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The duck-billed platypus and short-beaked echidna are iconic species in Australia. Their morphology and physiology 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 molecular 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 biology. In this review we focus on the reproductive biology of monotremes and discuss works that unravel genes involved in lactation, testicular descent, gamete biology and fertilization, 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.
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Review Article
Sex Dev 2008;2:115–127
DOI: 10.1159/000143429
Reproductive Biology in Egg-Laying
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 Echidna Fertilization Lactation Ova
Platypus Reproduction Sex chromosomes Sperm
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 .au
© 200 8 S. Karger AG, Basel
1661–5425/08/0023– 0115$24.50/0
Accessible online at:
Grützner /Nixon /Jones
Sex Dev 2008;2:115–127
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
Monotreme Reproduction Sex Dev 2008;2:115–127
( 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-
Grützner /Nixon /Jones
Sex Dev 2008;2:115–127
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
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
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-
Monotreme Reproduction Sex Dev 2008;2:115–127
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
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
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.,
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
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.
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.
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... Also, whether these particles are responsible for agglutination. Grützner et al. 27 reported that the epithelial cells of the epididymis produce and secrete a specific protein(s) that is needed for the formation of sperm bundles in monotremes. The authors also reported that the dispersal of these bundles is dependent upon epididymal protein(s) interactions. ...
... In echidna and platypus, sperm are arranged parallel to each other to increase the bundle's forward velocity 28 . The motility of the bundle in echidna is approximately three times faster than that of lonesome sperm 27 . The formation of such sperm bundles in echidna is considered an evolutionary adaptation to assert dominance because the females are promiscuous and commonly mate with several males. ...
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Fertility in birds is dependent on their ability to store adequate populations of viable sperm for extended durations in sperm storage tubules (SSTs). The exact mechanisms by which sperm enter, reside, and egress from the SSTs are still controversial. Sharkasi chicken sperm showed a high tendency to agglutinate, forming motile thread-like bundles comprising many cells. Since it is difficult to observe sperm motility and behavior inside the opaque oviduct, we employed a microfluidic device with a microchannel cross-section resembling close to that of sperm glands allowing for the study of sperm agglutination and motility behavior. This study discusses how sperm bundles are formed, how they move, and what role they may have in extending sperm residency inside the SSTs. We investigated sperm velocity and rheotaxis behavior when a fluid flow was generated inside a microfluidic channel by hydrostatic pressure (flow velocity = 33 µm/s). Spermatozoa tended to swim against the flow (positive rheotaxis) and sperm bundles had significantly lower velocity compared to lonesome sperm. Sperm bundles were observed to swim in a spiral-like motion and to grow in length and thickness as more lonesome sperm are recruited. Sperm bundles were observed approaching and adhering to the sidewalls of the microfluidic channels to avoid being swept with fluid flow velocity > 33 µm/s. Scanning and transmission electron microscopy revealed that sperm bundles were supported by a copious dense substance. The findings show the distinct motility of Sharkasi chicken sperm, as well as sperm's capacity to agglutinate and form motile bundles, which provides a better understanding of long-term sperm storage in the SSTs.
... The monotreme lineage includes platypus (Ornithorhynchus anatinus) and two genera of echidnas, long-beaked echidna (Zaglossus spp.) and short-beaked echidna (Tachyglossus aculeatus) [14]. They lay softshelled eggs, and the youngs that emerge after hatching are altricial and immunologically naive [11,13,15]. These newly hatched youngs are vulnerable, blind and hairless and are fed solely by mother's milk. ...
... We, therefore, utilized the NMR spectroscopy and performed 2D 1 H- 15 N HSQC experiments to validate our earlier findings. For this, the 15 N-labelled rEchAMP was purified from C41(DE3) cells grown in M9 medium supplemented with 15 N-NH 4 Cl as per the procedure described in materials and methods. ...
Background: Antibiotic resistance is a problem that necessitates the identification of new antimicrobial molecules. Milk is known to have molecules with antimicrobial properties (AMPs). Echidna Antimicrobial Protein (EchAMP) is one such lactation specific AMP exclusively found in the milk of Echidna, an egg-laying mammal geographically restricted to Australia and New Guinea. Previous studies established that EchAMP exhibits substantial bacteriostatic activity against multiple bacterial genera. However, the subsequent structural and functional studies were hindered due to the unavailability of pure protein. Results: In this study, we expressed EchAMP protein using a heterologous expression system and successfully purified it to >95% homogeneity. The purified recombinant protein exhibits bacteriolytic activity against both Gram-positive and Gram-negative bacteria as confirmed by live-dead staining and scanning electron microscopy. Structurally, this AMP belongs to the family of intrinsically disordered proteins (IDPs) as deciphered by the circular-dichroism, tryptophan fluorescence, and NMR spectroscopy. Nonetheless, EchAMP has the propensity to acquire structure with amphipathic molecules, or membrane mimics like SDS, lipopolysaccharides, and liposomes as again observed through multiple spectroscopic techniques. Conclusions: Recombinant EchAMP exhibits broad-spectrum bacteriolytic activity by compromising the bacterial cell membrane integrity. Hence, we propose that this intrinsically disordered antimicrobial protein interact with the bacterial cell membrane and undergoes conformational changes to form channels in the membrane resulting in cell lysis. General significance: EchAMP, the evolutionarily conserved, lactation specific AMP from an oviparous mammal may find application as a broad-spectrum antimicrobial against pathogens that affect mammary gland or otherwise cause routine infections in humans and livestock.
... Grützner and coresearchers [31] reported that the epididymal epithelium of monotremes produces and secretes a specific protein(s) that is required for the formation of sperm bundles. In both short-beaked echidnas and platypus, Nixon et al., [32] found that an epididymal secreted protein, acidic cysteine-rich osteonectin; SPARC, contributes to sperm bundle formation and that the dispersal of these bundles is associated with the loss of this protein. ...
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To ensure survival, some unique features can be distinguished in birds that help them maintain reproduction. These features include the ability to store sperm for long periods within the utero-vaginal junction, a high sperm concentration per ejaculate, and polyspermy fertilization. Sperm face many challenges prior to fertilization. After copulation, most ejaculated sperm exit the female reproductive tract, and less than 1% continue in an attempt to achieve fertilization. In addition, egg size is substantially larger than sperm size because of the presence of the egg yolk. This results in a large number of sperm penetrating the egg away from the oocyte. These challenges have triggered evolutionary changes to maintain the existence of many species, such as the enormous relative size of the testis, which produces billions of sperm each day, and the ability to store viable sperm for long periods in the oviduct to ensure asynchronous fertilization. This chapter discusses several contemporary and sometimes controversial points regarding sperm behavior and their storage in the oviduct.
... The bundle formation involves the binding of sperm heads together. An epididymal secretory protein, acidic cysteine-rich osteonectin (SPARK), that is responsible for bundle formation was identified 40,41 . In addition, the dispersal of these bundles was associated with the loss of this protein 40 . ...
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A unique sperm behavior was observed in Egyptian chickens. Sperm showed a tendency to agglutinate forming motile thread-like bundles. Sperm agglutination behavior, kinematics, and some morphometric measures were studied in relation to sperm competition and fertility duration in Sharkasi and Dandarawi chickens. Sperm tendency to agglutinate was assessed by examining sperm morphology using scanning electron microscopy, Acridine orange-stained semen smears using fluorescence microscopy, and recording videos of sperm under phase contrast microscope. Sperm velocity and morphometric measures were evaluated using image-J software. To assess sperm competition, Sharkasi and Dandarawi hens were artificially inseminated by semen pools possessing equal number of Sharaksi and Dandarawi sperm. Artificial insemination was repeated ten times. The eggs obtained were incubated, and the hatchlings were discriminated as descending from Sharkasi or Dandarawi fathers according to their phenotype. To assess the fertility duration, Sharkasi and Dandarawi hens were inseminated by semen collected from roosters of the same strain. Eggs were collected for a period of 28 days post-insemination and incubated. Sharkasi spermatozoa showed higher tendency to agglutinate forming longer and thicker motile bundles. No significant differences were observed in sperm curvilinear and straight line velocity and in sperm morphometric measures between Sharkasi and Dandarawi chickens. Sharkasi roosters fathered 81.6% and 67.7% of the hatchlings produced by Sharkasi and Dandarawi mothers, respectively. The fertility period in Sharkasi and Dandarawi was 22 and 14 days, respectively. We suggest that the differences seen in sperm competitiveness and fertility duration can be attributed to sperm agglutination behavior.
... Asymmetric gonads are found in other vertebrate species (Yu, 1998), including platypus, which have a functioning ovary only on the left side (Grützner et al., 2008), as well as contrasting directional asymmetry found in frogs Zhou et al., 2011) and birds (Friedmann, 1927). Males in many avian species have a smaller right testis than the left (Lake, 1981). ...
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Osteoglossiformes are an order of “bony tongue” fish considered the most primitive living order of teleosts. This review seeks to consolidate known hypotheses and identify gaps in the literature regarding the adaptive significance of diverse reproductive traits and behavior of osteoglossiforms within the context of sperm competition and the wider lens of sexual selection. Many of the unusual traits observed in osteoglossiforms indicate low levels of sperm competition; most species have unpaired gonads and mormyroids are the only known vertebrate species with aflagellate sperm. Several osteoglossiform families have reproductive anatomy associated with internal fertilization but perform external fertilization, which may be representative of the evolutionary transition from external to internal fertilization and putative tradeoffs between sperm competition and the environment. They also employ every type of parental care seen in vertebrates. Geographically widespread and basally situated within teleosts, Osteoglossiforms present an effective study system for understanding how sperm competition and sexual selection have shaped the evolution of teleost reproductive behavior, sperm and gonad morphology, fertilization strategies, courtship and paternal care, and sexual conflict. We suggest that the patterns seen in osteoglossiform reproduction are a microcosm of teleost reproductive diversity, potentially signifying the genetic plasticity that contributed to the adaptive radiation of teleost fishes. This article is protected by copyright. All rights reserved.
... This, with the gradual caudal movement and loss of the residual cytoplasmic droplet, the acquisition of forward progressive motility and development of the fertilizing capacity, provides evidence of the complex role of the mammalian epididymis in post-testicular maturation of spermatozoa. Monotreme spermatozoa are released as sperm bundles at ejaculation (Fig. 6(b)) and are thought to only separate again into individual spermatozoa when they reach the site of fertilization in the oviduct (Gruzner et al., 2008). ...
In vertebrates, sperm structure is highly conserved with the head containing the hereditary information (DNA) neatly packaged into the nucleus and a neck or connecting piece usually attaching the head to the sperm tail or flagellum. The sperm tail is usually subdivided into a midpiece containing the mitochondria - the power generators of the cell - and a principal piece, the longest part of the spermatozoon, that uses energy from the mitochondria to move the tail. Distinguishing structural specialisations in the different vertebrate groups include: a helical head shape in sharks; side fins in bony fish sperm tails producing enhanced motility; an undulating tail membrane in amphibians; a larger mid-piece, well-developed long fibrous sheath and linear mitochondrial cristae in reptiles; helical head and mitochondrial helix in birds; and a more complex sperm maturation process in the epididymis, and tail modifications and sperm aggregations that influence motility in mammals. There is a close relationship between the evolution of structural and functional specialisations of vertebrate spermatozoa and the need to ensure successful fertilization. For example, evidence from comparative studies suggests that longer spermatozoa swim faster. Sperm motility ensures that the spermatozoon makes contact with the oocyte during fertilization and enzymes released from the acrosome on the sperm head, in addition to sperm motility, assist the fertilizing spermatozoon to reach the oocyte and incorporate its nucleus into the oocyte cytoplasm. Fusion of sperm and egg membranes is a prerequisite for fertilization and for the sperm nucleus to enter the cytoplasm of the oocyte. Other factors like mode of fertilisation, chemical characteristics of the seminal fluid, milieu of the female reproductive tract and sperm storage requirements may have also influenced the evolution of sperm specialisations.
... As the earliest offshoot of the mammalian lineage, monotremes (platypus and echidna) represent a unique evolutionary model with which to explore the enigmatic origins of post-testicular sperm maturation (Warren et al., 2008). Thus, whilst monotremes exhibit the main distinguishing characteristics of Mammalia, key aspects of their reproductive biology are more closely aligned with that of reptiles and birds (Grutzner et al., 2008;Jones et al., 2018). For instance, they lay eggs that are incubated externally and monotreme sperm have retained a vermiform shape analogous to that of the sauropsids (Lin and Jones, 2000) (Fig. 1). ...
Competition to achieve paternity has coerced the development of a multitude of male reproductive strategies. In one of the most well-studied examples, the spermatozoa of all mammalian species must undergo a series of physiological changes as they transit the male (epididymal maturation) and female (capacitation) reproductive tracts prior to realizing their potential to fertilize an ovum. However, the origin and adaptive advantage afforded by these intricate processes of post-testicular sperm maturation remain to be fully elucidated. Here, we review literature pertaining to the nature and the physiological role of epididymal maturation and subsequent capacitation in comparative vertebrate taxa including representative species from the avian, reptilian, and mammalian lineages. Such insights are discussed in terms of the framework they provide for helping to understand the evolutionary significance of post-testicular sperm maturation.
... Nevertheless, in terms of their reproductive systems they have evolved sophisticated cooperative sperm swimming abilities. The spermatozoa are filiform and bear more resemblance to avian and reptilian spermatozoa than to spermatozoa from other mammals, and during sperm maturation, the spermatozoa become organized into bundles (figure 3c) of approximately 100 cells each, which swim together in synchrony and move forward at about three times the speed of individual spermatozoa [77,78]. It has been argued [79] that this aspect of their biology provides important clues to the evolution of sperm maturation in the context of cooperative sperm swimming abilities. ...
Full-text available
While only a single sperm may fertilize the egg, getting to the egg can be facilitated, and possibly enhanced, by sperm group dynamics. Examples range from the trains formed by wood mouse sperm to the bundles exhibited by echidna sperm. In addition, observations of wave-like patterns exhibited by ram semen are used to score prospective sample fertility for artificial insemination in agriculture. In this review, we discuss these experimental observations of collective dynamics, as well as describe recent mechanistic models that link the motion of individual sperm cells and their flagella to observed collective dynamics. Establishing this link in models involves negotiating the disparate time- and length scales involved, typically separated by a factor of 1000, to capture the dynamics at the greatest length scales affected by mechanisms at the shortest time scales. Finally, we provide some outlook on the subject, in particular, the open questions regarding how collective dynamics impacts fertility. This article is part of the theme issue ‘Multi-scale analysis and modelling of collective migration in biological systems’.
... The early work covering monotreme reproduction was reviewed by Griffiths (1978) and then more specifically by Temple-Smith and Grant (2001). Further, there has been a renewed interest in the subject (Grützner et al., 2008) since the platypus genome was sequenced (Warren et al., 2008). ...
Many aspects of primate reproductive anatomy and physiology have been influenced by copulatory and postcopulatory sexual selection, especially so in taxa where multiple-partner matings by females result in the sperm of rival males competing for access to a given set of ova (sperm competition). However, the female reproductive system also exerts profound effects upon sperm survival, storage and transport, raising the possibility that female traits influence male reproductive success (via cryptic female choice). Current knowledge of sperm competition and cryptic choice in primates and other mammals is reviewed here. The relevance of these comparative studies to our understanding of human reproduction and evolution is discussed.
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As part of a radiotelemetric study of echidnas (Tachyglossus aculeatus) in south-east Queensland focusing on thermal relations, we were able to confirm and extend present knowledge of echidna reproduction. Mating was concentrated in July and August, as elsewhere, but we found that echidnas have the ability to conceive successfully a second time within the one season, apparently in response to losing the first young. Echidnas in this area of south-east Queensland may be able to attempt breeding every year. Our data supports published estimates of gestation in the range of 20 to 23 days. Females spent two to three weeks in a plugged 'incubation' burrow, maintaining a high and stable body temperature for a period encompassing the last few days of gestation, all of incubation and the first few days of the hatchling's life. The single young was carried in the female's pouch for 45-50 days, attaining a body weight of approximately 200g before being stowed in a different plugged 'nursery' burrow. We describe the first detailed timing of a female's visits to suckle her young. She visited regularly, every six days at first, gradually increasing in frequency to about every four days before the visits ceased and, presumably, the newly-independent young emerged at a calculated five and a half months of age.
Full-text available
We documented the breeding frequency of 25 wild female short-beaked echidnas, Tachyglossus aculeatus multiaculeatus, fate of young, and recruitment of subadults into a population over a 7-year period. Echidnas had 1 annual breeding period with courtship lasting 7-37 days. Females monitored were observed to mate only once per breeding season with 1 male. All females that mated produced a single fertile egg 23 days ± 1 SE after mating. Frequency of reproduction differed among individuals and years. Number of young hatched each season varied between 1 and 9. A total of 22 hatchlings was produced by 17 different females between 1990 and 1996. Seven young died before weaning, 8 were known to survive to weaning, and the fates of the remaining 7 were unknown. Number of new subadults found in the study site was comparable with the number of young known to have been produced each year.
The echidna is one of the world’s most extraordinary creatures. It is a living fossil whose relatives were walking the earth over 100 million years ago. Like the platypus, it is a mammal that lays eggs. And, like all mammals, it has fur and produces milk. This book describes the echidna’s lifestyle and the adaptations that have made it so successful. It draws on the latest research into these strange creatures, covering their evolution, anatomy, senses, reproduction, behaviour, feeding habits and metabolism. The authors reveal some fascinating new findings, showing how echidnas are masters of their environment, and not simply some sort of mammal ‘test model’ that went wrong. A final chapter on conservation includes information on captive diet and management.
The epididymal epithelial cells of the lizard (Lacerta vivipara) produce large amounts of specific proteins under androgenic control. Amongst them, a major protein family that binds to the head of spermatozoa, the lizard epididymal secretory protein (LESP) family, has been identified as a member of the lipocalin superfamily. LESPs are composed of 9 elements that present an identical molecular mass of 18 000 Da but have a large range of pHi (3.5 to 9). The structural analysis of this protein family was performed by the determination and comparison of both the aminoterminal sequence of each element and the complete sequence of three specific LESP cDNA clones. When not identical, LESP elements present randomly dispatched nucleotide and amino acid substitutions, indicating the existence of at least five LESP mRNA populations encoded by a multigenic family. We determined that these LESP genes are differentially expressed during the annual epididymal cycle.