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Journal Name:
Frontiers in Neuroscience
ISSN:
1662-453X
Article type:
Received on:
29 Nov 2014
Accepted on:
23 Apr 2015
23 Apr 2015
www.frontiersin.org
Citation:
Copyright statement:
This Provisional PDF corresponds to the article as it appeared upon acceptance, after rigorous
peer-review. Fully formatted PDF and full text (HTML) versions will be made available soon.
Molecular cloning, sequencing and tissue expression of vasotocin and isotocin precursor
221005, India;
Abstract
Basic and neutral lines of neurohypophyseal (NH) nonapeptides have evolved from the ancestral
10
peptide vasotocin (VT) by gene duplication at the base of the gnathostome lineage. In teleosts,
11
VT and IT are the basic and neutral peptides, respectively. In the present study, VT and IT
12
precursor genes of the catfishes (Siluriformes, Ostariophysi) Heteropneustes fossilis and Clarias
13
batrachus were cloned and sequenced. The channel catfish Icatalurus punctatus NH precursor
14
sequences were obtained from its EST database. The catfish NH sequences were used along with
15
the available Acanthopterygii and other vertebrate NH precursor sequences to draw phylogenetic
16
inference on the evolutionary history of the teleost NH peptides. Synteny analysis of the NH
17
gene loci in various teleost species was done to complement the phylogenetic analysis. In H.
18
fossilis, the NH transcripts were also sequenced from the ovary. The cloned genes and the
19
20
The genes are expressed in brain and ovary (follicular envelope) of H. fossilis with higher
21
transcript abundance in the brain. The addition of the catfish sequences in the phylogenetic
22
analysis revealed that the VT and IT precursors of the species-rich superorders of teleosts have a
23
distinct phylogenetic history with the Acanthopterygii VT and IT precursors sharing a less
24
evolutionary distance and the Ostariophysi VT and IT having a greater evolutionary distance.
25
The genomic location of VT and IT precursors, and synteny analysis of the NH loci lend support
26
to the phylogenetic inference and suggest a footprint of fish- specific whole genome duplication
27
(3R) and subsequent diploidization in the NH loci. The VT and IT precursor genes are most
28
likely lineage-specific paralogs resulting from differential losses of the 3R NH paralogs in the
29
two superorders. The independent yet consistent retention of VT and IT in the two superorders
1
30
might be directed by a stringent ligand-receptor selectivity, which was established early in the
31
gnathostome evolution.
32
Introduction
33
The neurohypophyseal (NH) nonapeptides are an evolutionarily old family which is believed to
34
have originated much before the vertebrates, as one of the first neurotransmitters in the archetype
35
metazoan nervous system (Grimmelikhuijzen et al., 2002). Starting from the most primitive
36
metazoan- the cnidarians, the presence of nonapeptide homologs has been documented in several
37
non-vertebrate phyla (Grimmelikhuijzen et al., 1982; Cruz et al., 1987; Proux et al., 1987;
38
Satake et al., 1999; Kawada et al., 2008; Takuwa- Kwodo et al., 2003; Stafflinger et al., 2008).
39
In vertebrates, studies are relatively extensive and the nonapeptide precursor genes have been
40
cloned from several vertebrate groups: cyclostomes (Lane et al., 1988; Gwee et al., 2009; Suzuki
41
et al., 1995), cartilaginous fish (Hyodo et al., 2004; Gwee et al., 2009), teleosts (Heierhorst et
42
al., 1989; Morley et al., 1990; Hyodo et al., 1991; Suzuki et al., 1992; Venkatesh and Brenner,
43
1995; Warne et al., 2000), lungfishes (Hyodo et al., 1997), coelacanth (Gwee et al., 2008),
44
amphibians (Nojri et al., 1987; Searcy et al., 2009), reptiles (Montefiano et al., 2001), and birds
45
(Hamann et al., 1992). This has complemented earlier works of characterization of peptide
46
principles of the neurohypophysis by chromatographic methods and has provided useful insights
47
into the distribution of the peptides in different vertebrate species, their precursor structure and
48
evolution. The nonapeptides are synthesized as part of a large precursor molecule that undergo
49
post-translational processing to release the nine amino acid hormone with two half cysteine
50
residues at the first and sixth positions forming a disulphide bridge, and a cysteine- rich protein
51
called neurophysin (Acher, 1996). Neurophysin aids in proper folding of the precursor and its
52
sorting into secretory vesicles (de Bree et al., 2000). In vertebrates, about 15 nonapeptides are
53
known till date. They are classified into two families on the basis of the amino acid at the 8th
54
position, as the basic and neutral peptide families. Each vertebrate possesses at least two
55
peptides, one each from the two families except cyclostomes, where only the basic peptide
56
representative vasotocin (VT) is documented (Gwee et al, 2009). This has led to a widely
57
accepted theory that the VT precursor gene is the ancestor of all vertebrate nonapeptide precursor
58
genes, and has given rise to the two lineages following an event of duplication early in the
59
vertebrate evolution probably at the base of the gnathostome lineage. Evolutionarily too, the two
60
peptide families follow distinct lineages with VT representing the common basic peptide in all
61
non- mammalian vertebrates to be replaced by vasopressin (VP) in mammals, while isotocin (IT)
62
is the neutral peptide in teleosts, mesotocin (MT) in lobe-finned fishes and non eutherian
63
tetrapods, and oxytocin (OT) in eutherians. In elasmobranchs, a host of different neutral peptide
64
homologs has been reported which is in contrast with the remarkable stability in the type and
65
numbers of nonapeptides in any other group of Vertebrata (Acher, 1996). Presence of more than
66
two neurohypophyseal peptides has been reported from groups like elasmobranchs, amphibians,
67
and metatherians (Chauvet et al., 1983, 1984 1985; Rouille et al., 1988, 1989; Parry et al; 2000;
68
Hyodo et al., 2004). In teleosts, VT and IT have been reported as the basic and neutral hormone
69
representatives, respectively. Larhammar et al. (2009) emphasized the need for simplifying the
70
nomenclature with the original names VP for the basic peptides and OT for all neutral peptides.
71
The two evolutionary lines of basic and neutral nonapeptides across vertebrates represent a
72
contrasting picture in the frequency of substitutions in the 9 amino acid (aa) hormone. In the
73
basic lineage, there have been lesser substitutions than that in the neutral line, as evident from the
74
distribution of the peptides in vertebrates. In fact, the number of cognate receptors for the two
75
peptide families is also contrasting. While the stable basic nonapeptide line has a diverse
76
repertoire of at least five types of cognate receptors (V1A, V1B, V2A, V2B and V2C), the
77
neutral nonapeptide series has a single type (OTR) in all vertebrate groups (Ocampa Daza et al.,
78
2012; Yagamuchi et al., 2012; Lagman et al., 2013). All types of receptors are present in the
79
early gnathostomes, the cartilaginous fish (Yagamuchi et al., 2012) suggesting that the ligand-
80
receptor selectivity might have been established very early in the vertebrate evolution and it is
81
the co-evolution of the hormone with its cognate receptors which has maintained the two
82
families of basic and neutral nonapeptides, and allowed only a basic amino acid substitution at
83
the 8th position in the basic line and a neutral amino acid substitution at the 8th position in the
84
neutral line. While, the ligand- receptor selectivity might have directed the evolution only in the
85
9 aa hormone part; the larger neurophysin region is not under this evolutionary pressure.
86
Therefore to know the evolutionary history of the NH precursors especially in the light of
87
recurrent local and global duplication events occurring throughout the vertebrate history, there is
88
a need to do a phylogenetic study over the entire precursor rather than considering the
89
90
91
Teleosts had undergone a third round of whole genome duplication, (3R) (Meyer and Schartl,
92
1999; Christoffels et al., 2004; Jallion et al., 2004) in addition to the two vertebrate-specific
93
whole genome duplications. After the 3R in teleosts, there was diploidization by the loss of 3R
94
paralogs. These losses of the 3R paralogs were different in various groups of teleosts (Garcia de
95
la serrana et al., 2014). Even in such a scenario, the consistency in the number and type of
96
97
public database are mostly sequences from the species-rich superorder Acanthopterygii while
98
only few nonapeptide precursor sequences from the other species-rich group, the superorder
99
Ostariophysi are available. The Acanthopterygii and Ostariophysi have undergone independent
100
adaptive radiations and are known to have suffered differential losses of 3R paralogs. Therefore,
101
it is pertinent to have sequence information from more species representing all groups in
102
103
104
Previous studies from our laboratory have shown a well developed and highly organized VT
105
system in the Asian air-breathing catfish Heteropneustes fossilis. VT was characterized in both
106
brain and ovary of the catfish with seasonal changes (Singh and Joy, 2008), and has been shown
107
108
hydration, ovulation and prostaglandin secretion (Singh and Joy, 2009; Singh and Joy, 2010;
109
Singh and Joy, 2011; Joy and Singh, 2013). IT is less effective in influencing these events.
110
Three VT receptors have been cloned from the catfish V1a1 (accession no.KF434615.1), V1a2
111
(accession no.KF434616.1) and V2A (accession no.KF434617.1), which display a wide tissue
112
distribution (Rawat et al., 2015). All the three receptors are expressed in the brain and ovary,
113
114
The catfish H. fossilis holds an important phylogenetic position as it is believed to be the sole
115
survivor of an old clade that went extinct at the K-T (Cretaceous-Tertiary transition period)
116
boundary (Jansen et al., 2006) when a major extinction event took place due to severe volcanic
117
activity (Deccan traps) in the Asian region. This might have severely polluted and deoxygenated
118
the inland water bodies and only fish with air-breathing apparatus survived. Air-breathing habit
119
also demands a shift in osmoregulation and fluid homeostasis in which apart from other systems,
4
120
the NH nonapeptides play a key role. In view of the important taxonomic and phylogenetic
121
position of H. fossilis and its air-breathing habit, molecular characterization of the NH peptide
122
genes as a follow on of earlier anatomical and physiological studies on VT has been attempted in
123
the present work. The sequence information was also extended to Clarias batrachus, another air-
124
breathing catfish, and used in the phylogenetic analysis. The catfish NH precursor sequence
125
information from the present study will add to the information pool of teleost nonapeptides from
126
a group other than the superorder Acanthopterygii and allow discussing the phylogeny of the
127
128
distribution profile for these genes was also carried out since VT was described in the catfish
129
ovary earlier.
130
131
132
Adult H. fossilis (4050 g) were collected from local fish markets in Varanasi. They were
133
maintained in the laboratory for 48 h under natural photoperiod (13.0 L: 11.0 D) and temperature (25
134
2 C) to overcome stress due to transportation and fed daily with goat liver ad libitum. Adult C.
135
batrachus were obtained in the resting phase for the cloning work. The fish were weighed and
136
sacrificed by decapitation. All experiments were performed in accordance with the guidelines of the
137
138
139
Guanidine thiocyanate - phenol solution (Qiagen), Revert-Aid H Minus First Strand cDNA
140
Synthesis Kit (Fermentas), 2X PCR Master Mix (Fermentas), DNase (Ambion) and veriquest SYBR
141
green qPCR master mix (affymetrix) were purchased through local suppliers. Agarose, Tris base,
142
Glacial acetic acid, EDTANa2 and other chemicals were of molecular grade, purchased from E-
143
Merck, India. LB broth, LB Agar, ampicillin, X-Gal and IPTG were purchased from Himedia, India.
144
Hyaluronidase type IV was purchased from Sigma-Aldrich, India. The primers used were
145
146
147
For cloning of VT, an approach of first isolating partial VT cDNA using degenerate PCR
148
(polymerase chain reaction) followed by 3 and 5 Rapid Amplification of cDNA ends (RACE)
149
to obtain the full length VT cDNA sequence was applied. Total RNA from the brain of
150
acclimatized female catfish in the resting phase of the reproductive cycle was prepared by the
151
single-step method of RNA isolation (Chomczynski and Sacchi, 1987) by acid guanidium
152
153
Five g total RNA was reverse- transcribed using random hexamer primers and Revert Aid M-
154
MuLV reverse transcriptase in a 20 L reaction volume (first strand cDNA synthesis kit,
155
Fermantas) using the manufacturers protocol. One L of the resulting cDNA was used to
156
amplify partial VT cDNA in 25 L reaction volume using 2 X PCR master mix (Fermentas) and
157
VT degenerate forward primer and VT degenerate reverse primer in a cycling condition of one
158
cycle of 95C, 5 min; then 35 cycles of 95C, 30 s; 57C, 30 s; 72C, 1 min; and a final 7-min
159
elongation at 72C. The degenerate primers were designed using the software iCODEHOP
160
161
sequences were used as inputs (Table 1). The resulting 145 bp partial VT amplicon was purified
162
using Nucleo-pore PCR clean- up gel extraction kit (Genetix) and sequenced taking services of
163
Eurofin genomics, Bangalore, India. For obtaining full length sequence, 3 RACE was done for
164
which total RNA from the brain was reverse-transcribed using oligo dT anchor primer and
165
Revert Aid M-MuLV reverse transcriptase. One L of the resulting cDNA was amplified using a
166
VT H FP (corresponding to the hormone moiety), designed from the partial sequence and anchor
167
primer to obtain a 700 bp 3 RACE amplicon, which was cloned and sequenced. This sequence
168
was utilized for designing two gene-specific nested reverse primers to do 5 RACE. Total RNA
169
from the brain was reverse-transcribed using the outer gene specific VT RP and Revert Aid M-
170
MuLV reverse transcriptase. The resulting cDNA was purified using the Nucleo-pore PCR
171
clean- up gel extraction kit (Genetix) to remove residual dNTPs. The purified cDNA was dA
172
tailed using dATP and terminal transferase. The dA tailed cDNA was used for PCR
173
amplification using the nested RP 2 primer and oligo dT anchor primer. The 5 RACE amplicon
174
was purified, cloned and sequenced. Full length VT cDNA sequence was submitted to the
175
176
For cloning IT precursor cDNA, total RNA from the brain was reverse-transcribed using an oligo
177
dT anchor primer and Revert Aid M-MuLV reverse transcriptase. One L of the resulting cDNA
6
178
was PCR amplified using a sense or forward primer IT H FP designed from the IT hormone
179
moiety and anchor primer, following the 3 RACE protocol. The resulting 700 bp amplicon was
180
sequenced taking services from Eurofin genomics. To get the 5 end of the precursor cDNA, 5
181
RACE was done. Total RNA from the brain was reverse- transcribed using an outer reverse or
182
antisense primer, RP1 (designed over a conserved region of the neurohypophyseal peptide
183
neurophysin) and Revert Aid M-MuLV reverse transcriptase. The resulting cDNA was purified
184
and dA tailed. The dA tailed cDNA was used for PCR amplification using an overlapping and
185
nested antisense primer RP2 and oligodT anchor primer. The resulting 200 bp 5 RACE
186
amplicon was ligated into pGEMT vector (promega kit), following the manufacturers protocol
187
and transformed into the E.coli DH5 competent cells. Transformed cells were plated into LB
188
Agar solid medium containing ampicillin, X- Gal and IPTG to select positive transformants and
189
blue- white screening was done for recombinant colonies. Plasmids from white colonies
190
(colonies transformed with recombinant plasmids) were extracted by alkaline lysis method and
191
sequenced. Sequence information from the IT precursor containing inserts (as known by in silico
192
translation of the insert sequences) was used to design a sense primer from the 5 UTR region
193
(IT UTR FP), followed by 3 RACE with this primer to obtain the entire IT precursor sequence,
194
which was submitted to the GenBank with the accession no. JX669009.1
195
Partial sequences of C. batrachus VT and IT precursors were obtained by 3 RACE with the
196
forward primers VT H FP and IT H FP (the same used for H. fossilis), to obtain a sequence read
197
from the hormone region till the 3 untranslated region. The primer details are given Table 1.
198
199
200
201
202
203
204
Table 1. List of primers used for cloning and semi-quantitative RT-PCR of vasotocin and
205
isotocin
Primers
Sequences
VT d FP
VT d RP
VT H FP
VT FP
VT RP
RP1
RP2
VT UTR FP
IT H FP
IT UTR FP
Oligo dT anchor primer
Anchor primer
IT FP
IT RP
actin FP
actin RP
TCCGCTTGTTACATCCARAAYTGYCC
ACATCCCAGTCCCTCTCCRCARCDAT
GTTACATCCAGAACTGCCCCAGA
TGTTACATCCAGAACTGCCCCAGA
CAGCCCAGTCCTTCTCCACAGCA
TTTATCTCCAGGACCGCAAG
CAGGACCGCAAGACATACAC
GTCCAGTGAGAGACAGACCTCCGG
ACATCTCCAACTGTCCCATC
CATCAGCTACTGAAGCTACTGATTCGT
GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV
GACCACGCGTATCGATGTCGAC
TCAATCTTCTGCATGCTGTGTCT
CACACGCCATGCACTGTCTATTG
TGGCCGTGACCTGACTGAC
CCTGCTCAAAGTCAAGAGCGAC
206
207
208
Since the VT and IT transcripts are less abundant in the ovarian follicles, a semi-nested 3 RACE
209
was done to get a single, sharp amplification of the gene transcripts. Briefly, it included a
210
primary PCR using VT UTR FP, IT UTR FP spanning the 5 UTR region of VT and IT genes,
211
212
transcription of RNA using oligodT anchor primer), followed by a secondary PCR using VT H
213
FP, IT H FP and anchor primer to amplify1 L of the primary PCR product. The amplicons from
214
215
216
The NH nonapeptide precursor sequences of species representing most vertebrate classes were
217
tried to be included in the phylogenetic analysis. The sequences were procured from the
218
GenBank. Details of the sequences of the species with accession numbers are given in
8
219
supplementary table 1. For procuring the NH precursors of Ictalurus punctatus, a BLAST search
220
against the Ictalurus EST database (Lu et al., 2010) was done using the H. fossilis VT and IT
221
sequences as queries. The identities of the putative VT and IT precursor mRNAs obtained were
222
confirmed by in silico translation of the sequences. The spotted gar Lepisosteus oculatus,
223
224
nonapeptide precursor sequences of this species were procured by BLAST searches in its
225
226
227
Phylogenetic trees were constructed using the Neighbor- Joining (NJ) and Maximum-Likelihood
228
(ML) method. Evolutionary distances were computed using the Poisson correction method for
229
rate of amino acid substitution. All phylogenetic studies were done using the software MEGA 6
230
231
sequences within and between species, the sequences were grouped according to the taxonomic
232
units for calculation of evolutionary distance between groups (Table 2). Keeping in view of the
233
enormous species diversity, the teleost sequences of the two superorders Acanthopterygii and
234
Ostariophysi were independently treated. Multiple sequence alignment used for the tree
235
construction and distance calculation was the same and was done in MEGA6 using Clustal W
236
parameters. Gonnet protein weight matrix with gap opening and gap extension penalties of 10
237
and 0.2 respectively was used for the alignment. The alignment is provided in a supplementary
238
file A.
239
240
241
242
243
244
245
246
247
248
Table 2. Details of the taxonomic groups and the species included in each for the phylogenetic
distance calculation between groups. The accession numbers of the sequences are given in
supplementary table 1
S.NO
Taxonomic group
Species
Cyclostome VT
Acanthopterygii VT
Ostariophysi VT
Heteopneustes fossilis, Clarias batrachus and Ictalurus punctatus, Danio rerio, Astyanas
mexicanus (LOC103042813, LOC103030472)
Cartilaginous fish VT
Lungfish VT
Coelacanth VT
Latimeria menadoensis
Acanthopterygii IT
Ostariophysi IT
10
11
Lungfish MT
12
Coelacanth MT
Latimeria menadoensis
12
Amphibian VT
13
Amphibian MT
14
Reptilian VT
Podarcis siculus
15
Reptile MT
Podarcis siculus
16
Avian VT
17
Avian MT
Taeniopygia guttata
18
Mammalian VP
19
Mammalian OT
10
249
250
The nonapeptide gene-containing chromosome blocks of spotted gar and representative teleost
251
species were obtained from the NCBI and Ensembl genome browser. Fugu, medaka, tilapia and
252
stickleback belong to the teleost superorder Acanthopterygii, and zebrafish and cavefish belong
253
to the superorder Ostariophysi. The selection of the chromosome blocks from Ensembl database
254
was based both on searching the genome assemblies for the nonapeptide genes by named
255
searches as well as BLAST searches using teleost nonapeptides as queries. This is to ensure that
256
nonapeptide loci are obtained even in the species where the genes are not annotated. The
257
chromosome blocks were obtained from the NCBI database mostly by navigating the genomic
258
context of the genes. Blocks obtained from both Ensembl and NCBI were tallied for each species
259
and a consensus assembly of genes on the blocks was arrived at by filling in for the non
260
annotated genes in one of the databases from information obtained from the other. Apart from
261
the nonapeptide genes, the assemblies were also searched for the genes linked with the
262
nonapeptide genes and the additional chromosomal blocks harbouring these genes (paralogous to
263
the linked genes), if distinct from the nonapeptide loci. Additionally, the loci of the linked genes
264
in human were obtained and used as a reference tetrapod assembly for comparison of conserved
265
synteny.
266
267
A two step qPCR was conducted to show tissue and seasonal expression of VT and IT genes. For
268
tissue expression profile, various tissues viz. brain, gills, liver, muscle, kidney and gonads were
269
collected. For the ovarian tissue sample, the follicles were collected from the post-vitellogenic
270
ovary and the follicular layer was separated from oocyte using hyaluronidase treatment (Mishra
271
and Joy, 2006). Briefly, the protocol included treatment of a batch of about 500 follicles with 0.3
272
% hyaluronidase (Type IV) for 5 min. The isolated follicular envelope (granulosa and theca
273
cells) and the denuded oocytes were used for the expression studies. For seasonal expression,
274
brain and ovary was collected during the different reproductive phases, resting (November
275
276
post-spawning (SeptemberOctober). About 100 mg tissues were used for extraction of total
277
RNA by the single-step method of RNA isolation. To remove genomic DNA contamination from
11
278
the preparation, DNAase (Ambion) treatment (2 units/ 10 g RNA) was given and subsequently
279
DNAase was heat inactivated at 75C in presence of EDTA. RNA purity was checked by
280
A260/A280 ratio. Samples having a ratio above 1.8 were only considered for reverse transcription.
281
Two g of the total RNA was reverse-transcribed using random hexamer primers and Revert Aid
282
M-MuLV reverse transcriptase in a 20 L reaction volume (first strand cDNA synthesis kit,
283
Fermantas) using the manufacturers protocol. The resulting cDNA was diluted 10 times and
284
1L was used in a PCR reaction of 20 L containing veriquest SYBR green 2X master mix and
285
VT/IT FP and IT FP/ RP using manufacturers protocol in an Applied Biosystem 7500 machine
286
with a thermal condition of 50 C for 2 min, 95 C for 10 min, followed by 40 cycles of 95 C for
287
15 sec and 60 C for 1min. The specificity of the PCR product was checked by dissociation curve
288
analysis of the amplicon and was also checked by agarose gel electrophoresis. The relative gene
289
expression in different tissues and across different reproductive seasons was expressed using the
290
comparative CT method with the catfish - actin (accession number FJ409641.2), used as the
291
endogenous control. The resting phase brain cDNA was taken as the calibrator sample for
292
relative quantity calculation (Livak et al., 2001). Each reaction was set up in duplicate and the
293
average CT value was taken for calculation. Graphs were plotted with the mean RQ (relative
294
quantity) values (2-CT) of five fish and represented as mean SEM. The results were analyzed
295
296
significance.
297
3. Results
298
299
The isolated full length cDNA of the HfVT precursor is 618 bp long with a cds from 60-524 bp and
300
encodes a VT precursor of 155 amino acids (aa; Fig.1; supplementary Fig.1A). The deduced VT
301
precursor codes for a signal peptide (1-20 aa), a 9 aa hormone moiety (21-29 aa), a 3 aa enzymatic
302
processing site (30-32 aa) and a 122 aa neurophysin (34-155 aa). The central part of the neurophysin
303
(40-118 aa) is highly conserved and belongs to the hormone_5 superfamily (NCBI CDD accession
304
pfam00184) of the neurohypophyseal hormones (NCBI CDD accession smart00003). There are 14
305
cysteine residues in this region (42, 45, 53, 59, 60, 66, 76, 86, 93, 100, 106, 107, 112 and 118). The
306
C- terminal part of the neurophysin (119- 155 aa) is poorly conserved except for the presence of a
307
leucine-rich region LLLRILH (141-146 aa) (Fig. 1) that is conserved across all vertebrate groups.
12
308
All these features conform to the typical features of the neurohypophyseal hormones. There is no
309
putative site for N- linked glycosylation in C-terminal domain corresponding to copeptin, making it
310
akin to other teleost neurohypophyseal hormone precursors, unlike lungfish and tetrapod hormone
311
precursors.
312
The isolated HfIT precursor cDNA is of 708 bp long with a cds from 56- 508 bp
313
hormone precursor of 151 aa (Fig. 1; supplementary Fig. 1B). Similar to the VT precursor, the
314
putative IT precursor encodes for a signal peptide (1-20 aa), a hormone moiety of 9 aa (21-29 aa),
315
enzymatic processing signal (30- 32aa) and a 118 aa neurophysin (34- 151 aa). Further, the central
316
part of neurophysin (40- 118 bp) is highly conserved and belongs to the hormone_5 superfamily
317
(NCBI CDD accession pfam00184) of the neurohypophyseal hormones (NCBI CDD accession smart
318
00003) and there are 14 cystine residues in this region (42, 45, 53, 59, 60, 66, 76, 86, 93, 100, 106,
319
107, 112, 118) (Fig. 1). The presence of an extended C-terminal part homologous to the C- terminal
320
part of the VT precursors and the mammalian vasopressin-associated copeptin makes it similar to
321
other IT precursor but dissimilar to other neutral peptide precursors like MT and OT precursors and
322
other cartilaginous fish neutral hormone precursors which lack an extended C-terminal region. The C-
323
terminal part of the neurophysin (119- 151 aa) is poorly conserved except for the presence of a
324
leucine- rich region LLKLLHL (138-144aa), which is a signature of the C-terminal region of the
325
neurohypophyseal hormone precursors. There is neither a dibasic cleavage site nor putative site for
326
327
328
The cloned partial sequences of C. batrachus (Cb) VT and IT cDNA extend from the N-terminal
329
neurophysin coding region to the 3 UTR (supplimentary Fig.1C and 1D). The putative precursors,
330
like the H. fossilis precursors, have all the essential features of the neurohypophyseal precursor
331
proteins, with a central conserved neurophysin having 14 cysteine residues. The poorly conserved C-
332
terminal regions have leucine-rich boxes, LLLRILH in the VT precursor and LLKLLHL in the
333
334
In silico translation of the clones, recognized as the neurohypophyseal hormone precursors from
335
the EST database of I. punctatus (Ip) by BLAST reveals that they indeed code for VT and IT
336
precursors. The sequence read of the putative VT precursor clone (acc. no. BM495247) starts
13
337
from 5 UTR region and ends in the C-terminal part of the neurophysin. On the other hand, the
338
putative IT precursor clone (acc. no. BE213165) sequence extends from the signal peptide region
339
340
The 3 UTR region of the H. fossilis VT gene has a long stretch of 19 CA repeats. The C.
341
batrachus VT gene has 4 only CA repeats in the 3 UTR at approximately the same position as
342
343
344
BLAST search revealed that the spotted gar has three neurohypophyseal nonapeptides, VT as the
345
basic nonapeptide (XM 006626529.2) and two nonapeptides in the neutral family, IT (XM
346
006626499.1) and a novel peptide (XM 006626523.1, 1-366 nt) with a unique hormone
347
CFISNCPLG is present. While the spotted gar IT has an extended C-terminal with a leucine-
348
rich core, the novel nonapeptide has a short C-terminal like all other neutral nonapeptide
349
350
351
The nonapeptide genes expressed only in the brain and ovary of the catfish (Fig. 2). In the ovary,
352
the expression was confined to the follicular envelope and not in oocytes. There was no
353
expression in the gill, liver, kidney, testis and muscle. The VT and IT mRNA expressions were
354
355
The sequence reads of the semi-nested 3 RACE amplicons of VT and IT in the ovary were
356
partial but good enough to indicate the presence of functional transcripts of the genes in ovarian
357
follicles (not shown). Also, the sequences were similar to the brain transcripts, pointing to the
358
same gene responsible for synthesis of the nonapeptides in both brain and ovary.
359
VT and IT expressions showed significant seasonal changes both in the brain and ovary (Fig. 3).
360
In the brain, the VT expression was low in the resting phase and increased in the preparatory,
361
pre-spawning and spawning phases. The expression remained high in the post-spawning phase
362
similar to the preparatory and pre-spawning phases. The expression of IT precursor was similar
363
to that of the VT precursor but it decreased sharply in the post-spawning phase as in the resting
364
phase.
14
365
The highest expression was observed in the spawning phase for both the genes (Fig. 3A and 3B).
366
In ovary too, the expression of both VT and IT increased in the reproductive phases compared to
367
the resting phase (Fig. 3C and 3D). However, while in the spawning phase the expression of the
368
VT precursor was higher as compared to the preparatory and pre-spawning phases (Fig. 3C), the
369
IT precursor expression remained equally high in all the reproductive phases (Fig.3D).
370
371
372
The phylogenetic inference on vertebrate NH peptide precursors was drawn from both tree
373
constructions as well as from the evolutionary distance calculation in between groups. Both the
374
NJ and ML trees are similar and only the ML tree is shown in Fig. 4. The NH sequences of
375
salmonids and catastomids (teleosts) were included in the initial analysis but were removed from
376
the tree as they did not make a definite cluster probably due to polyploidization that led to two
377
copies of VT and IT genes, and gene conversions (Hyodo et al., 1991; Suzuki et al., 1992). Both
378
the phylogenetic tree and evolutionary distance calculation in between the groups (Fig. 5) show
379
that the NH precursor paralogs of cartilaginous fish, coelacanth and mammals are
380
phylogenetically closer than their homologs in other groups. The cartilaginous fish VT and
381
neutral hormone precursors share a distance of 0.350, coelacanth VT and MT precursor share a
382
distance of 0.132, and the mammalian VP and OT precursors share a distance of 0.219. The
383
evolutionary distances of the NH precursors of these groups with that of other vertebrate groups
384
are larger (Fig. 5). Amongst the cartilaginous fish, elephant shark VT and OT precursors share a
385
386
The phylogenetic tree with the VT precursor of Agnatha as the outgroup also shows that the
387
teleost VT and IT precursors form a cluster with those of the spotted gar (Holostei) while other
388
gnathostome NH precursors form another cluster, which includes the cartilaginous fish VT and
389
neutral hormone precursors, and coelacanth and lungfish VT and MT precursors. However, the
390
bootstrap support for the node separating the teleost NH precursors and the other gnathostome
391
NH precursors is low.
392
The catfish NH precursors expectedly segregated into their respective clusters i.e. the HfVT,
393
CbVT, IpVT and HfIT, CbIT, IpIT, making two distinct clusters. Within these clusters, the
15
394
CbVT, HfVT and CbIT, HfIT are closer to each other than to IpVT and IpIT, respectively. The
395
spotted gar NH precursors made a common cluster; the two neutral NH peptide precursors (IT
396
precursor and the novel peptide precursor) are evolutionarily closer than the VT precursor. The
397
Acanthoperygii VT and IT precursors are phylogenetically closer than their orthologs in the
398
Ostariophysi. The evolutionary distance in between the groups confirms this. The evolutionary
399
distance between the Acanthopterygii VT and IT precursors is only 0.269 while that between
400
401
Ostariophysi IT precursors is 0.452. The ostariophysian VT precursor form a cluster having the
402
catfish VT precursors and one of the two VT precursors of cavefish (Astyanax mexicanus), while
403
zebrafish (Danio rerio) VT precursor does not form part of this cluster. The ostariophysian IT
404
precursors make a cluster that includes the catfish and zebrafish IT precursors, and one of the IT
405
precursors of the cavefish. The other cavefish VT and IT precursors, which do not form a part of
406
the ostariophysian clusters, make a separate cluster and share a close phylogenetic relation with
407
each other. Unlike the Acanthopterygii VT and IT precursors, the Ostariophysi VT and IT
408
precursors are phylogenetically distant and share an evolutionary distance of 0.422 (Fig. 5).
409
3.5.2 Linkage and synteny analysis of VT and IT in teleosts and spotted gar
410
Details of the chromosome blocks and linkage groups used in the analysis are summarized in
411
supplementary table 2. A schematic representation of conserved synteny in the spotted gar and
412
teleosts is presented in Fig. 6. In the spotted gar, VT, IT and the novel peptide precursors are
413
present in the same linkage group. But, the VT precursor gene (avp-like) and IT precursor genes
414
(oxt-like) are not arranged in tandem, unlike in human and Xenopus where the nonapeptide genes
415
are present in tandem. However, the oxt-like gene and the novel peptide precursor gene (both in
416
the neutral family) are arranged in tandem. Similarly, in acanthopterygian teleosts (fugu, tilapia,
417
medaka, sticleback), the avp- like (VP- like) and oxt- like genes are in the same linkage group
418
but are not present in tandem. In the ostariophysian teleosts, two contrasting situations exist. In
419
zebrafish, avp- like and oxt- like are present on two different chromosomes. In contrast, in the
420
cavefish where there are two genes each for VT and IT, one pair is present in the same linkage
421
group like in the acanthopterygian fish. The other avp-like and oxt-like genes are present on
422
different linkage groups so that in the cavefish, there are three linkage groups harbouring the
423
nonapeptide genes. The chromosome block in which the two genes are linked shares a conserved
424
synteny with the acathopterygian NH loci and zebrafish avp- like containing linkage group. The
16
425
other oxt-like gene is present in the paralogon of this block and shares a conserved synteny with
426
the zebrafish oxt-like containing block and the Acanthopterygii paralogon of the NH loci. The
427
genomic context of the other avp-like gene is considerably different from that of the teleost and
428
429
examination it seems to share a conserved synteny with the tetrapod nonapeptide loci as it is
430
present in the same linkage group as the genes fast kd 5 and ddrgk (genes linked with NH genes
431
432
433
4. Discussion
434
435
In the present study, we cloned a full length cDNA of 618 bp from H. fossilis that coded for a VT
436
precursor of 155 aa. There is no difference in the sequence of the brain and ovarian transcripts
437
and hence the same gene expresses in both the tissues. We could also clone C. batrachus partial
438
VT precursor gene from the N-terminal neurophysin coding region to the 3 UTR. The catfish
439
VT precursor gene does not show any major departure from the other known teleost sequences.
440
However, a repeat of CA dinucleotide at the 3 UTR is unique and the number of repeats is
441
varied in different catfishes. In H. fossilis, there are 19 repeats, while in C. batrachus, there are
442
only 4 repeats. The putative Ictalurus VT precursor gene from the database do not show any CA
443
repeat. The available sequence read ended with the coding region of the neurophysin and did not
444
extend to the 3 UTR region so that no CA repeat was observed. It may be inferred that the clone
445
has CA repeats in the 3UTR, as it belongs to the microsatellite enriched library. It is very likely
446
that the CA repeats may be a conserved feature of the VT precursor genes of catfishes and may
447
448
The deduced amino acid sequence of the VT precursors shows that, like all other members of
449
450
having a separate function. Apart from the signal peptide, the vasopressin family peptides have a
451
three domain structure. The nine amino acid hormone forms the smallest domain, the most
452
biologically active region, and is released with a C-terminal amidation after a three-step
453
enzymatic process. The second domain called neurophysin is joined to the hormone moiety with
454
a GKR, which is the site of the three-step enzymatic process of cleavage of the hormone and
455
amidation. The N-terminal 9 amino acid of the neurophysin is poorly conserved, while the rest is
17
456
conserved. The third domain is copeptin which is poorly conserved except for the presence of a
457
leucine-rich core (de Bree and Burbach, 1998). The catfish VT precursors like the vasopressin
458
family peptides have the three domain structure. Only in mammals, a second cleavage apart from
459
the hormone-neurophysin cleavage has been reported, which separates the copeptin from the
460
461
amphibians and lungfish (Hyodo et al., 1997) but unlike in teleosts. The catfish VT copeptin
462
domain does not have a N-linked glycosylation site nor is cleaved. The isolated IT precursor has
463
a multi-domain structure too and, like VT/VP precursors, has an extended C-terminal with a
464
leucine-rich core, while no other vertebrate pre-prohormone in the neutral line has got an
465
extended C-terminal.
466
467
468
In H. fossilis, VT and IT genes are expressed only in the brain and ovary. In a previous study, the
469
470
catfish (Singh and Joy, 2008). Brain VT showed seasonal variation associated with the annual
471
reproductive cycle of the catfish with an increase during the recrudescent phase and a decrease
472
after spawning. The expression of the VT and IT genes shows differences in tissue and seasonal
473
transcript levels. The transcript levels are high in the brain than ovary. In the ovary, the
474
expression was confined to the follicular layer since the denuded oocytes did not show any
475
signal. In the seasonal study, the VT and IT precursor transcripts in the brain and ovary show
476
significant variations with higher levels during the reproductive phase, suggesting a prominent
477
influence of the reproductive factors on the transcriptional activity of the genes. The expression
478
of both VT and IT precursor genes in the ovary points to local synthesis of the peptides for a
479
paracrine role in ovarian activity. This confirms the earlier findings from our laboratory showing
480
481
follicular layer (Singh and Joy, 2008). The ovarian VT levels vary with the annual ovarian cycle
482
closely associated with recrudescence and spawning (Singh and Joy, 2008). Moreover, steroid
483
484
modulate VT secretion as in the brain (Singh and Joy, 2009, 2011). In vitro studies with ovarian
485
follicles have showed that VT stimulated steroidogenesis, oocyte final maturation, oocyte
486
hydration, ovulation and prostaglandin secretion (Joy and Singh, 2013; Singh and Joy, 2010;
18
487
2011). Gwee et al., (2009) have shown VT expression in the ovary of elephant shark. Bobe et al.
488
(2006) have reported VT and IT mRNAs in the preovulatory ovaries of rainbow trout, while the
489
transcripts were not detected in the ovary during the other stages of reproduction. In mammals,
490
there are several reports of the presence of VP and OT in non-neuronal peripheral sites, where
491
they have a paracrine role (Wathes et al., 1984; Clements and Funder, 1988; Sernia et al., 1994).
492
Similarly, in other classes of vertebrates like aves, presence of VT has been reported in the
493
reproductive tissues (Saito and Grossmann, 1999). The peripheral expression of VT and its
494
homologue signifies a phylogenetic pattern and the phenomenon seems to be conserved in all
495
496
497
498
Phylogenetic inference from the present study shows that the precursor paralogs in some
499
vertebrate groups (cartilaginous fish, coelacanth, mammals) are closer than their homologs in
500
other groups. Similarly, studies in salmons (Hyodo et al., 1991; Suzuki et al., 1992) and flounder
501
(Warne et al., 2000) showed that the VT and IT precursors share high sequence identity. Gwee et
502
al., (2008, 2009) also showed that the sequence identity between the respective precursor
503
paralogs is very high in coelacanth and elephant shark. The studies also reported that the high
504
sequence similarity was mostly in the central portion of the neurophysin. Events of gene
505
conversion between the paralogous genes may be responsible for diluting the distance created by
506
the long evolutionary history and make them seem more closely related than what they actually
507
are, typifying concerted evolution. Concerted evolution is common in duplicated genes, for
508
example, as reported in rRNA genes, the highly conserved histone and ubiquitin gene families,
509
and the heat shock protein gene family (Nei and Rooney, 2005). For the NH precursors, gene
510
conversions seem to encompass the central neurophysin region and, therefore, the high sequence
511
similarity in the central portion. Further evidence in favour of frequent events of gene conversion
512
in between the nonapeptide paralogs is given for the bovine VP and OT precursor mRNA and
513
avian NH precursors, having their central neurophysin not merely similar but identical pointing
514
to a recent event of gene conversion (Ruppert et al., 1984; Levy et al., 1987). Gwee et al., (2008)
515
found a low GC3 content in all the three exons of coelacanth VT and MT precursors as
516
compared to bovine and human VP and OT genes, and attributed the sequence similarity in
19
517
coelacanth to purifying selection rather than due to gene conversion. In mammals and yeast, gene
518
conversion events are known to be GC-biased so that the GC content increases after gene
519
conversion (Duret and Galtier, 2009; Galtier, 2003; Marais 2003; Noonan et al., 2004).
520
However, a recent study showed that gene conversions might not always be GC-biased (Assis
521
and Kondrashov, 2011) and in such a scenario one of the evidence in favour of gene conversion
522
can be incompatibility between phylogenetic tree and known duplication history (Mansai and
523
Innan, 2010), as is the case in the present study. The neurophysin is only concerned with
524
precursor processing and axonal transport which is common to all nonapeptides in contrast to the
525
hormone moieties that evolved under the constraints of stringent and differential ligand- receptor
526
selectivity in the two families. Hence, gene conversion events encompassing only the
527
neurophysin might have occurred during evolution. This might also be facilitated by the 3-exon
528
structure of the nonapeptide genes where the central neurophysin is encoded by a separate exon,
529
530
Based on the gene structures of salmon VT and IT, Urano and Ando (2011) had proposed a
531
distinct and independent origin of the teleost IT as compared to the neutral hormone precursors
532
533
precursors with a leucine-rich core, like in VT/VP. However, the authors have added that the
534
molecular divergence of VT and IT in teleosts is complicated because of the 3R. The recent
535
addition of the spotted gar sequences in the database and the analysis in the present study has
536
prompted a re-apprisal. The presence of IT with the extended C-terminal in the spotted gar
537
suggests an origin of IT in the actinopterygian lineage before the 3R. The spotted gar has an
538
additional neutral peptide with a short C-terminal, which may be related directly to the
539
evolutionary line of other vertebrate neutral hormone precursors that has originated at the base of
540
the gnathostome lineage. In no teleost studied till date, a neutral peptide with a short C-terminal
541
has been reported. Therefore, it may be hypothesized that the neutral hormone precursor gene
542
with the short C-terminal might have originated very early in vertebrates, as documented in the
543
cartilaginous fish (Hyodo et al., 2004; Gwee et al., 2009) and this early neutral hormone
544
precursor might be the evolutionary forerunner of MT and OT. In teleosts, this neutral hormone
545
precursor gene might have been secondarily lost or needs to be traced in the teleost lineage.
20
546
An interesting result that surfaced due to the addition of the catfish nonapeptide precursor
547
sequences in the phylogenetic study is about the differences in the evolutionary distance shared
548
by the precursor paralogs of different groups within teleosts. While the Acanthopterygii VT and
549
IT precursors share a very small evolutionary distance (0.269) between them, the Ostariophysi
550
VT and IT precursors are phylogenetically distant (0.422). The distance between the VT
551
precursors and IT precursors of Acanthopterygii and Ostariophysi is also high (0.426 and 0.452,
552
respectively). Phylogenetic clustering of the teleost NH precursors further supports these results.
553
In the phylogenetic tree, the Acanthopterygii VT and IT precursors clustered together and
554
seemed to have diverged from a common lineage. While the Ostariophysi IT precursors formed a
555
definite cluster that included the catfish IT precursors, one of the IT precursors of the cavefish
556
and the zebrafish IT precursor. The VT precursors of the catfish and one of the cavefish make a
557
cluster which does not include the zebrafish VT precursor. The results from the distance
558
calculation between the groups and the phylogenetic tree suggest that underlying the apparent
559
homogeneity in all teleosts having VT and IT, there might be a difference in the origin of VT and
560
561
The analysis of the genomic loci of the NH hormone genes and the synteny analysis of the loci in
562
different groups of teleosts further support the above assumption. Gwee et al., (2008 and 2009)
563
showed that in coelacanth, lamprey and elephant shark (cartilaginous fish), the NH gene loci has
564
a conserved synteny with the tetrapod NH loci. In fugu, there is a rearrangement. In the present
565
study, the NH gene loci were studied in the spotted gar and teleosts belonging to different
566
groups, which are known to have suffered independent gene losses during the process of
567
diploidization (Garcia de la serrana et al., 2014). Our study indicates that, indeed, the loci and 3R
568
paralogons show the footprints of rearrangements and differential losses of the NH gene paralogs
569
in the different teleost lineages. On examination of the different genes linked with the
570
nonapeptide genes in the spotted gar chromosome segment and the human linkage groups (as a
571
tetrapod reference), it may be inferred that a rearrangement in the position of the genes in the
572
nonapeptide loci of the spotted gar occurred by the fusion of the two blocks of chromosomes
573
after the origin of IT in the actinopterygian lineage, disrupting the tandem arrangement of the
574
genes, before the 3R. After the 3R, in teleosts the two paralogous blocks (3R paralogons)
575
harbouring the nonapeptide genes suffered further rearrangement and differential gene losses
576
during the process of diploidization. The rearrangement was common judging from the
21
577
conserved synteny in both the NH gene loci and its paralogon in all teleosts. However, the gene
578
losses in these loci were differential in the two teleost superorders Acanthopterygii and
579
Ostariophysi. This led to the linked and non-linked avp-like and oxt-like genes in these different
580
groups. In the acanthopterygian lineage, both the genes might have been lost from the same
581
paralogon maintaining the linked situation of the two genes similar to the spotted gar i.e. the pre
582
3R genome. In the zebrafish, avp like and oxt-like genes were lost one each from two paralogons
583
so that a single copy of the NH hormone genes was maintained and they remained unlinked. It is
584
difficult to reconstruct the sequence of events that can explain the present picture of synteny in
585
the NH loci and also the inconsistent phylogenetic relation between the VT and IT precursors in
586
the different groups of teleosts. However, it may be inferred that in the base lineage of
587
acanthopterygians, after one pair of VT and IT precursor genes was lost from one of the 3R
588
paralogon, an event of gene conversion might have occurred between the remaining pair. This
589
might have occurred in the time period between the 3R and the explosive adaptive radiation and
590
could explain the phylogentic relation of the Acanthopterygii VT and IT precursors. The
591
Acanthopterygii VT precursors and Acanthopterygii IT precursors make two distinct clusters but
592
share a very close phylogenetic relation. In the Ostariophysi, the loss of the NH precursor
593
paralog was differential as compared to the Acanthopterygii with VT and IT precursor genes
594
being lost one each from the two 3R paralogons. Rapid speciation event might have occurred
595
after this. Gene conversion might not have occurred in the ostariophysian lineage. This explains
596
the unlinked state of the NH paralogs in the zebrafish and also the distant relation shared by the
597
VT and IT precursors of the Ostariophysi. The situation in the cavefish is difficult to explain. It
598
is likely that the VT precursor gene was lost from one of the 3R paralogons while in the other
599
both genes were retained so that a pair of linked genes remained. An independent event of gene
600
conversion might also have occurred in between the two linked genes explaining the close
601
phylogenetic relation between the two. The evolutionary history of the VT precursor gene of the
602
cavefish which is unlinked can be easily traced phylogenetically as it clusters with the catfish VT
603
precursors. However, the synteny analysis shows that it is present in a unique genomic context
604
which shares a conserved synteny with neither the tetrapod nor the teleost NH loci, making it
605
impossible to arrive at a possible origin of this gene. Genomic information of the NH loci from
606
more teleost species belonging to the Ostariophysi may be helpful in explaining this. Our future
607
direction of work would involve isolating the NH gene loci in the catfish so that the evolutionary
22
608
history of the VT and IT genes in the Ostariophysi may be inferred more clearly. With the
609
available information from the phylogeny and synteny analysis, it is clear that in the different
610
groups of teleosts the VT and IT precursors are lineage-specific paralogs arising from differential
611
loss of the 3R paralogs in the different superorders. The independent yet consistent retention of
612
613
diploidizations and gene conversions might have been directed by stringent ligand- receptor
614
615
6. Conclusion
616
The complete VT and IT precursors were cloned from H. fossilis, and partially from C.
617
batrachus. The deduced precursor proteins have the characteristics of the NH precursors. The
618
precursors express in the brain and follicular envelope of the ovary. The expression in both brain
619
and ovary shows seasonal variation with higher expression in the reproductive phases. The
620
phylogenetic analysis of the vertebrate NH precursors and the synteny analysis of the NH gene
621
loci show that the VT and IT precursors are lineage-specific paralogs in the superorders of
622
623
7. Acknowledgments
624
This study was supported by a Department of Science and Technology, New Delhi Research
625
Grant (No. SA/SO/AS-43/2009) to KPJ and RC. PB is grateful to the Council of Scientific and
626
Industrial Research, New Delhi for Junior and Senior Research Fellowships. We thank the
627
coordinator of DBT- BHU Interdisciplinary School of life Sciences for providing the qPCR
628
facility.
629
8. References
630
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631
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633
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634
Bobe, J., Montfort, J., Nguyen, T., Fosteir, A. (2006). Identification of new participants in the
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rainbow trout (Onchorhynchus mykiss) oocyte maturation and ovulation process using
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Chauvet, J., Hurpet, D., Michel, G., Chauvet, M. T., and Acher, R. (1984). Two multigene
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lysipressin and arginine vasopressin in the North American opossum Didelphis virginiana.
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Chauvet, M. T., Colne, T., Hurpet, D., Chauvet, J., and Acher, R.(1983). A multigene family for
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Hyodo, S., Ishii, S., Joss J. M. P., (1997). Australian lungfish neurohypophyseal hormone genes
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Hyodo, S., Tsukada, T., Takei, Y. (2004). Neurohypophyseal hormones of dogfish Triakis
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Jansen, S., Devaere, S., Weekers, P. H. H, Adriaens, A. (2006). Phylogenetic relationship and
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Joy, K, P., Singh, V. (2013). Functional interactions between vasotocin and prostaglandins
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isotocin cDNAs from the flounder Platichthys flesus; levels of hypothalamic mRNA
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Biol. 8, 357-366.
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Legends to figures
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fossilis, C. batrachus, I. punctatus and spotted gar. Hormone moiety and leucine- rich box in the
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C-terminal neurophysin are shaded and GKR (enzymatic processing signal) is boxed. The
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cysteine residues have been star marked. The C. batrachus sequences are partial at the N-
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Figure 2. Relative tissue expression levels of VT precursor (A) and IT precursor (B). Graphs
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were plotted with the mean RQ values (2-CT) of expression in brain, intact follicle and follicular
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envelope. The RQ values (mean SEM; n= 5 fish each) were calculated with the resting phase
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brain cDNA as the calibrator. Groups with different alphabets show significant variations in the
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expression levels.
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Figure 3. Seasonal expression levels of VT and IT precursors in brain (A, B) and ovary (C, D).
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The RQ values (2-CT) were calculated with the resting phase brain or ovary as the calibrator
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samples (mean SEM; 5 fish each). Groups with different alphabets show significant variations
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in the expression levels. RP- resting phase; PP- prepratory phase, PS- pre-spawning phase, SP-
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Maximum-Likelihood method. The bootstrap consensus tree inferred from 500 replicates is taken
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to represent the evolutionary history of the taxa analyzed. The analysis involved 67 amino acid
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sequences. All positions with less than 95% site coverage were eliminated. There were a total of
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97 positions in the final dataset. Evolutionary analyses were conducted in MEGA6. References
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Figure 5. Shows the number of amino acid substitutions per site from averaging overall sequence
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pairs between groups. Standard error estimate(s) shown above the diagonal were obtained by a
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bootstrap procedure (500 replicates). Analyses were conducted using the Poisson correction
873
model (Zuckerkandl and Pauling, 1965). The analysis involved 67 amino acid sequences. All
32
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positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment
875
gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 97
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positions in the final dataset. Evolutionary analyses were conducted in MEGA6 (Tamura et al.,
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2013).
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Figure 6 A schematic representation of the genomic loci of spotted gar and teleost NH precursor
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genes and their paralogons. Human assembly is represented as a reference of tetrapod genomic
880
context of the respective loci. The figure shows the linked situation of the genes in spotted gar
881
(Holostei), acanthopterygian teleosts and one of the loci in cavefish, and unlinked situation in
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teleosts belonging to Ostariophysi. Conserved syntenic blocks have bars with the same colours.
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Details of genes and proteins encoded are : ubox5- ring finger protein 37; fast KD5- fast kinase
884
domain containing protein5; lzts3b- leucine zipper, putative tumor suppressor family 3b;
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DDRGK1- DDRGK domain containing protein1; spra- sepiapterin reductase; smyd1- SET and
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MYND domain containing protein, fabp1- fatty acid binding protein1; thnsl2- threonine synthase
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like; avp- arginine vasopressin; oxt- oxytocin; znf367- zinc finger domain protein 367; slc35d2-
888
UDP- N- acetylglucosamine transporter; ccl28- c-c motif chemokine 28; prosapip1- prosap
889
interacting protein 1; dqx- DEAQ box RNA dependent ATPase; znf366- zinc finger protein 366;
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