imply recent origin of mammal
sex chromosomes. Genome Res 18:
965–973
(20 08).
Monotreme Reproduction Sex Dev 2008;2:115–127
127
Wallis MC, Waters PD, Delbridge ML, Kirby PJ,
Pask AJ, et al: Sex determination in platypus
and echidna: autosoma l location of SOX3
confirms the absence of SRY from mono-
tremes. Chromosome Res 15:
949–959
(2007).
Warren WC, Hillier LW, Marshall Graves JA,
Birney E , Ponting CP, et al: Genome analy sis
of the platy pus revea ls unique signatures of
evolution. Nature 453:
175–183 (20 08).
Waters PD, Delbridge ML, Deakin JE, El-
Mogharbel N, Kirby PJ, et al: Autosoma l lo-
cation of genes f rom the conserved mamma-
lian X in the platy pus (Ornith orhy nchu s
anatinus) : implications for mammalian sex
chromosome evolution. Chromosome Res
13:
401–410 (2005).
Watson JM, Mey ne J, Graves JAM: Chromo som-
al composition and position in the echidna
meiotic translocation chain, in Augee ML
(ed): Platypus a nd Echidnas, pp 53– 63 (Roy-
al Zoological Society of New South Wales,
Sydney 1992).
Watson JM, Meyne J, Graves JA: Ordered tan-
dem arrangement of chromosomes in the
sperm heads of monotreme mammals. Proc
Natl Acad Sci USA 93:
1020 0–10205 (1996).
Werdelin L, Nilsonne A: The evolution of the
scrotum and testicular descent in mammals:
a phylogenetic v iew. J Theor Biol 196:
61–72
(1999).
Wilhelm D, Koopman P: The mak ings of male-
ness: toward s an integrated v iew of male sex-
ual development. Nat Rev Genet 7:
620–631
(2006).
Woodburne MO, Rich TH, Springer MS: The
evolution of tr ibospheny and the a ntiquity of
mamma lian clades. Mol Phylogenet Evol 28:
360–385 (2003).
Zal ensk y A, Z alen skay a I: O rgan iza tion of c hro-
mosomes in spermatozoa: an additional lay-
er of epigenetic information? Biochem Soc
Trans 35:
609–611 (2007).
Zalensky AO, Allen MJ, Kobayashi A, Zalenska-
ya IA, Balhorn R, Bradbury EM: Well-de-
fined genome architecture in the human
sperm nucleus. Chromosoma 103:
577–590
(19 95).
Zimmermann S, Steding G, Emmen JM, Brink-
mann O, Nayernia K, et al: Targeted disrup-
tion of the Insl3 gene causes bilateral crypt-
orchidism. Mol Endocrinol 13:
681–691
(1999).
