When hepatopancreas and ovary explants were cultured in medium containing recombinant MeMIH-B, the vitellogenin gene MeVg1 expression level was upregulated in a dose-dependent man-ner, r
Trang 1approaches to study the reproductive functions of a
gonad-stimulating hormone from the shrimp
Metapenaeus ensis
Shirley Hiu-Kwan Tiu and Siu-Ming Chan
Department of Zoology, The University of Hong Kong, China
Neurosecretory structures in crustacean eyestalks are
known to produce the crustacean hyperglycemic
hor-mone (CHH), molt-inhibiting horhor-mone (MIH) and
gonad-inhibiting hormone (GIH) of the CHH⁄ MIH ⁄
GIH gene family These neuropeptides can regulate a
variety of physiologic processes, including molting,
carbohydrate metabolism, and reproduction [1–3]
GIH is one of the most studied neuropeptides of this
group because of its potential importance in shrimp
aquaculture In penaeid shrimp, GIH is produced in
the X-organs and stored in the sinus glands of eye-stalks [4–7] Although the precise mechanism is not known, GIH is postulated to inhibit reproduction by suppressing ovary growth or vitellogenesis [1,2] Eye-stalk ablation removes the source of GIH and results
in ovary growth In contrast, when eyestalk-ablated females were injected with eyestalk extract, the gonad stimulatory effect of eyestalk ablation was abolished [1,2] In addition to GIH, a factor found in the brain and thoracic ganglion of decapod has been implicated
Keywords
eyestalk neuropeptide hormone; RNA
interference; shrimp; vitellogenin gene
Correspondence
S.-M Chan, Department of Zoology, The
University of Hong Kong, Pokfulam Road,
Hong Kong
Fax: +852 2857 4672
Tel: +852 2299 0864
E-mail: chansm@hkucc.hku.hk
(Received 25 January 2007, revised 15 June
2007, accepted 2 July 2007)
doi:10.1111/j.1742-4658.2007.05968.x
Although the crustacean crustacean hyperglycemic hormone⁄ molt-inhibi-ting hormone⁄ gonad-inhibiting hormone neuropeptides have been studied extensively in the last two decades and several neuropeptides from the shrimp Metapenaeus ensis have been cloned, the functions of most of these neuropeptides remained putative In this article, we describe the use of recombinant protein and an RNA interference approach to study the reproductive function of the previously reported molt-inhibiting hormone (MeMIH-B) in M ensis When hepatopancreas and ovary explants were cultured in medium containing recombinant MeMIH-B, the vitellogenin gene (MeVg1) expression level was upregulated in a dose-dependent man-ner, reaching a maximum in explants treated with 0.3 nm recombinant MeMIH-B Shrimp injected with recombinant MeMIH-B showed an increase in vitellogenin gene expression in the hepatopancreas Moreover, a corresponding increase in the vitellogenin-like immunoreactive protein was detected in the hemolymph and ovary of these females Injection of MeMIH-B dsRNA into the female shrimp caused a decrease in MeMIH-B transcript level in thoracic ganglion and eyestalk These shrimp also showed reduction of vitellogenin gene expression in the hepatopancreas and ovary Furthermore, the hemolymph vitellogenin level was also reduced in these animals In summary, the results from recombinant protein and RNA interference experiments have demonstrated the gonad-stimulatory function of MeMIH-B in shrimp
Abbreviations
CHH, crustacean hyperglycemic hormone; GIH, gonad-inhibiting hormone; GSI, gonadosomatic index; MeVg1, Metapenaeus ensis
vitellogenin gene 1; MIH, molt-inhibiting hormone; RNAi, RNA interference; si, small interfering.
Trang 2in the stimulation of gonad maturation Injection of
protein extract from thoracic ganglion or the brain can
stimulate gonad maturation [8]
In sand shrimp Metapenaeus ensis, two forms of
MIH-like cDNA (i.e MeMIH-A and MeMIH-B) have
been cloned and characterized [4,5] MeMIH-B shows
only 68% amino acid similarity to MeMIH-A, and
amino acid sequence alignment indicates that MeMIH-B
is more closely related to GIH of the lobster Homarus
americanus [9] than to the mandibular organ-inhibiting
hormones of the crab Cancer pagarus [10] MeMIH-A
and MeMIH-B are non-sex-specific and are expressed
in the eyestalks of males and females The expression
of MeMIH-A is molt-stage-related, whereas the
expres-sion of MeMIH-B is correlated with the reproductive
cycle In addition to the eyestalk, MeMIH-B is also
expressed in the brain [4] MeMIH-B transcript level is
low in the initial phase of gonad maturation and
increases towards the end of maturation [4] These
findings suggest that the two neuropeptides should
have different functions As they share relatively high
sequence similarity, cross-bioactivity also occurs for
these two neuropeptides [4] For example, injection of
recombinant MeMIH-B also delays the process of
molting [4,11] At the time when we had characterized
MeMIH-B, only a few CHH type II neuropeptides
were reported [2,3] Despite its potential involvement
in reproduction, no further research on the
reproduc-tive function of MeMIH-B has been attempted, as
there is a lack of a good bioassay system for the
neu-ropeptide The recent cloning and characterization of
the gene encoding the major yolk protein, vitellogenin,
may provide a potential biomarker for analysis of
genes that regulate⁄ control reproduction [12]
Concurrently, the recently developed RNA
interfer-ence (RNAi) technique has been used to define the
bio-logical function of many genes This technique is based
on the gene-silencing effect of dsRNA [13] The
tech-nique has revolutionized ‘reverse genetic’ research by
introducing dsRNA to organisms or cells dsRNA can
knock down a gene and will produce a phenotypic loss
of function of that gene [14–16] Although the
com-plete mechanism has yet to be revealed, successful
RNAi has been reported for many animal models For
example, Caenorhabditis elegans can be soaked in
dsRNA or can be fed plasmids that make dsRNA and
consequently exhibit RNAi effects In many studies,
dsRNA can move across cell boundaries freely Thus,
it is not necessary to inject dsRNA directly into the
gonad to get progeny that exhibit RNAi effects [13]
As RNAi works in many organisms, it might also
work in shrimp Gene function analysis by RNAi may
be advantageous as compared to other conventional
approaches This article describes the production of recombinant protein and dsRNA for reproduction-related eyestalk neuropeptide gene, and use of an
in vitro explant culture system and an RNAi technique
to demonstrate the reproductive function of MeMIH-B
in M ensis
Results
Expression of MeMIH-B in shrimp Although we have previously studied the tissue distri-bution of MeMIH-B in the female shrimp, the expres-sion pattern of MeMIH-B in the central nervous system of different reproductive stages has not been fully investigated Moreover, to ascertain that MeMIH-B expression pattern is correlated with repro-ductive developmental stages in females, we have rein-vestigated the expression pattern of MeMIH-B in the eyestalks and other nervous tissues of the adult females
by northern blot analysis MeMIH-B transcripts could
be detected in the eyestalk, nerve cord, thoracic gan-glion and brain of shrimp at early to middle stages of gonad maturation (Fig 1A) In female eyestalks, MeMIH-B transcript level was low in immature shrimp with low gonadosomatic index (i.e GSI < 2)
As gonad development was in progress, a steady increase in MeMIH-B transcript level was observed Similarly, the expression pattern of MeMIH-B in the thoracic ganglia also followed that of the eyestalk (Fig 1B) For example, in both eyestalk and thoracic ganglion, the highest MeMIH-B transcript level was recorded at the late maturation stage in shrimp with GSI¼ 10 Similar to the previous results, expression
of MeMIH-B is sex-nonspecific, as the males also expressed MeMIH-B (Fig 1B)
Functional study of recombinant MeMIH-B
in vitro and in vivo The rMeMIH-B produced by pRSET expression was purified on an Ni2+-charged column To study the function of rMeMIH-B in reproduction, hepatopan-creas explants from females at early stage of gonad maturation (GSI < 2) were used A dose-dependent increase of MeVg1 expression was recorded when the concentration of rMeMIH-B was increased (i.e 0.3 pm, 3 pm and 30 pm) The maximum increase of MeVg1 transcript level was observed in the hepatopan-creas explants treated with 0.3 nm rMeMIH-B; further increase of rMeMIH-B (i.e 3 nm, 30 nm and 300 nm) resulted in a decrease in the overall MeVg1 expression level (Fig 2A) When the ovary explants were treated
Trang 3with 0.3 nm rMeMIH-B, an increase of about 25% of
MeVg1 expression was recorded (Fig 2B)
Next, we performed an in vivo injection of
rMeMIH-B into females to further confirm its
gonad-stimulatory effect To demonstrate the specificity of
rMeMIH-B in gonad maturation, a control group
injected with rMeMIH-A was included As compared
to the NaCl⁄ Pi control, injection of an equal amount
(6.6 nmol) of rMeMIH-A did not cause any change in
the overall expression of vitellogenin in the
hepatopan-creas and ovary (Fig 3A,B) In contrast, injection
of 6.6 nmol of rMeMIH-B stimulated an increase
(2–3-fold) in MeVg1 expression by the hepatopancreas
and ovary (Figs 3A,B) at 72 h, but only weakly for the
24 h time point (data not shown)
It is well accepted that the vitellogenin produced in the hepatopancreas serves as an extraovarian source for the final synthesis of vitellin The newly made vitel-logenin is expected to be secreted rapidly into the hemolymph and transported to the ovary for oocyte uptake To demonstrate that the increase in expression
of the MeVg1 gene could also result in the appearance
of vitellogenin in the hemolymph for transport, we also collected hemolymph samples of these injected shrimp and analyzed the increase in vitellogenin-spe-cific protein As shown in Fig 3C,D, when females were injected with rMeMIH-B (i.e 6.6 nmol), the hemolymph and ovaries of most animals contained a much higher level of vitellogenin (i.e 148 kDa) (Fig 3C, left panel) These vitellogenin-specific pro-teins are presumably derived from the translation of the MeVg1 gene from the hepatopancreas after rMeMIH-B stimulation The results from SDS⁄ PAGE and western blot analysis of the hemolymph and ovarian proteins from shrimp injected with 6.6 nmol of rMeMIH-B demonstrated an increase in the overall
1.0
A
B
0.75
0.5
0.25
1.0
0.75
0.5
0.25
Es MeMIH-B
Tg MeMIH-B
Fig 1 Expression of MeMIH-B in different tissues of early
(GSI < 2) mature females (N ¼ 5) (A) The relative expression levels
of MeMIH-B in nervous tissues (ES, eyestalk; Br, brain; Tg, thoracic
ganglia; Vn, ventral nerve) and non-nervous tissues (Hp,
hepatopan-creas; Mu, muscle; Ov, ovary); the bar indicates the SE (B) The
expression pattern of MeMIH-B (N > 20) at different gonad
matura-tion stages of the eyestalk (open bar) and thoracic ganglia
(diago-nally shaded bars) of females The percentage indicates the GSI of
the females M (B) indicates the expression pattern of MIH-B in
the same tissues in males (N ¼ 5) The lower panel is the northern
blot analysis of MeMIH-B expression in the eyestalk (Es) and
tho-racic ganglia during the gonad maturation cycle Each lane
repre-sents an RNA sample from the eyestalk or the thoracic ganglion of
one shrimp The last lane shows the RNA samples from a male.
The bar indicates the SE.
1.4
B A
1.2 1.0
0.8
0.6
0.4 0.2
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Concentrations of rMeMIH-B
Concentrations of rMeMIH-B
*
*
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
Fig 2 Histogram showing the relative expression levels of MeVg1
in (A) hepatopancreas and (B) ovary explants after exposure to dif-ferent concentrations (i.e from 0.3 p M to 0.3 l M ) of rMeMIH-B The sample size (or numbers of shrimp) is 10 for the in vitro assay Relative MeVg1 mRNA levels (A) are shown as means + SEM of
10 prawns The shrimp that show significant differences (P < 0.05)
in the relative MeVg1 mRNA levels are indicated by an asterisk.
Trang 40.5
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1.5
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B
C
D
treatment
0
0.5
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rMeMIH-B Ctrl rMeMIH-A
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Ctrl rMeMIH-A rMeMIH-B
148 kDa
Hycn
148 kDa NSP
MeVg1 rRNA
MeVg1 rRNA
Me MIH-A ctrl Me MIH-B
ctrl Me MIH-B
treatment
Me MIH-A ctrl Me MIH-B
treatment
Me MIH-A ctrl Me MIH-B
Fold changes of Vg level in hemolymph
Fold changes of Vg content in ovary
Fig 3 Effect of recombinant MeMIH-B and MeMIH-A on vitellogenin expression in shrimp (A) Left: relative expression levels of MeVg1 in hepatopancreas of females (N ¼ 10) at 48 h after injecting NaCl ⁄ P i , rMIH-A and rMIH-B Right: a typical northern blot analysis of the shrimp MeVg1 transcript level after injection of NaCl ⁄ P i , rMIH-A, and rMIH-B (B) Left: relative expression levels of MeVg1 in ovary of females (N ¼ 10) at 48 h after injection of NaCl ⁄ P i , rMIH-A, and rMIH-B Right: a typical northern blot analysis of the shrimp MeVg1 transcript level after injection of NaCl ⁄ P i , rMIH-A, and rMIH-B (C) Left: relative levels of vitellogenin in hemolymph of females (N ¼ 10) at 48 h after injec-tion of NaCl ⁄ P i , rMIH-A, and rMIH-B Right: western blot analysis (upper) of the hemolymph level of vitellogenin for shrimp injected with rMIH-B The 148 kDa protein is one of the vitellogenin subunits recognized by the shrimp antibody to vitellogenin [27] The lower panel shows the Coomassie blue staining of the hemocyanin (Hcy) corresponding to the same protein samples (D) Left: relative levels of vitelloge-nin in ovary of shrimp at 48 h after injection of NaCl ⁄ P i , MIH-A, and rMIH-B Right: western blot detection (upper) of vitellogenin (148 kDa)
in ovary of shrimp injected with rMIH-B NSP is the nonspecific protein unrelated to vitellogenin of the ovary samples In the northern blot (or western blot) analysis, each lane represents RNA (or protein) samples collected from individual shrimps.
Trang 5vitellogenin-specific protein (Fig 3C,D) Unlike the
rMeMIH-B-injected group, shrimp injected with
rMeMIH-A (6.6 nmol) did not show any changes in
the overall MeVg1 transcript level in the
hepatopan-creas or a significant inhepatopan-crease in MeVg1 protein level
in the hemolymph and ovary (Fig 3A–D)
Inhibition of vitellogenin expression after RNAi
We have performed preliminary experiments using a
nonspecific dsRNA (from Tiger frog virus), and the
results show no effect on MeMIH-B gene silencing
(data not shown) In the following study, individual
shrimp (N¼ 40; average GSI < 3) were injected with
3 lg of dsRNA for MeMIH-B, and RNA samples were
collected after 24, 48, 72, 96 and 120 h Northern blot
results from the eyestalk (Fig 4A) indicated no
signifi-cant reduction in MeMIH-B transcript level at all time
points However, when we used RT-PCR to analyze
the same samples, a significant reduction of MeMIH-B
transcript was observed (Fig 4B) In fact, by RT-PCR,
the MeMIH-B dsRNA appeared to knock down most
of the transcripts after 72 h of treatment (Fig 4B) In
addition, hybridization signals representing small-size
RNAs were strong and persisted from 24 to 120 h after
injection (Fig 4A) This suggests that dsRNAs are very
stable, as residual MeMIH-B dsRNA remained Unlike
in the eyestalk, there was a significant decrease in the
MeMIH-B transcript level in the nerve cord as early as
24 h after MeMIH-B dsRNA injection The knock-down also persisted 120 h after dsMIH-B injection (Fig 5A) MeMIH-B transcript level was lowest in nerve cord at 72 h after injection, but started to increase afterwards (Fig 5B) With regard to the effect
of MeMIH-B dsRNA on hepatopancreas MeVg1 expression, it was observed that there was a significant drop in MeVg1 transcript level in the hepatopancreas For example, at 24, 48 and 72 h after dsRNA treat-ment, drops of 20%, 71% and 23% of the overall MeVg1 transcript level were recorded (Fig 6A) Unlike in the hepatopancreas, the reduction of MeVg1 expression in ovaries of these female was small after the injection of MeMIH-B dsRNA For example, the reduction in MeVg1 transcript level in the ovary repre-sented only 6%, 7% and 22% decreases at 24, 48 and
72 h post-dsRNA treatment (Fig 6B)
Similar SDS⁄ PAGE analysis and western blot analy-sis were performed for these females In the hemolymph sample of the NaCl⁄ Pi-injected control, vitellogenin-spe-cific protein could be detected using antibody to vitel-logenin In contrast, no vitellogenin-specific protein was detected in the hemolymph of the dsRNA-injected females (Fig 7A) In the ovary, the amount of vitelloge-nin remained relatively constant However, only minute quantities of vitellogenin subunits (i.e 148, 97 and
78 kDa) were detected in the ovaries of the dsRNA-injected females (Fig 7B) These proteins were immuno-reactive to the antibody to vitellogenin of M ensis [27]
B -H I M e M
+ -+ -+ -+ -+
100 80 60 40 20
Fig 4 Effects of MeMIH-B RNAi in eyestalk of female shrimp (A) Northern blot detection of eyestalk MeMIH-B transcript level in control (–) and dsRNA-injected (+) females from animals at different time intervals (i.e 0, 24, 48, 72, 96 and 120 h); the arrow indicates the MIH-B transcript, and the smear indicates the residual dsRNA (B) Top panel: RT-PCR detection of MeMIH-B gene knockdown using MIH-B-specific primers Lower panel: relative change in MIH-B transcript level at different time intervals The bar diagram indicates the relative transcript level
of MeMIH-B after normalization with b-actin gene.
Trang 6Structure–function research on crustacean eyestalk
CHH family neuropeptides remains a challenging
endeavor This is mainly due to the existence of highly
similar neuropeptides in the same species [1,2] For example, it is now known that there are at least four
or five CHH-like genes in M ensis These genes may share high sequence similarity and⁄ or analogous function Some of these genes may be expressed in
MIH-B β-actin
+ -+ -+ -+ -+
-Ctr
120 96 72 48 24 0
100 80 60 40 20
120 96 72 48 24 0
Time after injection
Time after injection (h)
Fig 5 Effects of MeMIH-B RNAi in the ventral nerve cord of female shrimp (A) Northern blot detection of eyestalk MeMIH-B transcript level in control (–) and dsRNA-injected (+) females from animals at different time intervals; the arrow indicates the MIH-B transcript, and the smear indicates the residual dsRNA (B) RT-PCR detection of MeMIH-B transcript using specific primers The bar diagram indicates the rela-tive transcript level of MeMIH-B after normalization with b-actin gene.
1 V e M A R r
72h 48h 24h
72h 48h 24h
1 V e M A R r
y r a v o n i n i s e r p x e 1 V e M
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20
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100
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60
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+ -+ -+
-+ -+ -+ -MeVg1 expression in hepatopancreas
Fig 6 Expression of vitellogenin in hepatopancreas and ovary of dsRNA-injected (+) and control (–) females (A) Upper: northern blot detec-tion of hepatopancreas MeVg1 transcript level in shrimp Lower: bar indicates the relative decrease in expression level of MeVg1 (B) Upper: RT-PCR detection of ovary MeVg1 transcript level in shrimp Lower: bar indicates the relative decrease in expression level of MeVg1.
Trang 7non-neuronal (or non-eyestalk) tissues [3] Likewise,
additional MIH subtypes have also been found in one
species For example, in addition to the known MIH
and GIH subtypes, there is also a report suggesting the
existence of novel MIH subtypes In the GenBank
data-base (http://www.ncbi.nlm.nih.gov/), there are at least
four different entries for MIH-like neuropeptides in the
tiger prawn Penaeus monodon As there is a high degree
of sequence similarity and structural conservation of
these genes and gene products, confirmation of the true
identity and function of these neuropeptides remains
difficult Unfortunately, most of these MIH-like
peptides are produced in very low quantities, and they
function as inhibitory regulators in a physiologic
process, making it difficult to develop a good bioassay
The production of large quantities of recombinant
protein for bioassay may circumvent the lack of active
material for structure–function studies However, the
inhibitory nature of these hormones remains a challenge
for the successful development of a bioassay system
Attempts at developing biological assays for
inhibi-tory factors such as MIH have been reported, and the
inhibitory function on molting has been demonstrated
convincingly Most biological assays of the gonad
inhi-bition of this neuropeptide rely on its ability to inhibit
ovary development and reduce oocyte size, cause a decrease in total ovary protein incorporation, and sup-press ovary total protein synthesis [11,17,18] Biologi-cal assays using these criteria are nonspecific and provide little information on the mechanism of GIH regulation of reproduction Previously, we have pro-duced rMeMIH-B (formerly MeeMIH-B), but little progress was made in developing a biological assay for the recombinant protein This is mainly attributed to the lack of a biomarker for the reproductive process With the recent cloning of the vitellogenin gene in dif-ferent crustaceans [19–23], a more precise role for GIH can be defined with the vitellogenin as a biomarker Recently, there was a report on the effect of sinus gland extract and neuropeptide on vitellogenin gene expression in M japonicus In that study, the effect of
a CHH peptide and two MIH-like peptides on ovary vitellogenin gene expression was investigated; the results indicated that CHH causes inhibition, whereas the MIH-like neuropeptides have no effect on vitello-genin gene epression [23] Unlike the CHH of M japo-nicus, MeMIH-B (a type II neuropeptide) has a stimulatory effect on vitellogenin synthesis Taken together, the results suggest that a CHH-like (or type I) neuropeptide may be inhibitory for gonad maturation,
3 2 1
d t c j n i -A R s d t c j n i -S P 3
2 1
d t c j n i -A R s d t c j n i -S P
y r a v O h
m y l o m
g i n i a t s B C g
i n i a t s B C
t o l b n r e t s e W t
o l b n r e t s e W
3 2 1
a D k 8 1 a D k 7 a D k 6
a D k 8 1 a D k 7 a D k 6
Fig 7 Western blot analysis of the hemolymph and ovary total protein of dsRNA-injected females (A) Hemolymph sample of NaCl ⁄ P i -injected and dsRNA injected females Lanes 1–3: NaCl⁄ P i -injected and 4–6 dsRNA-injected animals Individual lanes represent protein sam-ples collected from injected shrimps, and the arrows indicate the vitellogenin-specific protein (148, 97 and 76 kDa) using antibody to vitellog-enin [19,27] (B) Ovary sample of NaCl ⁄ P i -injected and dsRNA-injected females Lanes 1–3: Ovary from the corresponding NaCl ⁄ P i -injected individual and 4–6 dsRNA-injected animals Each lane represents protein samples collected from individuals, and the arrow indicates the vitel-logenin-specific protein determined using antibody to vitellogenin [19].
Trang 8whereas the MIH-like neuropeptide (i.e MeMIH-B,
type II) may be a vitellogenin-stimulatory factor of
shrimp Because of the existence of multiple forms of
the CHH family neuropeptides, there may be a
dis-crepancy in defining the function of many cDNAs
cloned using a molecular biology approach For
example, although the MIH-like gene of Litopenaeus
vannamei was reported [24], detailed analysis revealed
that the MIH-like deduced protein was more closely
related to the CHH as described in other crustaceans
Since our report of a second form of MIH subtype
cDNA in M ensis, the naming of this MeMIH-B as a
GIH was based on its similarity to the lobster GIH, as
other MIH subtypes in shrimp had not been reported
[4] In re-evaluating the expression pattern of
MeMIH-B in the eyestalk and CNS, we further
recon-firmed that its expression level increases during active
vitellogenesis Our result suggests that a high transcript
level of the MeMIH-B gene (or the protein) may
be needed for vitellogenin production during active
vitellogenesis
The use of recombinant protein to study the
func-tion of crustacean neuropeptides has been reported
for a number of species [4,11,19,25] Recombinant
MeMIH-B has been produced and used in a
biologi-cal assay for molt inhibition [4] Except for the role
of CHH in the increase of glycemia, the use of
recombinant protein to study molt-inhibiting function
and gonad maturation remains a challenge for
crusta-cean endocrinologists [2,4] The difficulty is due to the
lack of a good biological marker for consistent
results As the hepatopancreas and ovary express
MeVg1, explants from these tissues were used in the
explant culture Given the fact that the
hepatopan-creas culture lasts for 3–4 h and active vitellogenin
expression can be detected in shrimp, the explant
culture system was successfully developed This is
the first demonstration of the stimulatory effects of a
neuropeptide in vitellogenin gene stimulation As
rMeMIH-B can stimulate MeVg1 expression in
hepa-topancreas and ovary in vitro, the result may provide
information on the initial mechanism of hormone
action In other words, the in vitro results indicate
that MeMIH-B may act directly on the
hepatopan-creas and ovary to inhepatopan-crease the rate of vitellogenin
gene expression, indicating that both the
hepatopan-creas and ovary are the targets of MeMIH-B This
result will provide the basis for identifying and
characterizing the receptor for the neuropeptide
Furthermore, rMeMIH-B acted on the
hepatopan-creas and ovary in a dose-dependent manner As the
optimal concentration (i.e 30 nm in vitro and
6.6 nmol in vivo) for the stimulatation of MeVg1
expression is low, the result also suggests that rMeMIH-B is highly potent in stimulating vitellogenin gene expression As subadult (i.e < 15 g) and adult females also responded to rMeMIH-B in a similar dose-dependent manner, the results would be useful for us to develop a strategy to induce gonad matura-tion in shrimp aquaculture
RNAi is defined as the gene-silencing effect medi-ated by dsRNA RNAi technology was developed in the mid-1990s, based on the antisense RNA techno-logy developed in the 1980s RNAi can silence or knock down the expression of a gene, and the phe-nomenon appears to be universal, as it has been reported in both plants, animals, and even cultured cells There are two major types of RNAi, with slight differences in the mechanism They are mediated by either: (a) dsRNA; or (b) small interference (si)RNA The longer dsRNA may generate a large population
of siRNA (with 21–23 nucleotides), and the use of longer dsRNA may be advantageous over siRNA In this study, the longer dsRNA was produced and used
in the RNAi experiments It has been reported that the longer dsRNA of approximately 600–800 nucleo-tides works best for most genes The GIH-specific dsRNA, however, was synthesized from the 238 bp coding region of the mature peptide As the coding sequences of all the neuropeptides are short (< 350 nucleotides) and there are scattered repetitive sequences in the noncoding region of MeMIH-B, selection of the effective gene region to produce dsRNA is limited and restricted only to the coding region As the siRNA produced by the endogeneous Dicer (assuming a mechanism similar to the verte-brates) is small, the siRNA has to be specific to cause
an effect For example, the RNAi will not work even with a 1–2 bp mismatch Thus, another highly similar gene (MeMIH-A) will not be affected This may explain why there are hybridization signals in the eye-stalk, as the eyestalk is also known to produce MeMIH-A The apparent lack of MeMIH-B knock-down (northern blot result) may simply indicate that another very similar but abundant neuropeptide (i.e MeMIH-A) may hybridize to the MeMIH-B probe When the more specific RT-PCR was used, the decrease in MeMIH-B transcript level was evident The amount of dsRNA injected in the animal may vary, depending on the expression level of the gene; a much higher dose of dsRNA was injected into the shrimp L schmitti to silence the CHH gene [26] In our study, the amount of dsRNA injected into each animal was about 3–5 lg for each shrimp (23–28 g)
At present, the mechanism of RNAi in shrimp is not known, but we expected that these dsRNA molecules
Trang 9could circulate by way of hemolymph and would be
taken up by a variety of tissues In the target tissues
that express MeMIH-B (i.e neuronal cells of the
eye-stalk and⁄ or central nervous system), the dsRNA
enters the cell to initiate gene knockdown The
dsRNA appears to be stable in the target tissues For
example, a strong hybridization signal representing the
residual MeMIH-B dsRNA still remains in the
eye-stalk at 120 h after injection (Figs 4A and 5A)
In conclusion, the use of recombinant protein or
RNAi alone may not be sufficient to confirm the
func-tion of a neuropeptide The combined use of
recombi-nant protein and RNAi described in this study has
provided unequivocal evidence for a stimulatory
func-tion of MeMIH-B in vitellogenesis We can also apply
similar approaches to study the structure–function
relationships of other CHH⁄ MIH ⁄ GIH neuropeptide
members
Experimental procedures
Animals
Shrimp were purchased from a local seafood market They
aquarium for 2 days before rMeMIH-B or dsRNA
injec-tion The GSI was calculated as the percentage of ovary
weight per total body weight
Production of the recombinant MeMIH-B
The cDNA encoding the mature peptide of the MeMIH-B
was amplified by PCR using restriction enzyme-linked
gene-specific primers (forward, 5¢-GACGAATTCTTCGG
CCTTCGC-3¢; reverse, 5¢-AGGAGATCTAAGCTTACCA
CGCTCCACCAGGG-3¢) that contained restriction sites
EcoRI and BamHI, respectively The PCR product was first
digested with EcoRI and HindIII, and was then ligated into
the cloning vector pRSET-B containing a T7 lac promoter
site (Invitrogen, Carlsbad, CA, USA) The constructs were
transformed into Escherichia coli XL1 Blue cells, and the
by alkaline lysis DNA minipreparation The insertion of
the desired gene in the plasmid was verified by automated
DNA sequencing The clones were transformed into E coli
induced by the addition of isopropyl
1-thio-b-d-galactopyr-anoside (Sigma, St Louis, MO, USA) to a final
concentra-tion of 1 mm The culture was allowed to continue for 4 h
The bacteria were pelleted by centrifugation (5000 g for 15
min) and resuspended in 50 mL of binding buffer (20 mm
cells were homogenized with a polytron, and centrifuged at
5000 g for 15 min The pellet was then resupended in
50 mL of denaturing binding buffer (8 m urea in binding
Follow-ing centrifugation as above, the supernatant was collected
(Qiagen, Hilden, Germany) affinity column that was pre-equilibrated with denaturing binding buffer The column was then washed three times with washing buffer (8 m urea,
pH 7.9) The fusion protein was eluted with elution buffer
imid-azole, pH 7.9) The denatured recombinant protein was refolded by both dilution and dialysis The concentration of urea present in the solubilized protein was decreased step-wise by addition of an equal volume of renaturing buffer
pH 7.9) for every 3 h until the concentration of urea was decreased to 1 m The diluted recombinant protein was then dialyzed in a dialysis bag (Sigma; cut-off 6–7 kDa) in a
three changes of buffer The recombinant protein was
over-night A Bradford protein assay (Bio-Rad, Hercules, CA, USA) was performed to determine the concentration of the
expressed using the same strategy and was used as negative control in the following in vitro and in vivo bioassay
Functional study of rMeMIH-B by explant assay and in vivo injection
The functional study of rMIH-B involving a shrimp in vitro explant culture system was based on a previously developed method [20–22] Briefly, hepatopancreas and ovary were dissected, cut into small fragments, and placed in the wells
of 24-well plate containing 1.5 mL of Medium 199 (Sigma) prepared in crab saline [28] Different concentrations of rMeMIH-B were added to the explants, and the culture
culture period, the tissues were collected for total RNA extraction followed by northern blot hybridization [25] or RT-PCR For northern blot analysis, the nylon membrane was hybridized in hybridization buffer (50% formamide)
overnight The probe (derived from partial MeVg1 cDNA) was synthesized as per kit instructions (Roche, Mannheim, Germany) After hybridization, the membrane was washed
15 min The signals were detected by adding the antidigoxi-genin–AP conjugate Chemiluminescent substrate CDP-Star (Roche, Mannheim, Germany) was added, and the mem-brane was exposed to X-ray film For PCR, the PCR mix
Trang 10(20 lL) consisted of 10 mm Tris⁄ HCl (pH 8.0), 1.5 mm
and reverse primers The PCR conditions were one cycle of
of DNA synthesis PCR products were analyzed on a 1.5%
agarose gel, and Southern Blot was performed to determine
the specific amplification of the cDNA
For in vivo injection of rMIH-B, adult females in the
nonreproductive stage were injected with 20 lL of either
6.6 nmol or 0.66 nmol of rMIH-B at the arthropodial
membrane of the periopod and returned to the culture
tanks At 24, 48 and 72 h after injection, the
hepatopan-creas and ovary of the shrimp were dissected for total
RNA preparation, and the hemolymph samples were
Functional study of MeMIH-B by RNAi
To prepare a DNA template for the synthesis of dsRNA,
DNA corresponding to the mature peptide of MeMIH-B
was amplified by PCR using T7 promoter-linked primers
(Life Technologies, Carlsbad, CA, USA) PCR conditions
were analyzed by 2% agarose gel electrophoresis The
tar-get PCR product band was purified with the
transcription of MeMIH-B dsRNA, 1–2 lg of purified T7
promoter-linked MeMIH-B DNA was used as template in
an in vitro transcription reaction with the T7 Megascript
RNAi Kit (Ambion, Austin, TX, USA), according to the
manufacturer’s recommendations In 20 lL of reaction
mix-ture, MeMIH-B DNA template was mixed with appropriate
buffer, 2 lL of ATP solution, 2 lL of CTP solution, 2 lL
of GTP solution, 2 lL of UTP solution, and 2 lL of
18 h During the transcription, the two RNA strands were
hybridized to form dsRNA
For RNAi experiments, shrimp (25–35 g) with similar
GSI values were purchased from a local seafood market
They were acclimated in a culture tank overnight prior to
injection Then, 50 lL of MeMIH-B dsRNA (3 lg in
arthro-dial membrane of a periopod The controls received an equal
tanks for culture before being killed for total RNA prepara-tion from different tissues The relative level of MeMIH-B expression in the nerve cord was used as an indication of the RNAi effect between the treatment and control groups
Statistical analysis of northern blots and western blots
Northern blot or western blot signals from the films (or blots) were scanned with the free software imagej (Image processing and analysis in Java: http://rsb.info.nih.gov/ij/)
to obtain quantitative numbers representing expression lev-els of the gene or protein The expression level was normal-ized with either the rRNA (for the northern blot) or hemocyanin (for the western blot) Either simple t-test or
Acknowledgements
This research was supported by the Research Grant Council of the Hong Kong Special Administrative Region, China (HKU 7214⁄ 05M) awarded to S.-M Chan
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