Title: Two gonadotropin releasing hormones and a method to isolate the same
Abstract: The present invention relates to two novel Gonadotropin releasing hormones muGnRH I and muGnRH II of amino acid SEQ ID 1 as QHWSAWRLPG, and SEQ ID 2 QHWSWGILPG respectively, useful for induced breeding in fish both in combination and alone, by activating production of Gonadotropin, and a method of isolating the same from Indian Murrel brain, and further, a method of inducing breeding in fishes using the said novel gonadotropin releasing hormones.
Patent Number: 6,977,242 Issued on 12/20/2005 to Chatterjee, et al.
| Inventors:
|
Chatterjee; Abhijit (West Bengal, IN);
Ray; Partha (West Bengal, IN);
Dasgupta; Subrata (West Bengal, IN);
Hattacharya; Samir (Kolkata, IN);
Pasha; Santosh (Delhi, IN)
|
| Assignee:
|
Council of Scientific and Industrial Research (New Delhi, IN)
|
| Appl. No.:
|
354433 |
| Filed:
|
January 28, 2003 |
| Current U.S. Class: |
514/15; 514/15; 514/8; 514/14; 514/2; 530/313; 530/327; 530/398; 930/130 |
| Intern'l Class: |
A61K 038/22; C07K 014/57.5 |
| Field of Search: |
514/15,8,14,2
530/313,327,398
930/130
|
References Cited [Referenced By] Primary Examiner: Tate; Christopher R.
Assistant Examiner: Chism; B. Dell
Attorney, Agent or Firm: Ladas & Parry LLP
Parent Case Text
This application claims the benefit of U.S. Provisional Application(s) No(s).:
60/353,041 Jan. 30, 2002 and incorporates the same by reference.
Claims
1. An isolated gonadotropin releasing hormone selected from the group consisting
of muGnRH I (SEQ ID NO:1) and muGnRH II (SEQ ID NO:2).
2. The hormone as claimed in claim 1, wherein said hormone is obtained from Indian
Murrel brain.
3. The hormone muGnRH II (SEQ ID NO:2) as claimed in claim 1 which is more active
than gonadotropin-releasing hormone muGnRH I (SEQ ID NO:1).
4. The hormone muGnRH I (SEQ ID NO:1) as claimed in claim 1 which increases gonadotropin
release by about 30 times as compared to the level of gonadotropin release in the
absence of muGnRH I (SEQ ID NO:1) and muGnRH II (SEQ ID NO:2).
5. The hormone muGnRH II (SEQ ID NO:2) as claimed in claim 1 which increases
gonadotropin release by about 35 times as compared to the level of gonadotropin
release in the absence of muGnRH I (SEQ ID NO:1) and muGnRH II (SEQ ID NO:2).
6. A composition comprising isolated gonadotropin releasing hormones muGnRH I
(SEQ ID NO:1) and muGnRH II (SEQ ID NO:2) which exhibit synergy in induced breeding
of fish.
7. A composition comprising the muGnRH I (SEQ ID NO: 1) and muGnRH II (SEQ ID
NO:2) as claimed in claim 1 in a ratio of about 1:1 which increases gonadotropin
release by about 50 times as compared to the level of gonadotropin release in the
absence of muGnRH I (SEQ ID NO: 1) and muGnRH II (SEQ ID NO:2).
8. The hormone muGnRH I (SEQ ID NO:1) as claimed in claim 1, wherein said hormone
shows no species barrier in induced breeding of fish and wherein there is an increase
in gonadotropin release ranging between 20-50 times as compared to the level of
gonadotropin release in the absence of muGnRH I (SEQ ID NO:1) and muGnRH II (SEQ
ID NO:2).
9. The hormone muGnRH II (SEQ ID NO:2) as claimed in claim 1, wherein said hormone
shows no species barrier in induced breeding of fish and wherein there is an increase
in gonadotropin release ranging between 20-50 times as compared to the level of
gonadotropin release in the absence of muGnRH I (SEQ ID NO:1) and muGnRH II (SEQ
ID NO:2).
10. The hormone muGnRH I (SEQ ID NO:1) as claimed in claim 1, wherein said hormone
is more active than other GnRHs selected from the group consisting of mammalian
GnRH, Salmon GnRH, GnRH superactive analogues, and commercially available GnRH
Ovaprim or a combination thereof.
11. The hormone muGnRH II (SEQ ID NO:2) as claimed in claim 1, wherein said hormone
is more active than other GnRHs selected from the group consisting of mammalian
GnRH, Salmon GnRH, GnRH superactive analogues, and commercially available GnRH
Ovaprim or a combination thereof.
Description
FIELD OF THE PRESENT INVENTION
The present invention relates to two novel Gonadotropin releasing hormones muGnRH
I and muGnRH II of amino acid SEQ ID 1 as QHWSAWRLPG, and SEQ ID 2 QHWSWGILPG respectively,
useful for induced breeding in fish both in combination and alone, by activating
production of Gonadotropin, and a method of isolating the same from Indian Murrel
brain, and further, a method of inducing breeding in fishes using the said novel
gonadotropin releasing hormones.
BACKGROUND AND PRIOR ART WORK
Gonadotropin Releasing Hormone (GnRH) is now the best available biotechnological
tool for the induced breeding of fish. GnRH is the key regulator and central initiator
of reproductive cascade in all vertebrates.
It's a decapeptide and first isolated from pig and sheep hypothalami with the
ability to induce pituitary release of luteinising hormone (LH) and follicle stimulating
hormone (FSH). Since then only one form of GnRH has been identified in most placenta!
mammals including human beings as the sole neuropeptide causing the release of
LH and FSH.
However, in non-mammalian species (except guinea pig) twelve GnRH variants
have now been structurally elucidated, among them seven different forms have been
isolated from fish species. Depending on the structural variants and their biological
activities number of chemical analogues have been prepared and one of them is sahnon
GnRH analogue profusely used now in fish breeding and marketed commercially throughout
the world. Question is why fishes require an induction by this neuropeptide hormone
to release germ cells from male and female partners so that fertilization of oocytes
by sperms may occur in the aquatic ambience.
In fact, most of the economically important culturable fish in land-locked water
do not breed until they are induced by the hormone. Fish in marine or riverine
water do not need induction by hormone for their breeding. But to culture fish
with the intention of increasing the yield of production in either ocean or river
is not possible. Culture requires a controllable area, that's why pisciculture
to enhance the yield is restricted to land-locked aquatic bodies where fish do
not usually breed or spawn without the induction of hormone.
Since fish meat has been found to be the best quality food among all animal
meats, there is an enormous increase in fish producing industries in global scenario
recently where the major rate-limiting factor is breeding. The induced breeding
of fish is now successfully achieved by the development of GnRH technology. In
this overview, a brief description is given to conceive the biotechnology of this
highly important area of our food supplement.
Structure of GnRH—GnRH was first isolated from the mammalian hypothalamus
as a decapeptide(Burgus et al., 1972; Mastuo et al., 1971). Since then, several
structural_variants of GnRH have been described which can be seen in FIG. 1 (shown
under the heading brief description of accompanying drawings).
There has been a striking conservation in GnRH peptide length, the first four
—NH2 terminus and two —COOH terminus amino acids are remarkably conserved
in different vertebrates. Whatever changes occurred are between 5 and 8 amino acid
residues, position 8 is most variable followed by position 6,5 and 7. This highly
variable position 8 suggests its role in the variation of biological activity between
the species and its critical function in recognizing GnRH receptors on the pituitary
cell membrane. To exert its biological function it has to first recognize its receptor
on the pituitary gonadotroph cell membrane and when it has to do so its linear
structure changes into a loop. The NH2- and COOH-terminal domain of GnRH are closely
opposed when GnRH binds to its receptor and this is the proposed result from a
P-II type rum involving resides 5-8. The P turn creates a hairpin loop that aligns
N and C termini (Sealfon et al., 1997) (FIG. 2). (Shown under the heading
brief description of accompanying drawings).
GnRH from Indian fish—Although India has several varieties of indigenous
culturable bony fish, practically nothing is known about their GnRH. This is really
a very unfortunate situation for which we still have to depend on the supply from
other countries to investigate the reproductive biology of our economically important
culturable fish as GnRH is the key regulator of reproductive control mechanism.
Several years ago we initiated this research and faced serious problems.
For the isolation of this peptide we would need an assay system to identify the
desired molecule at each step of purification. First piscine GnRH isolated purified
and structure determined was Salmon GnRH (Sherwood and her associates in 1983),
and the most current GnRH purified and characterized by two different groups Carolsfeld
et al and Robinson et al., was in 2000. Between the span of this time, number of
GnRH variants have been isolated by depending on the immunoreactivity of this molecule.
During Salmon GnRH isolation, Sherwood et al took the help of anti-GnRH antibody
from mammalian source, later they developed Salmon anti-GnRH antibody which permitted
to isolate different GnRH forms from other sources.
Meanwhile Jamaluddin et al, (Gen comp Endocrinology May 1989; 74(2): 190-8)
of applicants laboratory developed a very dependable GnRH bioassay. Basic principle
of this bioassay is to get the release of GTH from murrel primary pituitary cell
culture and determine the amount of GTH released by GTH-RIA.
OBJECTS OF THE PRESENT INVENTION
The main object of the present invention is to isolate and identify novel gonadotropin
releasing hormones for induced breeding in fishes.
Another main object of the present invention is to develop a method of isolating
and sequencing novel gonadotropin releasing hormones from Indian Murrel brain.
Yet another object of the present invention is to develop gonadotropin-releasing
hormones for enhanced release of gonadotropin in fishes.
Still another object of the present invention is to develop gonadotropin-releasing
hormones showing synergistic effect.
Still another object of the present invention is to develop various fractions
of hypothalami with gonadotropin releasing hormone activity.
Still another object of the present invention is to determine the effect of
calcium on the gonadotropin hormone releasing activity of GnRH.
Still another embodiment of the present invention is to develop a method of
inducing breeding in fishes using the said novel gonadotropin releasing hormones.
SUMMARY OF THE PRESENT INVENTION
The present invention relates to two novel Gonadotropin releasing hormones muGnRH
I and muGnRH II of amino acid SEQ ID 1 as QHWSAWRLPG, and SEQ ID 2 QHWSWGILPG respectively,
useful for induced breeding in fish both in combination and alone, by activating
production of Gonadotropin, and a method of isolating the same from Indian Murrel
brain, and further, a method of inducing breeding in fishes using the said novel
gonadotropin releasing hormones.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
Accordingly, the present invention relates to two novel Gonadotropin
releasing hormones muGnRH I and muGnRH II of amino acid SEQ ID 1 as QHWSAWRLPG,
and SEQ ID 2 QHWSWGILPG respectively, useful for induced breeding in fish both
in combination and alone, by activating production of Gonadotropin, and a method
of isolating the same from Indian Murrel brain, and further, a method of inducing
breeding in fishes using the said novel gonadotropin releasing hormones.
Further, the present invention relates to two novel Gonadotropin releasing
hormones muGnRH I and muGnRH II of amino acid sequence QHWSAWRLPG and QHWSWGILPG
respectively, useful for induced breeding in fishes both in combination and alone,
by activating production of Gonadotropin.
In an embodiment of the present invention, said hormones are obtained from Indian
Murrel brain.
In another embodiment of the present invention, muGnRH II is a more active gonadotropin
releasing hormone than muGnRH I.
In yet another embodiment of the present invention, muGnRH I increase gonadotropin
release by about 30 times.
In still another embodiment of the present invention, muGnRH II increase gonadotropin
release by about 35 times.
In still another embodiment of the present invention, wherein both muGnRH I,
and
muGnRH II in combination in the ratio of about 1:1 increase gonadotropin release
by about 50 times.
In still another embodiment of the present invention, said hormones show synergy
in induced breeding of fishes.
In still another embodiment of the present invention, both muGnRH I and muGnRH
II in combination increase gonadotropin release by about 50 times.
In still another embodiment of the present invention, said hormones show no species
barrier in induced breeding of fishes with increase in gonadotropin release ranging
between 20-50 times.
In still another embodiment of the present invention, said hormones are more
active
than other GnRHs comprising mammalian GnRH, Salmon GnRH, GnRH superactive analogues,
and commercially available GnRH Ovaprim, both in combination and alone.
In an embodiment of the present invention, a method of isolating and sequencing
two novel Gonadotropin releasing hormones muGnRH I and muGnRH II of amino acid
sequence QHWSAWRLPG and QHWSWGILPG respectively, useful for induced breeding in
fishes both in combination and alone, by activating production of Gonadotropin.
In another embodiment of the present invention, homogenizing hypothalami in acidic conditions.
In yet another embodiment of the present invention, filtering said homogenate
to remove fat and cell debris.
In still another embodiment of the present invention, adding acetone in a stepwise
manner to the said filtrate to obtain three precipitates namely AC I, AC II, and
AC III.
In still another embodiment of the present invention, estimating Gonadotropin
Releasing Hormone (GnRH) activity in the said precipitates.
In still another embodiment of the present invention, fractionating only active
precipitate AC II further using Molecular Exclusion Chromatography to obtain fractions
SG I and SG II.
In still another embodiment of the present invention, estimating Gonadotropin
Releasing Hormone (GnRH) activity in the fractions of previous step.
In still another embodiment of the present invention, fractionating only active
fraction SG II further, using Ion Exchange Chromatography with anionic column to
collect fractions MQ I, MQ II, MQ III, and MQ IV.
In still another embodiment of the present invention, estimating Gonadotropin
Releasing Hormone (GnRH) activity in the fractions of previous step, with only
fractions MQ I and MQ II having the same.
In still another embodiment of the present invention, fractionating MQ I further,
using Ion Exchange Chromatography with cationic column to obtain fractions MS I,
MS II, MS III, and MS IV.
In still another embodiment of the present invention, estimating Gonadotropin
Releasing Hormone (GnRH) activity in the fractions of previous step with only fraction
MS II having the same.
In still another embodiment of the present invention, fractionating MS II fraction
using Reverse Phase Chromatography to obtain fractions SRP I, SRP II, and SRP III.
In still another embodiment of the present invention, estimating Gonadotropin
Releasing Hormone (GnRH) activity in the fractions of previous step with only fraction
SRP II having the same.
In still another embodiment of the present invention, fractionating MQ II further,
using Reverse Phase Chromatography to obtain fractions QRP I, QRP II, and QRP III.
In still another embodiment of the present invention, calculating Gonadotropin
Releasing Hormone (GnRH) activity in the fractions of previous step with only fraction
QRP II having the same.
In still another embodiment of the present invention, sequencing QRP II and SRP
II fractions of above-mentioned step and naming them as muGnRH I and muGnRH II respectively.
In still another embodiment of the present invention, wherein fraction SG II
increase
gonadotropin release by about 20 times.
In still another embodiment of the present invention, wherein fraction SG II
increase
gonadotropin release by about 20 times.
In still another embodiment of the present invention, wherein fraction MQ I increase
gonadotropin release by about 10 times.
In still another embodiment of the present invention, wherein fraction MQ II
increase
gonadotropin release by about 40 times.
In still another embodiment of the present invention, wherein fraction MS II
increase
gonadotropin release by about 45 times.
In still another embodiment of the present invention, wherein calcium increase
gonadotropin releasing activity of muGnRH I and II.
A method of inducing breeding in fishes using the said novel gonadotropin releasing
hormones said method comprising steps of exposing fishes to the said Gonadotropin-releasing
hormones to help release gonadotropin, and inducing breeding in fishes using said gonadotropin.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
FIG. 1 shows comparison of primary amino acid sequences of naturally occurring
vertebrates and protochordate GnRHs. Underlined amino acids represent differences
in forms compared to mammalian GnRH.
FIG. 2 shows hairpin loop of GnRH.
FIG. 3 shows test of murrel GnRH activity in different fractions obtained through
acetone fraction. After preparation of three different fractions, 100 micrograms
protein was added in vitro pituitary cell culture and release of GtH was measured
by RIA.
FIG. 4 shows elution profile of mammalian GnRH through Sephadex G-25 column.
AC II fraction was dissolved in 1 N acetic acid and eluted through Sephadex G-25
column (58×1.5 cm) at a flow rate of 24 ml/hr.
FIG. 5
a shows elution profile of murrel Sephadex G-25 P-II through FPLC
Mono Q column. SG II fraction was dissolved in buffer A-20 mM Tris (pH 8.0) and
was injected into the FPLC anion exchange column (Mono Q). At first the column
was washed with buffer A and then eluted with buffer B-20 mM Tris (pH 8.0) with
1M NaCl.
FIG. 5
b shows elution profile of murrel Mono Q unbound peak (MQ I) through
FPLC Mono S column. MQ I was dissolved in buffer A-50 mM ammonium acetate (pH 4.8)
and was injected into the FPLC cation exchange column (Mono S). at first the column
was washed with buffer A and then eluted with buffer B-50 mM Ammonium acetate(pH
4.8) with 0.5M NaCl.
FIG. 6 shows elution profile of Mono QP-II (a) and Mono SP-II (b) through FPLC
Pep-RPC column. GnRH enriched fraction (Mono QP-II) and (Mono SP-II) was dissolved
individually in solvent A-0.1% TFA in water and was injected individually into
the FPLC peptide reverse phase chromatography (Pep-RPC). Firstly the column was
washed with solvent A and then eluted with solvent B-0.1% TFA in acetonitrile.
FIG. 7 shows amino acid sequences of muGnRH I, and muGnRH II.
PURIFICATION OF MURREL GNRH
(i) Extraction and Solvent Fractionation:
The hypothalami were homogenized using 1 N acetic acid containing 1 mM PMSF in
a Potter-Elvehjem homogenizer under ice. To avoid frothing, homogenization was
done very cautiously and slowly. The pH of the crude homogenate was maintained
at pH 3.2. The extracted material was filtered through double-fold cheesecloth
to remove fat and cell debris.
It was then lyophilized and stored at -80° C. until further use. The dried
extract was reconstituted in 0.1 N acetic acid and equal volume of chilled acetone
was added and kept at -20° C. for 15 min.
A thick precipitate (AC I) appears at the bottom of the suspension while a clear
supernatant is decanted off and to it double the volume of chilled acetone was
added and kept at -20° C. for 30 min following which another thin precipitate
layer (AC II) appeared. Once again, the clear supernatant was decanted and double
the volume of chilled acetone was added to it and kept at -20° C. for 14 h.
This resulted in the appearance of another thin precipitate layer (AC III) and
further addition of chilled acetone to the clear supernatant did not result in
the formation of any precipitate even after keeping it for 3 days at -20°
C. These three acetone fractions were extracted with petroleum ether (40-60°
C.) to remove acetone, lipids and other hydrophobic substances.
The final precipitates were centrifuged at 8,000 g for 15 min at 4° C. and
the residues were stored at -80° C. until further use. GnRH enriched fraction
was identified as AC II on the basis of GTH release from pituitary cell culture
in vitro (FIG.
3).
Molecular Exclusion Chromatography:
GnRH was further purified by subjecting AC II to molecular exclusion chromatography
on a Sephadex G 25 column. Elution of the column with 0.05 M PBS resulted in two
distinct peaks, SG I and SG II (FIG.
4). Mammalian GnRH was consequently
run to identify the pattern on the column. On the basis of GTH release from pituitary
cell incubation in vitro, GnRH was found to be located in SG II. As shown in table
1 below.
| TABLE 1 |
| |
| Biological activities of Sephadex G-25 eluted fractions in |
| terms of GtH release from murrel pituitary cell incubations |
| |
|
GtH released |
| |
System* |
(ng/6 × 104 cells) |
| |
|
| |
Control (C) |
2.8 ± 0.18 |
| |
C + SG I |
9.3 ± 0.93 |
| |
C + SG II |
37.9 ± 2.18 |
| |
|
(ii) Ion Exchange Chromatography:
This was further purified by FPLC Mono Q (anionic) and Mono S (cationic) ion-exchange
chromatography. The pooled, dialysed and lyophilised active peak of the molecular
exclusion chromatographic step (SG II) was loaded on a Mono Q column; washing the
Mono Q column with 20 mM Tris (pH 8.0) gave rise to a unbound peak (MQ I) while
on gradient elution with NaCl (1.0 M) containing 20 mM Tris (pH 8.0), three bound
peaks (MQ II, MQ III and MQ IV) were obtained (FIG. 5
a).
Assay of GnRH activity on the basis of GTH release from in vitro pituitary
cell culture identified the bound MQ II to possess the maximum GnRH activity while
MQ III and MQ IV showed negligible activity (As shown below in Table 2a).
| TABLE 2a |
| |
| Biological activities of FPLC Mono Q and Mono S eluted fractions |
| in terms of GtH release from murrel pituitary cell incubations |
| |
|
GtH released |
| |
Systema |
(ng/6 × 104 cells) |
| |
|
| |
Control (C) |
2.5 ± 0.17 |
| |
C + MQ I |
21.6 ± 0.98 |
| |
C + MQ II |
77.5 ± 2.92 |
| |
C + MQ III |
2.7 ± 0.29 |
| |
C + MQ IV |
2.3 ± 0.34 |
| |
|
| |
a2 μg protein from each peak was added to the incubation systems.
Values are mean ± SEM of four experiments |
Surprisingly the unbound peak of the Mono Q elution (MQ I) also showed
significant amounts of GnRH activity thus indicating the presence of a cationic
form of GnRH.
MQ I was therefore loaded onto a Mono S (cationic) column; washing the column
with 50 mM ammonium acetate (pH 4.8) gave rise to a single unbound peak (MS I)
while on subsequent gradient elution with 0.5 M NaCl, three bound peaks (MS II,
MS III and MS IV) were obtained (FIG. 5
b). Assay of GnRH activity in all
the four peaks identified MS II to be highly enriched in GnRH (As shown below in
table 2b).
| |
TABLE 2b |
| |
|
| |
|
GtH released |
| |
Systema |
(ng/6 × 104 cells) |
| |
|
| |
Control (C) |
2.4 ± 0.16 |
| |
C + MS I |
3.4 ± 0.23 |
| |
C + MS II |
83.1 ± 3.46 |
| |
C + MS III |
3.0 ± 0.18 |
| |
C + MS IV |
2.6 ± 0.09 |
| |
|
| |
a2 μg protein from each peak was added to the incubation systems.
Values are mean ± SEM of four experiments |
(iii) Reverse Phase Chromatography:
We now had two forms of GnRH in hand—one anionic form (MQ II) and the other
cationic form (MS II). Both were further purified through Pep-RPC column chromatography.
After loading the column with either MQ II or MS n, in both the cases, the unbound
proteins were eluted with 0.1% trifluoroacetic acid in water while the bound material
was eluted with 0.1% trifluoroacetic acid in acetonitrile gradient. MQ II resolved
into one unbound peak (QRP I) and two bound peaks (QRP H and QRP HI) (FIG. 6
a);
MS H gave rise to one unbound (SRP I) and two bound (SRP II and SRP ID) peaks (FIG.
6
b). GnRH was found to be located in QRP H and SRP H (FIG. 6 insets) and
were termed muGnRH I (anionic) and muGnRH H (cationic).
(iv) Functional Activity of the Purified GnRHs
Both muGnRH I and muGnRH II are highly active in releasing GTH from murrel pituitary
cells, muGnRH II being slightly more active. Moreover, these two GnRHs act almost
equally in different species of carp indicating no species barrier (As shown below
in Table 3).
| TABLE 3 |
| |
| A comparison of gonadotropin release from the pituitaries |
| of different carps in response to murrel GnRH I and II |
| |
|
Incubations |
GtH II released |
| |
Carp species |
(2 μg GnRH fractions) |
(ng/6 × 104 cells) |
| |
|
| |
Catla catla |
Control (C) |
2.6 ± 0.32 |
| |
|
C + mGnRH I |
74.5 ± 2.32 |
| |
|
C + mGnRH II |
82.6 ± 3.02* |
| |
Cirrhinus mrigala |
Control (C) |
2.1 ± 0.24 |
| |
|
C + mGnRH I |
72.0 ± 2.13 |
| |
|
C + mGnRH II |
80.1 ± 2.61* |
| |
Cyprinus carpio |
Control (C) |
2.8 ± 0.22 |
| |
|
C + mGnRH I |
73.8 ± 2.51 |
| |
|
C + mGnRH II |
81.7 ± 3.12* |
| |
|
| |
*p < 0.01 as compared to control |
Surprisingly, when added in combination, muGnRH I and II exhibit highly
significant increase in GTH release as a result of combination (As shown below
in Table 4).
| |
TABLE 4 |
| |
|
| |
Incubations |
GtH released |
| |
(2 μg GnRH fractions) |
(ng/6 × 104 cells) |
| |
|
| |
Control (C) |
2.6 ± 0.18 |
| |
C + mGnRH I |
73.3 ± 2.2* |
| |
C + mGnRH II |
79.2 ± 3.12* |
| |
C + mGnRH 1 + mGnRH II |
115.0 ± 3.23** |
| |
mammalian GnRH |
49.9 ± 2.02 |
| |
salmon GnRH (synthetic) |
56.2 ± 2.15 |
| |
GnRH superactive analogue |
68.0 ± 2.32 |
| |
(des-Gly10,[D-Alaa]-LHRH) |
| |
|
| |
Values are mean ± SEM of five experiments. |
| |
*p < 0.0 when compared with mammalian or salmon GnRHs |
| |
**p < 0.01 as compared with GnRH superactive analogue |
In fact, whether alone or in combination, muGnRHs had significantly greater activity
as compared to mammalian GnRH, salmon GnRH or the GnRH superactive analogue.
In an embodiment of the present invention addition of calcium to GnRH I and II
enhance the gonadotropin releasing activity of GnRH I and II.
Both these synthesized GnRHs showed much greater activity in the laboratory
and field experiments as compared to salmon GnRH, its analogues and other superactive
analogues and commercial product "Ovaprim". Amino acid sequence of these two decapeptides
(murrel GnRH I and II) could not be given here as we have applied for patent. This
Indian Product (murrel GnRH I plus II) therefore has great promise for national
and international market.
The above mentioned results placed Indian murrel GnRHs (I plus II) as a highly
promising global competitor since Salmon GnRH analogue (Ovaprim) prepared by Syndel
Laboratory, Canada is dominating the market and extensively sold all over the world
to breed culturable fish.
Both the murrel GnRHs (GnRH I and II) are novel, no naturally occurring GnRH
discovered till date has such sequences (FIG.
7). The activity of the synthetic
GnRHs has been examined in the laboratory (pituitary cell culture) and field to
breed carps.
A method of inducing breeding in fishes using the said novel gonadotropin releasing
hormones said method comprising steps of exposing fishes to the said Gonadotropin-releasing
hormones to help release gonadotropin, and inducing breeding in fishes using said gonadotropin.
SEQUENCE LISTING
<100> GENERAL INFORMATION:
<160> NUMBER OF SEQ ID NOS: 2
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO: 1
<211> LENGTH: 10
<212> TYPE: PRT
<213> ORGANISM: channa punctatus
<400> SEQUENCE: 1
Gln His Trp Ser Ala Trp Arg Leu Pro Gly
1 5 10
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO: 2
<211> LENGTH: 10
<212> TYPE: PRT
<213> ORGANISM: channa punctatus
<400> SEQUENCE: 2
Gln His Trp Ser Trp Gly Ile Leu Pro Gly
1 5 10
*
