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“Comparative sequence analysis of canine
progesterone receptor-B-Upstream
Segment and the relationship of this
segment with the estrous cycle in Canidae”
Drs. Kim van Dam
Supervisors: MSc Ana Gracanin
Dr. Ir. Jan A. Mol
September 2009
Department of Clinical Sciences of Companion Animals
Faculty of Veterinary Medicine
Utrecht University
Table of contents
Abstract ……………………………………………………………………………….2
Introduction ...…………………………………………………………………………3
Part A: Comparative sequence analysis of canine progesterone receptor-BUpstream Segment
 Introduction …………………………………………………………………...4
 Aim of research ……………………………………………………………….6
 Material and methods………………………………………………………….7
 Results…………………………………………………………………………8
 Discussion …………………………………………………………………...11
Part B: Review: Relationship between estrous cycle and sequence of progesterone
receptor-B-Upstream Segment in Canidae
 Introduction ………………………………………………………………….13
 Aim of this review.…………………………………………………………...13
 Literature search……………………………………………………………...14
 Discussion …………………………………………………………………...18
Conclusion……………………………………………………………………………21
References …………………………………………………………………………...22
Appendix …………………………………………………………………………….25
1
Abstract
The dog seems to have unique features concerning activation function (AF) 3 domain
and putative phosphorylation sites of the progesterone receptor-B-Upstream Segment
(BUS) in comparison with mice, rats, rabbits, horses, cattle and human. The first aim
of this research is to assess if the dog is still unique when compared to wolves, seals,
ferrets, cats and pigs. A comparative sequence analyses was performed. In the wolf,
the same differences as the dog were found in the AF3 domain and putative
phosphorylation sites. Therefore, the differences found in AF3 domains and putative
phosphorylation sites do not seem to be unique for the dog, but might be unique for
wolf-like canids or Canidae in general.
According to many authors, canids or Canidae in general have a unique reproduction
cycle. One of the remarkable features of this cycle is the prolonged luteal phase,
during which progesterone is the dominating hormone. The question rises if there is a
connection between the unique sequence of BUS and the apparently unique prolonged
luteal phase of Canidae. Answering this question is the second aim of this study.
Therefore, a short review was performed. Besides Canidae, also some members of the
Ursidae and Phocidae families showed a prolonged luteal phase. These species were
all monoestrous females with spontaneous ovulation. The sequence analysis of the
seal showed no changes in the AF3 domain and the changes in the putative
phosphorylation sites differed from the changes in the dog and wolf. Therefore, no
connection between BUS and the estrous cycle could be assessed in Canidae.
2
Introduction
One of the most common tumors of the bitch is the tumor of the mammary gland
(Johnston et al., 2001). A lot of research on the cause and development of mammary
gland tumors has been done. The steroid hormone progesterone plays a major role in
the development of mammae tumors in dogs. To exert its function, progesterone
needs a receptor, the progesterone receptor (PR). The PR might play an important role
in the development of canine mammary gland tumors, and will therefore be the main
focus of the current research. Gracanin et al. (unpublished) already did a comparative
analysis of the sequence of the canine PR. The canine PR had some unique features at
the N-terminal region compared with the PR of cattle, human, rabbits, horses, mice
and rats. Additional sequencing of one of the unique parts of the canine PR, a 30 bp
insertion, revealed that also the PR of the wolf had the same 30 bp insertion. Seals,
ferrets, cats and pigs do not have a 30 bp insertion. The first aim of this research is to
sequence and compare the two other unique features of the canine PR with the PR of
the wolf, the seal, the ferret, the cat and the pig to further assess if these features are
unique for the dog.
Another remarkable feature of the female dog, Canidae in general, is their prolonged
luteal phase. In this research, the luteal phase is called prolonged if the luteal phase
not ends within three weeks after the luteal phase of a non-pregnant female is started.
Therefore, female Canidae are exposed to long-lasting high concentrations of
progesterone during each estrous cycle. It might be possible that this prolonged
exposure to high long-lasting levels of progesterone evoked adaptations in the
progesterone-signaling cascade, which includes the PR. The second aim of this
research is to assess whether this might be true: is there a connection between the
reproductive cycle and the sequence of the PR in Canidae? However, more
information is necessary. Therefore, the reproductive cycle of several species are
compared with each other by means of a short review.
3
Part A: Comparative sequence analysis of the BUpstream Segment of the canine PR
Introduction
Isoforms of the progesterone receptor
In most vertebrates two isoforms of the PR are known, namely PR-A and PR-B
(Graham et al., 1997). Human, mice, rats, guinea pigs and cattle even have a third PR
isoform, namely PR-C (Chen et al., 2008). So far, the PR-C isoform has not been
detected in the dog. PR-A and PR-B are produced from one gene, but have their own
promoter and start codon. The sequence of the dog’s PR has been assessed by
Lantinga-van Leeuwen et al. (2000).
PR-A and PR-B have distinct functions. PR-B is a stronger transactivator on most
promoters than PR-A is, while PR-A functions as a transpressor of PR-B (Graham et
al., 1997). PR-A does not only inhibit PR-B, but also inhibits the activity of other
receptors, like the androgen (AR), the glucocorticoid (GR), the mineralocorticoid and
the estrogen receptors (ER) (Graham et al., 1997; Weigel, 1996; McDonnell et al.,
1994; Vegeto et al., 1993). The balance between PR-A and PR-B expression might
vary between tissue types and species (Lantinga-van Leeuwen et al., 2000) and in
human, the balance also varies between tumor types of the mammae (Graham et al.,
1995). Furthermore, estrogen and progesterone in the body determine the expression
of the PR (Graham et al., 1997). The final effect of progesterone on the tissues
depends on the balance between PR-A and PR-B activity.
Structure of the progesterone receptor
The PR belongs to the superfamily of steroid hormone receptors (also known as the
nuclear or intracellular receptors). The receptor consists of several domains (Fig. 1).
The conserved domains are located at the C-terminal region of the PR gene. The
conserved domains are the DNA-binding domain (DBD), the hinge region (HR) with
its nuclear localization signal, and the hormone- or ligand-binding domain
(HBD/LBD) (Lavery and McEwan, 2005; Rochette-Egly, 2003; Stryer, 2000; Graham
et al., 1997; Alberts et al., 1994). DNA can only bind to the DBD, if simultaneously
the appropriate hormone is bound to the LBD. PR-A and PR-B have a DBD and LBD.
Furthermore, PR-A and PR-B contain an inhibitory function (IF) region. The IF
region might regulate the auto-inhibition and transpression of the PR (Abdel-Hafiz et
al., 2002; Hovland et al., 1998; Huse et al., 1998).
→ PR-B
1
→PR-A
165
BUS
______
AF3
933
DBD
_______________ ______
IF
AF1
H
LBD
__ _______________________
NLS
AF2
Figure 1: Structure of the human progesterone receptor. Numbers indicate the position of the amino
acids. BUS, B-upstream segment; AF, activation function region; IF, inhibitory function
region; DBD, DNA-binding domain; NLS, nuclear localization signal; H, hinge region;
LBD, ligand binding domain. (Adapted from Gracanin et al. (unpublished); Chen et al.
(2008); Rochette-Egly (2003).
4
Less conserved regions are the activation function (AF) domains (Gracanin et al.,
unpublished; Stryer, 2000; Alberts et al., 1994). PR-A and PR-B have an AF1 domain
and an AF2 domain. One of the functions of the AF domains is to interact with
coactivators. The AF2 domain needs a ligand to be bound, while the AF1 domain can
function without a ligand (Lavery and McEwan, 2005). A third activation domain
(AF3 domain) is situated at the N-terminus. The domain only occurs in the PR-B and
is a strong activator (Tung et al., 2001; Sartorius et al., 1994; Meyer et al., 1992).
That might explain why PR-B is most of the time a stronger transactivator than PR-A.
Several theories exist about the function of the AF3 domain (Graham et al., 2002).
One of the theories suggests that the AF3 domain inhibits the effect of the IF region
on the transcription activity of PR-B. PR-B might have a different conformation when
it is in solution. The AF3 domain is thought to stabilize the N-terminus (Tung et al.,
2001). Because of the different conformation, the IF region becomes concealed. The
IF region is not able to function. The IF region does not have any influence on the
activity of the AF3 domain; it only affects the AF1 and AF2 domain (Graham et al.,
2002).
Besides the AF domains, the PR contains numerous post-translational modification
(PTM) sites. Post-translational modification affects the stability and subcellular
localization of the receptor. In addition, it influences the interactions of the receptor
with other proteins. Various ways of PTM exist, like phosphorylation, sumoylation,
ubiquitylation and acetylation (Faus and Haendler, 2006). Acytelation is not known in
the PR. The only known ubiquitylation site is situated at the C-terminal end of the PR.
Sumoylation inhibits the transcription activity of PR. Phosphorylation happens at
numerous phosphorylation sites. The N-terminal end of the PR-B, which lacks in PRA, contains the following (putative) phosphorylation sites in humans: S20, S25, S81,
S102, S130 and S162 (these and the following names are based on the location of the
amino acids in human). Most of the phosphorylation sites are phosphorylated by
proline-dependent kinases (that include cyclin-dependent kinases (CDKs). CDKs
phosphorylate its sites constitutively (in the absence of ligand) or in response to
hormone. Next to sites that become phosphorylated by CDKs, S81 becomes
phosphorylated by casein kinase II (CK-II). In human PR-B this is a constitutional
phosphorylation site (Rochette-Egly, 2003; Weigel, 1996).
The PR-B-Upstream Segment in the dog
The focus of this research is on the N-terminal region of the PR-B, also known as the
B-upstream segment (BUS). This segment includes the AF3 domain and several
phosphorylation sites. Gracanin et al. (unpublished) compared the sequence of BUS in
the dog with the BUS sequence of other species (rats, mice, rabbits, horses, cattle and
humans). Three important and unique differences of BUS were found in dogs. First,
BUS of dogs had an insertion of 30 base pairs. This insertion lacked in other species.
More species were sequenced (wolf, seal, ferret, cat, pig), and it appeared that besides
the dog, also BUS contained a 30 base pair insertion in the wolf. As can be seen in
figure 2, the dog (Canis lupus familiaris) and the grey wolf (Canis lupus lupus) are
related very close to each other.
Secondly, it was found that the sequence of the usually conserved AF3 domain was
different in comparison with other species. There are three motifs that are important
for the functioning of the AF3 domain (Tung et al., 2001). The three motifs in the dog
were different from these motifs in the other species. The first motif of the AF3
(AF3(1) domain was also different in the cow. The usually conserved 55LxxLL was
5
replaced by SxxLL in the dog and LxxLI in cattle. AF3(2) usually consists of
115
LxxLL, but the sequence in the dog was PxxAL. The last motif concerning the AF3
domain usually consists of 140W, but in the dog it consisted of an R. These findings
are consistent with the findings of Chen et al. (2008).
Finally, Gracanin et al. (unpublished) found that BUS in the dog missed three
important putative phosphorylation sites: S25 and S81.
Aim of this research
Gracanin et al. (unpublished) investigated the uniqueness of BUS in dogs in
comparison with BUS in cattle, humans, rabbits, horses, mice and rats. It appeared
that BUS in dogs had unique features. However, is this still true when the AF3
domain and putative phosphorylation sites are compared with more species? This
leads to the following hypothesis: the sequence of the AF3 domain and the putative
phosphorylation sites of BUS in the dog are unique. The aim of the current research is
to assess if this hypothesis is correct or not. Again, the AF3 domain and the putative
phosphorylation sites of BUS in the dog will be compared with that in other species:
the wolf, the seal, the ferret, the cat, the pig and the human.
Abovementioned species are chosen because of their variable relationship with the
dog (Fig. 2). The wolf is very closely related to the dog. Wolves and dogs belong to
the group of the Canis lupus (wolf-like canids), which belong to the family of Canidae
(Bardeleben et al., 2005). The seal belongs to the Phocidae and the ferret (Mustulae
putorius furo) to the Mustelidae, which are, together with the Canidae and Ursidae,
carniformae. Because there is no genomic DNA of Ursidae available in the internal
database, the Ursidae are not a part of this research. Carniformae are together with the
feliformae, of which the cat (Felis sylvestris catus/domesticus) is an example,
carnivores. The carnivores belong to the Laurasiatheria, just like pigs (Sus scrofa
domesticus). Pigs are omnivores. Laurasiatheria are mammals, just like human (Homo
sapiens) are, which belong to Euarchontoglires. Human are primates and belong to the
family of Hominidae.
6
Dog
Canidae
//
Wolf
Ursidae 2
//
Phocidae
//
Ursidae 1
//
Mustelidae
//
Felidae
//
Equidae
//
Suidae
//
Bovidae
//
Hominidae
//
Leporidae
//
Muridae
Figure 2: Schematic cladogram. Indicates the relationship between dogs and wolfs, which belong to
the Canidae family; seals, which belong to the Phocidae; ferrets, belonging to the Mustelidae;
cats, belonging to Felidae; horses, belonging to Equidae; pigs, which are Suidae; cattle,
which are Bovidae; human, belonging to the Hominidae; rabbits, belonging to Leporidae and
rats and mice belonging to the Muridae family. ‘//’ indicate that some branches are missing
in this simplified cladogram. This figure is adapted from Flynn et al. (2005) and Wilson et
al. (2005).
Materials and methods
DNA samples and sequencing
To sequence BUS, genomic DNA of the wolf, seal, ferret and pig was amplified with
a polymerase chain reaction (PCR). The genomic DNA was obtained from the internal
genomic database (appendix ‘Genomic DNA’). The breed belonging to the genomic
DNA was unknown, except for the dog.
The BUS sequences of the dog and human were obtained from GenBank. Access
number of the dog’s PR-B (Canis familiaris): AF177470.1; access number of the
human PR-B (Homo sapiens): AP001533.4.
Because of limited time, BUS of the cat was not sequenced. The sequence used for the
multiple species alignment was extracted from the cat’s (partially known) genome
from GenBank (accession number ACBE01336570.1). This was done by using a
nucleotide BLAST program from NCBI; in this program, the sequence of the human
BUS was entered.
7
The primers used for the PCR, were developed by Gracanin et al. (unpublished). In
addition, most cycling conditions were assessed by Gracanin et al. (unpublished). The
properties and cycling conditions of the used primers can be found in appendix
‘Primers and cycling conditions’. Diverse thermal cycle apparatus were used for the
PCRs. A Phusion Hot Start DNA polymerase (F-540S; Finnzymes, the Netherlands)
was used for amplification. Because BUS is abundant with GC nucleotides, Phusion
Buffer GC 5x (F-518; Finnzymes, the Netherlands) combined with DMSO 3% was
added.
To separate the PCR products, a gel-electrophoresis was performed using a 1,5%
solution of agarose into 0,5x TBE. Ethidium-bromide was added, to visualize the
result with GelDoc 2000 (Bio-Rad, the Netherlands). Except for the wolf and the pig,
it was necessary to perform a second PCR, because sometimes the band of interest
was very weak or the PCR gave more than one product. Therefore, the band of
interest was selected from the gel and used for a second PCR. Again, after the second
round of PCR the DNA was isolated from the gel. DNA was extracted from the gel by
means of the ‘QIAquick gel extraction kit’ of QIAGEN® (Qiagen Inc., Valencia, CA,
USA) according to the guidelines of the manufacturer. To prepare the DNA for
sequencing, the ABIPRISM BigDye Terminator v3.0 Ready Reaction Cycle
Sequencing Kit (Applied Biosystems, USA) was used. Also this time, the basic
protocol was followed. For the sequencing itself, the ABI3130xl Genetic Analyzer
from Perkin Elmer Applied Biosystems (USA) was used.
Sequence assembly and alignment
The various sequences obtained per specie were assembled to one main sequence.
This was done with the computer program SeqManTM II 5.08© (1989-2004) of DNASTAR* Inc. Standard parameters were used. However, the assembling parameters had
to be adjusted, because of some short sequences. The parameters were changed to
match size 12, a minimum match percentage of 50 and a minimum sequence length of
30. The minimum match percentage used for the wolf was 30.
The nucleotide sequences of BUS were translated to protein sequences by EditSeqTM
5.08© (1989-2004, DNA-STAR* Inc.). All the sequences were substituted to
MegAlignTM 5.08© (1993-2004, DNA-STAR* Inc.), including those of the dog, the
cat and the human. After this, a multiple species alignment was carried out by the
ClustalW method (using standard parameters).
Results
Gracanin et al. (unpublished) already found unique features of BUS in the dog. The
aim of the current research was to further investigate the uniqueness of the dog’s BUS
concerning the AF3 domains and the putative phosphorylation sites.
Before sequencing the PCR products, the products were separated by electrophoresis
(Fig. 3). A second PCR was necessary, except for the wolf and the pig, and also these
products were separated with an electrophoresis (Fig. 4). The second round of PCR
gave one clear band. The expected size of the PCR products with the used primers
was 583 bp.
8
Ferret
Control
Seal
Control
Pig
Control
Ferret
Control
Control
Seal
Wolf
Dog
Control
Figure 3: Amplification of PR-BUS. The PCR
products were separated by electrophoresis. Control: 100bp ladder; ▪
indicates 500 bp; → indicates BUS.
Figure 4: Amplification of PR-BUS
after the second PCR round.
The PCR products were
separated by electrophoresis.
Control: 100bp ladder; ▪
indicates 500 bp; → indicates
BUS.
The sequence of BUS was not complete for all the species (Fig. 5). The last four
nucleotides of the wolf’s BUS were unknown, while other nucleotides of other species
were uncertain. Because of this, the last putative phosphorylation site in the wolf and
seal remained unknown, just like some other amino acids within BUS.
Dog
Wolf
Ferret
Seal
Cat
Pig
Human
ATGACGGAGCGGACGGGAAAGGATGCCCGGGCTCCCCACGTGGCGGGAGGCGCGCCCTCCCCCGC
ATGRCGGAGSGGACGGSAAASGATGCCCGGGCTCCCCACGTGGCGGGAGGCGCGCCCTCCCCCGC
ATGACAGAGCCGAGGGCAAAGGATTCCCAGGCTTCCCACGTGGCAGGCGGCGCGCCC------AC
ATGACAGAGCCGARGGCAAAGGAT--CCAGGCTTCC-ACGTGGCAGGCGGCGCGCCC------AC
ATGACAGAGCTGAAGGCAAAGGAACCCCAGGCACCCCACGTGGCGGGCGCCGCGCCCTCCTCCAC
ATGAMTGAGCKGAAGGCAAAGGRWCCCCGGGCTCCCCACGTGGCGGGCAGAGCGCCCT----CCC
ATGACTGAGCTGAAGGCAAAGGGTCCCCGGGCTCCCCACGTGGCGGGCGGCCCGCCCT----CCC
65
65
59
56
65
61
61
Dog
Wolf
Ferret
Seal
Cat
Pig
Human
GCC-GGCCGCAGAGCCCGAGTCCCGACGTCGAGACGGCGGCCGCCTCCGGGCGAGTCAGACCTCG
GCC-GGCCGCAGAGCCCGAGTCCCGACGTCGAGACGGCGGCCGCCTCCGGGCGAGTCAGACCTCG
ACC-GGTAGGATCTCCTCTTTCGGGMCGCCGGGACGCTGGCTCCTTCCRGGCGAGTCAGAYCTCG
ACC-GGTAGGATCTCCTCTTTCGGGACGCCGGGACGCTGGCTCCTTCCAGGCGAGTCAGATCTCG
ACT-GCTCGGAGCGCCTCTGCTAGGACTCCGGGACGCTGGCCCTTTCCAGGAGAGTCAGACCTCG
CCA---CCCAGCTCGGGACGCTGGGACGCCCAGACACAGGCCCCTTTCAGGCGAGCCAGACCTCG
CCGAGGTCGGATCCCCACTGCTGTGTCGCCCAGCCGCAGGTCCGTTCCCGGGGAGCCAGACCTCG
129
129
123
120
129
123
126
Dog
Wolf
Ferret
Seal
Cat
Pig
Human
GACGCCCCGCGGGTCGCCGCAGCCGCAGCCGCAGCCGCAGCCGCAGCCTCAGCCGCGCCCTCCGC
GACGCCCCGCGGGTCGCCGCAGCCGCAGCCGCAGCCGCAGCCGCAGCCTCAGCCGCGCCCTCCGC
GACSCCTCGC------------------------------CTGTAGTTTCGGCCATACCYATC-GACCCCTCGC------------------------------CTGTAGTTTCGGCCATACCCATC-AACTCCTCCC------------------------------CTATAGTCTCAGCTATACCTATC-GAAGCGTCGC------------------------------CCGCAGCCTCGGCCATACCCCTC-GACACCTTGC------------------------------CTGAAGTTTCGGCCATACCTATC--
194
194
156
153
162
156
159
Dog
Wolf
Ferret
Seal
Cat
Pig
Human
GCCCTCGGACCGGCTGCTCTTCTCCCGGCGCGGCCAGGGCGCGGACC---CTGGCGGGAAGGCGC
GCCCTCGGACCGGCTGCTCTTCTCCCGGCGCGGCCAGGGCGCGGACC---CTGGCGGGAAGGCGC
-TCCTTRGACSGGCTGCTCTTCCCTCSKCCCWGYCAGGGACAGRACS---CGGACSGGAAGACYC
-TCCTTAGACGGGCTGCTCTTCCCTCGTCCCTGCCAGGGACAGAACG---CGGACGGGAAGACCC
-TCTCTGGACCCGTTGCTCTTCCCTAGGCCTTGCCAGGGACAAGACC---CGGACCCGAAGACAC
-TCCCTGGACGGGCTACTCTTCCCTGGGCCCTGCCAGGGACAGGAAC---CAGACGGGAAGACGC
-TCCCTGGACGGGCTACTCTTCCCTCGGCCCTGCCAGGGACAGGACCCCTCCGACGAAAAGACGC
256
256
217
214
223
217
223
Dog
Wolf
Ferret
Seal
Cat
Pig
Human
AGGACGCGCAGCCGCGGCCGGACGTGGCCCGGGCGGATCCGAGACTCGAAGCCGCGAGCGGGGCG
AGGACGCGCAGCCGCGGCCGGACGTGGCCCGGGCGGATCCGAGACTCGAAGCCGCGAGCGGGGCG
ARGAYCAGCAGCCGCTGTCAGACGTGRAGGSGGCGYATMCCAGAGTWGAAGCCRCAAGCRGTGCA
AGGACCAGCAGCCGCTGTCAGACGTGGAGGGGGCGTATCCCAGAGTAGAAGCCACAAGCAGTGCA
AGGACCAGCAGCCGCTGTCAGACGTGGAGGGGGCGTATCCCAGAGTTGAAGCCACAAGCAGTGCA
AGGACCAGCAGTCGCTGTCAGACGTGGAGGGGGCGTATCCCAGAGTTGAAGCTACAGAGGGTGCT
AGGACCAGCAGTCGCTGTCGGACGTGGAGGGCGCATATTCCAGAGCTGAAGCTACAAGGGGTGCT
321
321
282
279
288
282
288
9
Dog
Wolf
Ferret
Seal
Cat
Pig
Human
GGAGCCGACAGCCCCGGGCCCCCGCGCCAGGACCGAGGGCCGCTGCACGGCGCTCCGAGCACAGC
GGAGCCGACAGCCCCGGGCCCCCGCGCCAGGACCGAGGGCCGCTGCACGGCGCTCCGAGCACWGC
GGAGYTGRCARCTCTAGAMCYCCAGAAAAAGACAGAGGGCTGCTGGACAGTGTCTTGGACACGCT
GGAGCTGACAGCTCTAGACCTCCAGAAAAAGACAGAGGGCTGCTGGACAGTGTCTTGGACACGCT
GGAGCTGGCAGCTCTAGACCCCCAGAAAAGGACAGAGGGCTGCTGCACAGTGTCTTGGACACGCT
GGAGGTGGCAGCTCTAGACCCTCGGAAAAAGACACCGGGCTGCTGGACAGTGTCTTGGACACGCT
GGAGGCAGCAGTTCTAGTCCCCCAGAAAAGGACAGCGGACTGCTGGACAGTGTCTTGGACACTCT
386
386
347
344
353
347
353
Dog
Wolf
Ferret
Seal
Cat
Pig
Human
GCTGCGCCCCGCCGGCCCGGGGCAGGGCCG---CAGCTCTCCGGCCTGGGAGCCCCGCAGCCCGC
GCTGCGCCCCGCCGGCCCGGGGCAGGGCCG---CAGCTCTCCGGCCTGGGAGCCCCGCAGCCCGC
ACTGGAGCCCKCAGGCCCGGGGCAGAGCCACGCCAGCCCTCCTGCYTGTGAGYCCACYAGCCCTT
ACTGGAGCCCGCAGGCCCGGGGCAGAGCCACGCCAGCCCTCCTGCTTGTGAGCCCACCAGCCCTT
ACTGGAGCCTTCAGCTTCCGGGCAGACCCACGCCAGCTCTCCTGCCTGTGAGGCCGCCAGCCCTT
ACTAGCGCCCTCAGGTCCCGGGCAGAGCCACGCCAGCCCTCCCGCCTGCGAAGCCACCAGCCCTT
GTTGGCGCCCTCAGGTCCCGGGCAGAGCCAACCCAGCCCTCCCGCCTGCGAGGTCACCAGCTCTT
448
448
412
409
418
412
418
Dog
Wolf
Ferret
Seal
Cat
Pig
Human
GGTGCCCGTCTGGCCCCGAGCCGCCCGAAGATCCCCGGGGCGCCCGCAGCAGCCAGGGCGCGGCG
GGTGCCCGTCTGGCCCCGAGCCGCCCGAAGATCCCCGGGGCGCCCGCAGCAGCCAGGGCGCGGCK
GGTGCCTGTTTRGCTCCRAGCTTCCCGAAGAMCCCCGGGTTGCCYCCACCACCCAGGGGGTGTCR
GGTGCCTGTTTGGCTCCAAGCTTCCCGAAGAACCCCGGGTTGCCCCCACCACCCAGGGGGTGTCG
GGTGCCTGTTTGGCTCTGAGCTTCCTGAAGACCCCCGGGTTGCCCCCACCACCCAGGTGGGGTTG
GGTGCTTGTTTGGCTCTGAGCTTCCCCAGGACGCTCGGGTTGCCCCTTCCACCCAGGGAGTATTG
GGTGCCTGTTTGGCCCCGAACTTCCCGAAGATCCACCGGCTGCCCCCGCCACCCAGCGGGTGTTG
513
513
477
474
483
477
483
Dog
Wolf
Ferret
Seal
Cat
Pig
Human
TGCCCGCTCATG
-GYCGBCT????
TCCCCGCTCATG
WCCCCGCTCATG
TCTTCGCTCATG
CCCCTGCTCATG
TCCCCGCTCATG
525
520
489
486
495
489
495
Figure 5: ClustalW multiple species alignment for the nucleotide sequence of BUS.
The ClustalW method was used to perform the multiple species alignment. The
alignment showed remarkable differences between the AF3 domains and the putative
phosphorylation sites in the various species (Fig. 6).
Dog
Wolf
Ferret
Seal
Cat
Pig
Human
MTERTGKDARAPHVAGGAPSPAPAAEPESRRRDGGRLRASQTSDAPRVAAAAAAAAAAAS
MXEXTXXDARAPHVAGGAPSPAPAAEPESRRRDGGRLRASQTSDAPRVAAAAAAAAAAAS
MTEPRAKDSQASHVAGGAPTP--VGSPLSGRRDAGSFXASQXSDX----------SPVVS
MTEPXAKDP-GFHVAGGAPTP--VGSPLSGRRDAGSFQASQISDP----------SPVVS
MTELKAKEPQAPHVAGAAPSSTLLGAPLLGLRDAGPFQESQTSNS----------SPIVS
MXEXKAKXPRAPHVAGRAPSPTQLGT--LGRPDTGPFQASQTSEA----------SPAAS
MTELKAKGPRAPHVAGGPPSP-EVGSPLLCRPAAGPFPGSQTSDT----------LPEVS
*
*
60
60
48
47
50
48
49
Dog
Wolf
Ferret
Seal
Cat
Pig
Human
AAPSAPSDRLLFSRRGQGADP-GGKAQDAQPRPDVARADPRLEAASGAGADSPGPPRQDR
AAPSAPSDRLLFSRRGQGADP-GGKAQDAQPRPDVARADPRLEAASGAGADSPGPPRQDR
AIPIS-LDXLLFPXPXQGQXX-DXKTQDQQPLSDVXXAXXRVEAXSXAGXXXSRXPEKDR
AIPIS-LDGLLFPRPCQGQNA-DGKTQDQQPLSDVEGAYPRVEATSSAGADSSRPPEKDR
AIPIS-LDPLLFPRPCQGQDP-DPKTQDQQPLSDVEGAYPRVEATSSAGAGSSRPPEKDR
AIPLS-LDGLLFPGPCQGQEP-DGKTQDQQSLSDVEGAYPRVEATEGAGGGSSRPSEKDT
AIPIS-LDGLLFPRPCQGQDPSDEKTQDQQSLSDVEGAYSRAEATRGAGGSSSSPPEKDS
*
*
119
119
106
105
108
106
108
Dog
Wolf
Ferret
Seal
Cat
Pig
Human
GPLHGAPSTALRPAGPGQGRSS-PAWEPRSPRCPSGPEPPEDPRGARSSQGAACPLM
GPLHGAPSTALRPAGPGQGRSS-PAWEPRSPRCPSGPEPPEDPRGARSSQGAAXX??
GLLDSVLDTLLEPXGPGQSHASPPACEXTSPWCLFXSXLPEXPRVAXTTQGVSSPLM
GLLDSVLDTLLEPAGPGQSHASPPACEPTSPWCLFGSKLPEEPRVAPTTQGVSXPLM
GLLHSVLDTLLEPSASGQTHASSPACEAASPWCLFGSELPEDPRVAPTTQVGLSSLM
GLLDSVLDTLLAPSGPGQSHASPPACEATSPWCLFGSELPQDARVAPSTQGVLPLLM
GLLDSVLDTLLAPSGPGQSQPSPPACEVTSSWCLFGPELPEDPPAAPATQRVLSPLM
*
*
175
173
163
162
165
163
165
Figure 6: ClustalW multiple species alignment for the amino acid sequence of BUS. Respectively,
AF3(1), AF3(2) and AF3(3) motifs are underlined. Putative phosphorylation sites are
indicated by *.
As far as the sequences were known, the sequence of BUS in the dog and wolf were
completely similar. The sequence of BUS in the dog and wolf differed from all other
10
species concerning the AF3 domain. The AF3(1) and the AF3(2) motifs usually
included LxxLL, like in the seal, the ferret, the pig, the cat and the human. In the dog
and wolf, the AF3(1) motif contained SxxLL and the AF3(2) contained PxxAL. The
third motif of the AF3 domain consisted of a W in all the species, except for the dog
and the wolf; here the motif consisted of an R.
Concerning the putative phosphorylation sites, a lot of sites differed amongst the
species. S20 was only missing in the seal and the ferret. S25 lacked not only in the dog,
but also in the wolf, the cat and the pig. The only site which uniquely missed in the
dog and the wolf was S81. S102 lacked in all species, except for the human. It appeared
that the dog also missed the last putative phosphorylation site, S162. Besides the dog,
also the pig missed the putative phosphorylation site S162. The sequence of this site in
the wolf and the seal were unknown.
Discussion
The hypothesis of this research was that the sequence of the AF3 domain and the
putative phosphorylation sites of BUS in the dog were unique. Gracanin et al.
(unpublished) already compared BUS of the dog with that in cattle, humans, rabbits,
horses, mice and rats. The results of Gracanin et al. (unpublished) and of the current
research were presented in table 1.
Table 1: Results of this research and of Gracanin et al. (unpublished). AF: activation function domain.
S: putative phosphorylation site. The letters in the table indicate amino acids. The sequence of
human BUS is used as reference; only the motifs or sites that differed from the sequence in
human are shown.
Species →
Dog
Wolf
SxxLL
SxxLL
PxxAL
PxxAL
R
R
Seal
Ferret
Cat
Pig
Mouse
Rat
Rabbit
Cattle
Horse
Motif/Site↓
AF3(1):
LxxLL
AF3(2):
LxxLL
AF3(3):
W
S20
S25
S81
S102
S130
S162
LxxLI
T
E
P
G
E
P
G
R
C
?
?
T
R
E
T
R
R
P
R
R
R
E
A
R
R
A
L
P
Because the whole sequence of BUS in the wolf had the same sequence as that in the
dog, the differences do not seem to be unique for the dog anymore. So the hypothesis
is condemned. However, the findings could mean that BUS might be unique for wolflike canids or maybe even for Canidae in general. It is necessary to do more research
in order to confirm this suggestion.
11
Concerning the AF3 domain, only in the dog and wolf this domain differed
completely. Except for cattle, all the motifs that are important for the functioning of
the AF3 domain were conserved in the other animals. In cattle only AF3(1) differed.
Tung et al. (2001) found that in human PR-B the three AF3 motifs are necessary for
efficient transcription. If one of the AF3 domains mutated, AF3 activity was lost by
more than 70%. When AF3(1) and AF3(2) both mutated, the activity reduced by more
than 85%. The results of this research showed altered motifs of AF3 in dogs and
wolves. If the same applies to dogs and wolves as applies to human, this finding could
mean that AF3 activity is drastically reduced in dogs and wolves. PR-B might almost
have the same transcription activity as PR-A. Gracanin et al. (unpublished) also found
a changed motif of AF3(1) in cattle. This might mean that also in cattle the PR-B has
less transcriptional activity, but not as less as in dogs and wolves, because the other
motifs were not changed. To assess these suggestions, it would be interesting to repeat
the research of Tung et al. (2001) again in dogs and/or wolves and cattle.
Concerning the putative phosphorylation sites, there were many differences between
the species. However, only the dog, the wolf and cattle missed S81. S130 was conserved
in all the species, while S102 lacked in all the species except for the human. In the seal,
the ferret, the cat and the pig an arginine (R) was found at this site, but in the dog and
wolf there was a glycine (G).
The lack of putative constitutive phosphorylation sites in the dog and wolf might
influence the progesterone-signaling cascade by changing the transcription activity of
the PR. However, Takimoto et al. (1996) found that mutations at serine residues in
human BUS had no effect on the transcriptional activity of PR-B. To asses if this also
applies to canine BUS, more research is necessary.
Another interesting similarity between dogs and wolves, apart from their BUS, is their
estrous cycle. Many researchers speak of ‘the unique estrous cycle of the
dog/Canidae’. With the results of this research, it seemed that the AF3 domain in
wolf-like canids, or maybe even Canidae in general, might be unique. Also Gracanin
et al. (unpublished) found an insertion of 30 base pairs, only in the dog and wolf. It is
also known that progesterone is a main risk factor in the development of
mammaetumors in dogs and that the PR is necessary to mediate the effects of
progesterone. During the ‘unique’ estrous cycle of dogs, bitches are exposed to longlasting high concentrations of progesterone. It might be that this long-lasting exposure
to high concentrations of progesterone could have lead to changes in the progesterone
signaling cascade, including the PR. Putting this together, could there be a connection
between the reproduction cycle and the sequence of the AF3 domain and
phosphorylation sites in Canidae?
12
Part B: A review: The reproductive cycle of
mammals
Introduction
Features of the reproduction cycle vary a lot between animals. For this research,
knowledge about the exposure time to progesterone during the cycles of the female is
necessary. With this information, it can be assessed if Canidae are unique in having an
estrous cycle with long-lasting exposure to progesterone. The type of cycle and
ovulation are of interest, because they determine the features of the luteal phase. If a
luteal phase occurs, the duration of the luteal phase is important to know. With this
information, it might be possible to asses whether there might be a connection
between the reproduction cycle and the sequence of BUS of the PR in Canidae.
There are two types of reproduction cycles, the estrous cycle and the menstrual cycle.
Species with an estrous cycle can be mono- or polyestrus. The different stages of the
reproduction cycle can be classified in many ways (Johnston et al., 2001). In this
research the reproduction cycle will be divided into a follicular phase, in which the
new follicles develop and oocytes maturate, and a luteal phase, in which functional
corpora lutea (CL) are present. The CL develop after the oocytes ovulate. Ovulation
occurs spontaneous or after stimulation of the cervix (induced ovulators). However, in
dogs the CL already begin luteinizing before ovulation has taken place (Reynaud et
al., 2005). During the luteal phase, the CL produce progesterone. In polyestrous
species with spontaneous ovulation, the luteal phase ends within three weeks when the
female is not pregnant in order for a new follicular phase to start. Hormones, like
prostaglandin F 2α (PGF2α), are produced in the uterus and/or in the ovary or CL and
lead to degradation of the CL (McCracken et al., 1999; Senger, 1999). The article of
McCracken et al. (1999) gives more information the exact mechanisms. Other species
do not seem to have such a mechanism; their luteal phase is prolonged.
After luteolysis, plasma progesterone concentrations subsequently decrease below a
defined value. During the reproductive cycle, the progesterone level in the blood is the
highest during the luteal phase. In dogs, progesterone reaches a peak concentration of
15-90 ng/ml in the blood during the luteal phase. At the end of the luteal phase and
during the follicular phase, basal concentration of progesterone is less than one ng/ml
(Johnston et al., 2001).
The abovementioned features indicate the total exposure time of a female to
progesterone during her reproduction cycles. This is important to know in order to
assess if Canidae are unique in having an estrous cycle with long-lasting exposure to
progesterone, because the features indicate.
Aim of this review
The aim of this review is to gain further insight into the reproduction cycle of the
species investigated in part A; therefore a short review is presented. Hopefully it can
be assessed whether there might be a connection between the estrous cycle of Canidae
and the sequence of their AF3 domain and putative phosphorylation sites.
The focus is on the following properties of the cycle: the type of reproduction cycle,
type of ovulation and length of luteal phase.
13
The reproductive cycle
Many authors use different classifications for the reproductive cycle. This makes it
difficult to compare the duration of the luteal phase of different species. McCracken et
al. (1999) divided the common mammal species in reproductive cycle groups (Fig. 7).
It gives more insight into the luteal phases of the different animals.
Figure 7 : Reproductive cycles of canines, reflex ovulators, domestic animals, rodents and primates.
Luteolysis is indicated; luteolysis controls the lifespan of the corpus luteum during the cycle.
E: estradiol-17ß; LH: luteinizing hormone; P: progesterone (McCracken et al., 1999).
14
Canidae
According to McCracken et al. (1999) (and other authors like Concannon et al., 2009;
Gobello et al., 2001; Asa et al., 1998) dogs and wolves have a quite unique cycle.
Dogs and wolves are placed in a separate group, ‘the group of the canine cycle’ (Fig.
7). The animals in this group have a monoestrous cycle, just like many other Canidae;
they come into estrous once or twice a year (Concannon et al., 2009; Johnston et al.,
2001; Asa, 1998; Seal et al., 1979). All the domestic dogs (Canis lupus familiaris),
with some exceptions e.g. the Basenji dog, come into estrous twice a year. The bitch
and the she-wolf (Canis lupus lupus) ovulate spontaneously following a peak of
luteinizing hormone (LH) (Concannon et al., 2009; Johnston et al., 2001; Asa, 1998;
Seal et al., 1979). Jackals, coyotes and foxes are also spontaneous ovulators
(Concannon et al., 2009). During and after this pre-ovulatory LH surge, progesterone
increases rapidly until high levels have been reached. The CL persist during the whole
luteal phase; the luteal phase is prolonged in comparison with the luteal phase of
nonpregnant females of other species (Concannon et al., 2009; Gobello et al., 2001).
Many researchers have tried to unravel the mechanism of luteolysis in dogs. Until
now, hormones, like PGF2α, do not seem to cause luteolysis, in contrast to luteolysis
in many polyestrous species. It is still unknown how luteolysis is initiated. According
to Hoffmann et al. (2004) immune mediated events might play an important role in
luteolysis of the CL.
The luteal phase of nonpregnant and pregnant dogs and wolves is approximately of
equal duration, namely 65 days (range 55-75 days) in dogs and 59-63 days in wolves
(Concannon et al., 2009; Asa, 1998; Seal et al., 1979). There are no differences
between the serum progesterone concentrations of pregnant and nonpregnant estrous
cycles in dogs and wolves (Johnston et al., 2001; Asa, 1998). Nonpregnant females of
the coyote and the Arctic, the red and the Andean fox also have a prolonged luteal
phase (Asa, 1998).
Ursidae
Ursidae also belong to the caniformae. Knowledge is limited about the physiology of
reproduction in Ursidae. The few studies that have been done were based on very
small numbers of animals. Some of these studies used bears that lived in captivity; the
cycles of these animals may differ from the bears that live in the wild.
It seems that the different members of the Ursidae family have various types of
reproductive cycles. Therefore, Ursidae cannot be classified in one of the groups of
McCracken et al. (1999). Schwarzenberger et al. (2004) report that the sun bear
(Ursus or Helarctos melayanus) is a polyestrous bear. According to Concannon et al.
(2009) and Onuma et al. (2001) this bear ovulates spontaneously. However,
Schwarzenberger et al. (2004) found reasons to believe that this bear actually might
be an induced ovulator with spontaneous ovulation occurring now and then. Boone et
al. (2004) and Sato et al. (2001) suggested that this might be the same for the
American black bear (Ursus americanus). Boone et al. (2004) reported that it is
believed that the American black bear is a monoestrous bear, but that this is based on
limited research. According to Concannon et al. (2009) the giant panda (Ailuropoda
melanoleuca) is a monoestrous bear with spontaneous ovulation. However,
researchers do not agree whether the giant panda belongs to the Ursidae or not (Yu et
al., 2004). Nevertheless, most researchers do agree the giant panda is related very
close to the Ursidae.
15
The luteal phase of the nonpregnant sun bear takes 90 days and the luteal phase of
pregnant sun bears lasts 95-107 days (Schwarzenberger et al. (2004). No other
information is available concerning the luteal phase. According to Sato et al. (2001),
all Ursidae have a prolonged luteal phase in comparison with many polyestrous
species, when fertilization does not occur. However, the researchers were not able to
assess if the females had been pregnant and lost their embryo or fetus for any reason
whatsoever during early pregnancy.
Phocidae
Little is known about the reproductive physiology of Phocidae. However, Atkinson
(1997) wrote a review about the reproductive biology of seals. In addition, the
handbook of Dierauf and Gulland (2001) gives an overview of all the known
information about the reproduction of Phocidae.
It is not possible to place the seal in one of the groups of McCracken et al. (1999).
This is because there are some differences concerning the reproductive features of
seals belonging to the Phocidae family. However, as far as known, most of the
Phocidae are monoestrous. The Hawaiian monk seal (Monachus schauinslandi) is an
exception; this seal is polyestrous. Ovulation takes place spontaneously. Also in some
of the Phocidae, like the harbour (Phoca vitulina vitulina) and the hooded seal
(Cstophora cristata), a prolonged luteal phase exists if fertilization does not occur.
The CL persist. The luteal phase is however shorter than the luteal phase of pregnant
animals, in contrast to dogs. Progesterone levels stay high for 4-5 months during the
luteal phase in non-pregnant harbour and hooded seals. Pregnancy lasts 9-11 months
in harbour seals and 12 months in hooded seals. Also in seals it is unknown what
initiates luteolysis.
As far as known, the Hawaiian monk seal is the only Phocid who seems to produce
hormones like PGF2α, which soon cause luteolysis in nonpregnant females
(Concannon et al., 2009). Progesterone concentrations in nonpregnant Hawaiian
monk seals decrease 17-20 days after ovulation and a new cycle starts.
Mustelidae
Ferrets and mink have a polyestrous cycle and are induced ovulators (Fig. 7;
Concannon et al., 2009; Lindeberg, 2008; Quesenberry, 2004; Johnston et al., 2001;
McCracken et al., 1999). Ovulation only occurs after (multiple) stimulation of the
cervix by mating. If there is no stimulation of the cervix, and because of that, no
ovulation, the female ferret will show persistent estrous until the end of the mating
season (Lindeberg, 2008; Quesenberry, 2004). When mating does take place,
stimulation of the cervix leads to an (or multiple) LH surge(s) followed by ovulation
and development of CL. If the mating is not fertile, the luteal phase is of equal
duration as the duration of the luteal phase in pregnant females, namely 39-43 days
(Concannon et al., 2009; Lindberg, 2008; Quesenberry, 2004; McCracken et al.,
1999). Factors that finally cause luteolysis in the nonpregnant female ferret are
unknown. It seems that no hormones like PGF2α are involved in luteolysis
(Concannon et al., 2009).
Felidae
Also cats have a polyestrous cycle and are induced ovulators (Fig. 7). However,
spontaneous ovulation happens in the queen (Concannon et al., 2009; Johnston et al.,
2001). When mating does not occur, the queen goes into a new period of estrous after
a postestrous period (Fig. 8) (in contrast to ferrets).
16
Postestrus
Prolonged luteal
phase
(Proestrus)
Estrus
Gravidity, parturition
Anestrus
Figure 8: Estrous cycle of the queen (Department of Reproduction of Companion Animals, Faculty of
Veterinary Medicine Utrecht University).
If mating does take place, the queen becomes pregnant. However, if she is not
pregnant, like after a non-fertile mating or spontaneously ovulation, the CL will
persist. The luteal phase lasts 40-45 days (Concannon et al., 2009; McCracken et al.,
1999). Also now, a prolonged luteal phase is seen, in comparison with other species.
The mean duration of pregnancy is 64,4 days (range: 52-74 days; Johnston et al.,
2001). After many researches, no hormones like PGF2α seem to initiate luteolysis in
the cat (Concannon et al., 2009; Tsutsui et al., 2009; Johnston et al., 2001). The cause
of luteolysis is still unknown. Tsutsui et al. (2009) have tried to asses that the feline
placenta secretes progesterone in order to maintain pregnancy. During the luteal phase
of nonpregnant females, such progesterone secretion would be absent. However,
Tsutsui et al. (2009) were not able to detect placental progesterone in the peripheral
blood. It might be possible that the placenta locally supports pregnancy in an
autocrine or paracrine way, and thereby maintaining the CL, without measurable
progesterone concentrations in the peripheral blood.
Suidae, Equidae and Bovidae
According to McCracken et al. (1999) the pig, the horse and cattle belong to the group
of domestic animals (Fig. 7). These females are polyestrous (Senger, 1999; Downey,
1980). Ovulation is a spontaneous event (Senger, 1999; Downey, 1980). Ovulation
follows an LH surge. The luteal phase is quite long in comparison with the follicular
phase. If the female is not pregnant, the production of hormones, e.g. PGF2α, leads to
the degradation of the CL and a next cycle starts (McCracken et al., 1999; Senger,
1999; Downey, 1980).
Muridae
The rat and the mouse belong to one of the groups of McCracken et al. (1999) (Fig.
7). The cycle is polyestrous (Quesenberry, 2004). The follicular period is very short,
only 4 to 5 days (Quesenberry, 2004). The follicular period and spontaneous ovulation
continue to repeat themselves until cervical stimulation occurs. After stimulation of
the cervix by mating, the CL develop. In case of a sterile mating, initiation of
luteolysis soon begins due to production of hormones, e.g. PGF2α (Concannon et al.,
2009; Zakar and Hertelendy, 2007). A prolonged luteal phase is rarely seen
(Quesenberry, 2004; Richardson, 1997; Harkness et al., 1995).
17
Leporidae
Rabbits are polyestrous animals with induced ovulation (McCracken et al., 1999).
Ovulation occurs after (multiple) stimulation of the cervix by mating. When ovulation
stays out, a new cycle starts. If ovulation takes place, CL develop. In case of a
nonfertile mating, the CL will degrade soon due to production of hormones like PGF2α
(Concannon et al., 2009; Zakar and Hertelendy, 2007). However, a prolonged luteal
phase is seen in rabbits (Quesenberry, 2004). The duration of the luteal phase in
nonpregnant rabbits is shorter than the duration of the luteal phase in pregnant rabbits.
Hominidae
The reproduction cycle of human is not of the estrous type. Humans have a menstrual
cycle because of the period in which menses occurs (Fig. 7; Farage et al., 2009;
McCracken et al., 1999). Old World nonhuman primates also have a menstrual cycle.
The follicular phase starts with the beginning of the menses (Farage et al., 2009). At
the end of the follicular phase, ovulation takes place spontaneously one day after the
LH surge (Farage et al., 2009). The period of the follicular and luteal phase is
approximately of equal length. If fertilization does not take place, luteolysis of the CL
occurs, because of the production of hormones, e.g. PGF2α (Concannon et al., 2009).
After luteolysis, the concentration of progesterone in the blood drops. This decline in
progesterone concentration leads to endometrial shedding: the menses. A new cycle
starts.
Discussion
In order to assess a connection between the estrous cycle of Canidae and the sequence
of their AF3 domain and putative phosphorylation sites, a short review was done to
gain further insight into the reproduction cycle of the species investigated in part A
and in the research of Gracanin et al. (unpublished). The focus was on a few specific
features of the reproductive cycle, because these features seemed of most importance
in order to asses or reject the hypothesis. Based on the type of reproductive cycle, the
type of ovulation and the length of the luteal phase it would be possible to
approximate the total exposure time of a female to progesterone during her cycles.
Female Canidae, some Phocidae and some Ursidae are exposed to long-lasting high
concentrations of progesterone during their cycles. These females have a monoestrous
cycle with spontaneous ovulation. No hormones like PGF2α are produced to degrade
the CL in case fertilization did not occur. Therefore, the CL persist and the luteal
phase is prolonged in comparison with most polyestrous species. Other species have
various types of reproduction cycles, which prevent such a long-lasting exposure to
high progesterone concentrations. All these species are however polyestrous. In some
reproductive cycles, ovulation or CL formation only occurs after mating, while in
other cycles hormones like PGF2α are produced, which cause luteolysis in
nonpregnant females. Because of these features, the exposure time of these females to
high progesterone concentrations during their cycles is only short.
Canidae, Ursidae and Phocidae all belong to the caniformae (Fig. 2; Flynn et al.,
2005). This could be an explanation of the resemblance of estrous cycles of those
families. Ursidae evoluted very fast (Yu et al., 2004) and also the members of the
Phocidae family diverged a long time ago (Hidgon et al., 2007). Therefore, the
18
different species of the Ursidae and Phocidae family have different features, which
might also be the case for the features of the reproduction cycle.
Mustelidae also belong to the caniformae. However, their reproduction cycle does not
resemble that of Canidae and monoestrous Phocidae and Ursidae. Their cycle
resembles the cycle of the polyestrous Ursidae and Felidae. It is remarkable that also
the rabbit, which is a member of the Leporidae, has a polyestrous cycle with induced
ovulation. Leporidae are not closely related to Ursidae and Felidae (Fig. 2). It would
be interesting to investigate how it could happen that Leporidae have such a different
reproduction cycle than it’s closer relatives, like Muridae, do have. Therefore, more
research is necessary.
The reproduction cycles of Suidae, Equidae, Bovidae, Muridae and Hominidae are
very different from the cycle of Canidae, concerning the type of cycle and ovulation
and the duration of the luteal phase. In figure 2 it can be seen that these species are not
closely related to Canidae.
The aim of this part of the research was to asses whether there might be a connection
between the estrous cycle of Canidae and the sequence of their AF3 domain and
putative phosphorylation sites. The results of part A and part B of this research and
that of Gracanin et al. (unpublished), were presented in table 2.
Table 2: Results of Part A and B of this research and of Gracanin et al. (unpublished). ME:
monoestrous cycle; PE: polyestrous cycle; S: spontaneous ovulation; I: induced ovulation; +,
(+), -: prolonged luteal phase is always present, is rare, is absent respectively. The other
letters in the table indicate amino acids. ‘?’ indicates that data is unknown.
Species→
Feature↓
Type of
cycle
Type of
ovulation
Prolonged
luteal
phase
AF3(1):
LxxLL
AF3(2):
LxxLL
AF3(3):
W
S20
S25
S81
S102
S130
S162
Dog
Wolf
Ursidae Seal Ferret Cat Pig Cattle Horse Human Mouse Rat Rabbit
ME
ME
S
S
+
+
ME
PE
S
I
+
+
SxxLL
SxxLL
ME
PE
S
PE
PE
PE
PE
PE
PE
PE
PE
PE
I
I
S
S
S
S
S
S
I
+
-
(+)
(+)
-
-
-
-
-
-
-
R
R
R
?
LxxLI
PxxAL PxxAL ?
R
R
E
P
G
E
P
G
C
?
?
?
?
?
?
?
?
T
R
?
T
R
E
T
R
E
A
R
R
R
P
L
P
A
19
Based on this limited data, there seems to be no relationship between the estrous cycle
and the AF3 domain and putative phosphorylation sites in Canidae. However, it might
be possible that the AF3 domain of monoestrous animals with spontaneous ovulation
is unique compared with that of species with another type of cycle. There was no
change found in the AF3 domain of the seal, but the exact breed of this seal was
unknown. Features of reproductive cycles of seals vary, so it will be necessary to
know the exact breed. It might be that it was a polyestrous seal. If the suggestion that
the AF3 domain in monoestrous species is different in comparison to that of
polyestrous species is correct, it might be that the AF3 is different in monoestrous
Phocidae. Ursidae would also be interesting to include in a research like this, but no
genomic DNA was available for this research. However, only limited information is
available concerning the reproduction physiology of Phocidae and Ursidae. In order to
assess a possible connection this information is necessary.
In addition, it was not possible to assess a connection between the cycle and the
putative phosphorylation sites. The more related the species are, the more the changes
in the putative phosphorylation sites resemble each other. Amino acids at the sites
were similar in the dog and wolf; also, the amino acids in the ferret and the seal were
similar, just like the amino acids in mice and rats were similar.
What could be the reason for dogs and wolves having a different sequence of the AF3
domain? The AF3 domain is a strong activator of PR-B. That means that the
transcription activity of PR-B is high. Progesterone leads to the expression of PR-B.
PR-B is necessary for proper lobuloalveolar development of the mammary gland
during pregnancy. It might be possible that the sequence of the AF3 domain is
different in dogs and wolves to compensate for the exposure to long-lasting high
concentrations of progesterone during their estrous cycle. That would mean that if the
AF3 domain was not changed, the lobuloalveolar growth of the mammary gland
would be enormous, every cycle again, compared to that in other species without a
prolonged luteal phase. The different AF3 domain of dogs and wolves may lead to a
different transcriptional activity of PR-B and therefore a different lobuloalveolair
growth. Therefore, the aforementioned suggestive comparative sequence analysis with
the inclusion of other monoestrous species with spontaneous ovulation, like members
of the Ursidae and Phocidae family, could give useful information to investigate if
this hypothesis might be correct.
20
Conclusion
The hypothesis that the sequence of the AF3 domain and the putative phosphorylation
sites of BUS in the dog were unique was not correct. The AF3 domains were the same
in all the investigated species, except in cattle, dogs and wolves. In cattle the AF3(1)
motif differed from other species. In the dog and the wolf, all three AF3 motifs
differed from the AF3 motifs of other species. Concerning the putative
phosphorylation sites many variants were found in all the species; many species
missed putative phosphorylation sites.
The dog and wolf showed the same sequencing of BUS. Therefore, the dog is not
unique. However, it might be that the differences in the AF3 domain are unique for
wolf-like canids or Canidae in general.
A connection between the various features of the estrous cycle and the changes in
BUS in Canidae could not be assessed. Some members of the Ursidae and Phocidae
families showed a prolonged luteal phase, like Canidae. These females all have a
monoestrus cycle. According to the sequence analysis, no changes were found in the
AF3 domain of the seal and the sequence of the putative phosphorylation sites also
differed from these sites in the dog and wolf. Therefore, no connection between BUS
and the estrous cycle could be assessed in Canidae. More knowledge about
reproductive features and inclusion of more species, like Ursidae, is necessary in
order to assess this assumption.
21
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24
Appendix
Genomic DNA
For the sequence analysis of the PR, genomic DNA samples of various species from
the internal database were used (table 3). The dog was used as a control.
Table 3: Features of genomic DNA. DSD: Duitse Staande Draadhaar dog.
Genomic DNA →
Species ↓
Dog (DSD)
Dog (Kooiker)
Wolf
Ferret
Ferret
Seal
Seal
Pig
Pig
Sample ID
ng/µl DNA
A260/280
A260/230
4842
11081
114
1490
9930
1282
1283
3767
3768
36,8
23
173,1
11,2
3,3
93,5
22,9
108,4
78,6
1,89
1,85
1,87
2,29
1,45
1,87
1,84
1,88
1,92
1,75
1,55
2,09
1,42
0,57
2,03
1,80
2,21
1,97
Primers and cycling conditions
Primer sets and cycling conditions used in this research are presented in table 4. The
dog was used as a control.
Table 4: Cycling conditions of the primer sets. U: upper/forward primer; l: lower/reverse primer.
Product size is based on canine BUS. s: seconds; m: minutes; * indicates conditions used for
the seal and the ferret; ** indicates conditions used for the wolf and the pig.
Primer sets →
Features ↓
Sequence cBUS1_u
cBUS2_l
BUS4_u
BUS2_l
Initiation
Denaturation
Annealing
cBUS1_u
cBUS2_l
Extension
Final extension
# cycles (step 2-4)
Product size (bp)
Species
72°C ~ 5 m
35
536
Dog, wolf
BUS4_u
BUS2_l
TCATGACGGAGCGGACGGGAAA
GCCCTCCGGCCGGCTCAT
AGGAGAGGGGAGTCCCGGTCGTCAT
CCGCAGCCGTCGCCAGCCTTG
98°C ~ 30 s
98°C ~ 10 s
64°C ~ 15 s
98°C ~ 30 s
98°C ~ 10 s
60°C ~ 10 s*
70°C ~ 10 s**
72°C ~ 10 s
72°C ~ 5 m
30
583
Dog, wolf, seal, ferret, pig
25
26