WO2003064460A2 - Two gonadotropin releasing hormones and a method to isolate the same - Google Patents

Two gonadotropin releasing hormones and a method to isolate the same Download PDF

Info

Publication number
WO2003064460A2
WO2003064460A2 PCT/IN2003/000015 IN0300015W WO03064460A2 WO 2003064460 A2 WO2003064460 A2 WO 2003064460A2 IN 0300015 W IN0300015 W IN 0300015W WO 03064460 A2 WO03064460 A2 WO 03064460A2
Authority
WO
WIPO (PCT)
Prior art keywords
gonadotropin
mugnrh
gnrh
hormones
fractions
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IN2003/000015
Other languages
French (fr)
Other versions
WO2003064460A3 (en
WO2003064460A8 (en
Inventor
Abhijit Chatterjee
Partha Ray
Subrata Dasgupta
Samir Bhattacharaya
Santosh Pasha
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Council of Scientific and Industrial Research CSIR
Department of Biotechnology of Ministry of Science and Technology India
Original Assignee
Council of Scientific and Industrial Research CSIR
Department of Biotechnology of Ministry of Science and Technology India
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Council of Scientific and Industrial Research CSIR, Department of Biotechnology of Ministry of Science and Technology India filed Critical Council of Scientific and Industrial Research CSIR
Priority to EP03701712A priority Critical patent/EP1470153B1/en
Priority to DE60323052T priority patent/DE60323052D1/en
Priority to JP2003564080A priority patent/JP4394954B2/en
Priority to AU2003202800A priority patent/AU2003202800B2/en
Publication of WO2003064460A2 publication Critical patent/WO2003064460A2/en
Publication of WO2003064460A8 publication Critical patent/WO2003064460A8/en
Publication of WO2003064460A3 publication Critical patent/WO2003064460A3/en
Anticipated expiration legal-status Critical
Priority to NO20043551A priority patent/NO20043551L/en
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S930/00—Peptide or protein sequence
    • Y10S930/01—Peptide or protein sequence
    • Y10S930/13—Luteinizing hormone-releasing hormone; related peptides

Definitions

  • 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.
  • GnRH Gonadotropin Releasing Hormone
  • GnRH 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 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).
  • the main object of the present invention is to isolate and identify novel gonadotropin releasing hormones for induced breeding 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.
  • 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.
  • 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
  • the present invention relates to two novel Gonadotropin releasing hormones muGnRH I and muGnRH II of amino acid sequence QHWSAWRLPG and
  • said hormones are obtained from Indian Murrel brain.
  • muGnRH II is a more active gonadotropin releasing hormone than muGnRH I.
  • muGnRH I increase gonadotropin release by about 30 times.
  • muGnRH II increase gonadotropin release by about 35 times.
  • both muGnRH I, and muGnRH II in combination in the ratio of about 1:1 increase gonadotropin release by about 50 times.
  • said honnones show synergy in induced breeding of fishes.
  • both muGnRH I and muGnRH II in combination increase gonadotropin release by about 50 times.
  • said hormones show no species barrier in induced breeding of fishes with increase in gonadotropin release ranging between
  • 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.
  • homogenizing hypothalami in acidic conditions hi yet another embodiment of the present invention, filtering said homogenate to remove fat and cell debris.
  • GnRH Hormone
  • fractionating MQ I further, using Ion
  • fractionating MQ II further, using
  • fraction SG II increase gonadotropin release by about 20 times.
  • 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.
  • fraction MQ II increase gonadotropin release by about 40 times.
  • fraction MS II increase ⁇ gonadotropin release by about 45 times.
  • a method of inducing breeding in fishes using the said novel gonadotropin releasing hormones comprising steps of exposing fishes to the said Gonadotropin- releasing hormones to help release gonadotropin, and inducing breeding in fishes using said gonadotropin.
  • 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, lOOmicro grams 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 x
  • Fig 5a shows elution profile of murrel Sephadex G-25 P-II through FPLC Mono Q column.
  • SG II fraction was dissolved in buffer A-20mM 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 5b shows elution profile of murrel Mono Q unbound peak (MQ I) through FPLC Mono S column.
  • MQ I was dissolved in buffer A-50mM 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).
  • Fig 7 shows amino acid sequences of muGnRH I, and muGnRH II. Purification of murrel GnRH (i) Extraction and solvent fractionation:
  • 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.
  • AC 13 A thin precipitate layer
  • AC IH another thin precipitate layer
  • 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.
  • 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.
  • 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
  • muGnRH I and II exhibit highly significant increase in GTH release as a result of combination (As shown below in Table 4).
  • a method of inducing breeding in fishes using the said novel gonadotropin releasing hormones comprising steps of exposing fishes to the said Gonadotropin- releasing hormones to help release gonadotropin, and inducing breeding in fishes using said gonadotropin.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Endocrinology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Reproductive Health (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Gynecology & Obstetrics (AREA)
  • Pregnancy & Childbirth (AREA)
  • Peptides Or Proteins (AREA)
  • Farming Of Fish And Shellfish (AREA)
  • Feed For Specific Animals (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Design And Manufacture Of Integrated Circuits (AREA)
  • Fodder In General (AREA)

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.

Description

NOVEL TWO GONADOTROPIN RELEASING HORMONES AND A METHOD
TO ISOLATE THE SAME
Technical Field 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 Art
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 salmon 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 1989 May; 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. hi 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 honnones 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. hi 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 in.
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 IN.
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 IN.
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 LI 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 LI 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, lOOmicro grams 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 x
1.5 cm) at a flow rate of 24 ml/hr.
Fig 5a shows elution profile of murrel Sephadex G-25 P-II through FPLC Mono Q column. SG II fraction was dissolved in buffer A-20mM 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 5b shows elution profile of murrel Mono Q unbound peak (MQ I) through FPLC Mono S column.
MQ I was dissolved in buffer A-50mM 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 l5 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 13) 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 14h. This resulted in the appearance of another thin precipitate layer (AC IH) 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
Figure imgf000009_0001
(ii) Ion exchange chromatography:
This was further purified by FPLC Mono Q (anionic) and Mono S (cationic) ion-exchange chromatography. The pooled, dialysed and lyophihsed active peak of the molecular exclusion chromatographic step (SG H) 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
IN) 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
IN 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
Figure imgf000009_0002
a 2 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 (pH4.8) 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. 5b). 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
Figure imgf000010_0001
a 2 μg protein from each peak was added to the incubation systems. Values are mean ±
SEM of four experiments
(Hi) Reverse Phase chromatography: We now had two forms of GnRH in hand — one anionic form (MQ H) 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. 6a); MS H gave rise to one unbound (SRP I) and two bound (SRP II and SRP ID) peaks (Fig. 6b). 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 R
Figure imgf000010_0002
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
Figure imgf000011_0001
Values are mean ± SEM of five experiments. p< 0.01 when compared with mammalian or salmon GnRHs p < 0.01 as compared with GnRH superactive analogue h fact, whether alone or in combination, muGnRHs had significantly greater activity as compared to mammalian GnRH, salmon GnRH or the GnRH superactive analogue. Iα an embodiment of the present invention addition of calcium to GnRH I and II enhance the gonadotropin releasing activity of GnRH I and H
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 H) could not be given here as we have applied for patent. This Indian Product (murrel GnRH I plus ϋ) 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.

Claims

12Claims
1. Two 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.
2. The hormones as claimed in claim 1, wherein said hormones are obtained from Indian Murrel- brain.
3. The hormones as claimed in claim 1, wherein muGnRH II is a more active gonadotropin-releasing hormone than muGnRH I.
4. The hormones as claimed in claim 1, wherein muGnRH I increase gonadotropin release by about 30 times.
5. The hormones as claimed in claim 1, wherein muGnRH II increase gonadotropin release by about 35 times.
6. The hormones as claimed in claim 1, wherein said hormones show synergy in induced breeding of fishes .
7. The hormones as claimed in claim 1, wherein both muGnRH I, and muGnRH II in combination in the ratio of about 1:1 increase gonadotropin release by about 50 times.
8. The hormones as claimed in claim 1, wherein said hormones show no species barrier in induced breeding of fishes with increase in gonadotropin release ranging between 20- 50 times.
9. The hormones as claimed in claim 1, wherein 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.
10. 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, said method comprising:
(a) homogenizing hypothalami in acidic conditions, (b) filtering said homogenate to remove fat and cell debris,
(c) adding acetone in a stepwise manner to the said filtrate to obtain three precipitates namely AC I, AC II, and AC III,
(d) estimating Gonadotropin Releasing Hormone (GnRH) activity in the precipitates of step (c), 13
(e) fractionating only active precipitate AC II further using Molecular Exclusion Chromatography to obtain fractions SG I and SG π,
(f) estimating Gonadotropin Releasing Hormone (GnRH) activity in the fractions of step (e), (g) 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 IN, (h) estimating Gonadotropin Releasing Hormone (GnRH) activity in the fractions of step (g), with only fractions MQ I and MQ II having the same, (i) fractionating MQ I of step (h) further, using Ion Exchange Chromatography with cationic column to obtain fractions MS I, MS II, MS III, and MS IN, (j) estimating Gonadotropin Releasing Hormone (GnRH) activity in the fractions of step (i) with only fraction MS II having the same, (k) fractionating MS II fraction using Reverse Phase Chromatography to obtain fractions SRP I, SRP π, and SRP III,
(1) estimating Gonadotropin Releasing Hormone (GnRH) activity in the fractions of step (k) with only fraction SRP II having the same, (m) fractionating MQ II of step (h) further, using Reverse Phase Chromatography to obtain fractions QRP I, QRP II, and QRP III, (n) calculating Gonadotropin Releasing Hormone (GnRH) activity in the fractions of step (m) with only fraction QRP II having the same, and (o) sequencing QRP II and SRP II fractions of steps (n) and (1) and naming them as muGnRH I and muGnRH II respectively.
11. A method as claimed in claim 10, wherein fraction SG II increase gonadotropin release by about 20 times.
12. A method as claimed in claim 10, wherein fraction SG II increase gonadotropin release by about 20 times.
13. A method as claimed in claim 10, wherein fraction MQ I increase gonadotropin release by about 10 times.
14. A method as claimed in claim 10, wherein fraction MQ II increase gonadotropin release by about 40 times.
15. A method as claimed in claim 10, wherein fraction MS II increase gonadotropin release by about 45 times. 14
16. A method as claimed in claim 10, wherein calcium increases gonadotropin-releasing activity of muGnRH I, and muGnRH LI.
17. A method of inducing breeding in fishes using gonadotropin releasing hormones of claim 1, 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.
PCT/IN2003/000015 2002-01-30 2003-01-30 Two gonadotropin releasing hormones and a method to isolate the same Ceased WO2003064460A2 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP03701712A EP1470153B1 (en) 2002-01-30 2003-01-30 Two gonadotropin releasing hormones and a method to isolate the same
DE60323052T DE60323052D1 (en) 2002-01-30 2003-01-30 TWO GONADOTROPINE RELEASING HORMONES AND METHOD FOR INSULATION
JP2003564080A JP4394954B2 (en) 2002-01-30 2003-01-30 Two gonadotropin-releasing hormones and methods for isolating them
AU2003202800A AU2003202800B2 (en) 2002-01-30 2003-01-30 Two Gonadotropin releasing hormones and a method to isolate the same
NO20043551A NO20043551L (en) 2002-01-30 2004-08-25 Two gonadotropin releasing hormones and one method of isolating them

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US35304102P 2002-01-30 2002-01-30
US60/353,041 2002-01-30

Publications (3)

Publication Number Publication Date
WO2003064460A2 true WO2003064460A2 (en) 2003-08-07
WO2003064460A8 WO2003064460A8 (en) 2003-12-04
WO2003064460A3 WO2003064460A3 (en) 2004-02-19

Family

ID=27663168

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2003/000015 Ceased WO2003064460A2 (en) 2002-01-30 2003-01-30 Two gonadotropin releasing hormones and a method to isolate the same

Country Status (8)

Country Link
US (2) US6977242B2 (en)
EP (1) EP1470153B1 (en)
JP (2) JP4394954B2 (en)
AT (1) ATE405579T1 (en)
AU (1) AU2003202800B2 (en)
DE (1) DE60323052D1 (en)
NO (1) NO20043551L (en)
WO (1) WO2003064460A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110226188A1 (en) * 2010-03-19 2011-09-22 National Pingtung University Of Science & Technology Artificial decapeptide for inducing vitellogenesis in fish

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10313458A1 (en) * 2003-03-25 2004-11-18 Henkel Kgaa Detergents or cleaning agents
WO2014148603A1 (en) * 2013-03-22 2014-09-25 日本水産株式会社 Method for inducing spawning of piscivorous fish for marine products
JP6909160B2 (en) * 2015-11-27 2021-07-28 日本水産株式会社 Fish feed using cell-penetrating peptide and cell-penetrating peptide

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BHATTACHARYA E.A.: "Biotechnology input in fish breeding" INDIAN JOURNAL OF BIOTECHNOLOGY, vol. 1, no. 1, 13 February 2002 (2002-02-13), pages 29-38, XP002262286 *
HALDER S ET AL: "INDUCED SPAWNING OF INDIAN MAJOR CARPS AND MATURATION OF A PERCH AND A CATFISH BY MURREL GONADOTROPIN RELEASING HORMONE, PIMOZIDE AND CALCIUM" AQUACULTURE, AMSTERDAM, NL, vol. 97, no. 4, 1991, pages 373-382, XP008022209 *
MUKHOPADHYAY E.A.: "Gonadotropin releasing hormone stimulation of cyclic 3',5'-AMP in the pituitary cell of a teleost (Channa punctuatis, Bloch) requires extracellular calcium: its relationship to gonadotropin release" GEN.COMP.ENDOCRINOL., vol. 97, 1995, pages 353-365, XP002262288 *
MUKHOPADHYAY E.A.: "Intracellular events in response to Gnrh causing gonadotropin release from pituitary cells of a channis fish, channa punctatus" COMP.BIOCHEM.PHYSIOL., vol. 118c, no. 2, 1997, pages 129-136, XP002262287 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110226188A1 (en) * 2010-03-19 2011-09-22 National Pingtung University Of Science & Technology Artificial decapeptide for inducing vitellogenesis in fish
US8536124B2 (en) * 2010-03-19 2013-09-17 National Pingtung University Of Science & Technology Artificial decapeptide for inducing vitellogenesis in fish

Also Published As

Publication number Publication date
NO20043551L (en) 2004-08-25
DE60323052D1 (en) 2008-10-02
EP1470153A2 (en) 2004-10-27
JP4394954B2 (en) 2010-01-06
AU2003202800B2 (en) 2008-07-24
US7074758B2 (en) 2006-07-11
WO2003064460A3 (en) 2004-02-19
JP2009292836A (en) 2009-12-17
JP2005529070A (en) 2005-09-29
EP1470153B1 (en) 2008-08-20
US6977242B2 (en) 2005-12-20
US20030236185A1 (en) 2003-12-25
WO2003064460A8 (en) 2003-12-04
ATE405579T1 (en) 2008-09-15
US20050233951A1 (en) 2005-10-20

Similar Documents

Publication Publication Date Title
Ueno et al. Isolation and partial characterization of basic fibroblast growth factor from bovine testis
CA2018714C (en) Hypothalamic polypeptides with adenylate cyclase stimulating activity
Shibasaki et al. Corticotropin-releasing factor-like activity in human placental extracts
Rivier et al. Characterization of rat hypothalamic corticotropin-releasing factor.
Hofferer et al. Induction of ovulation and superovulation in mares using equine LH and FSH separated by hydrophobic interaction chromatography
Shimizu et al. Purification of mummichog (Fundulus heteroclitus) gonadotropins and their subunits, using an immunochemical assay with antisera raised against synthetic peptides
Kamei et al. Purification of follicle-stimulating hormone from immature Japanese eel, Anguilla japonica, and its biochemical properties and steroidogenic activities
Bulant et al. Processing of thyrotropin-releasing hormone prohormone (pro-TRH) generates pro-TRH-connecting peptides. Identification and characterization of prepro-TRH-(160-169) and prepro-TRH-(178-199) in the rat nervous system.
Sower et al. Update: brain and pituitary hormones of lampreys
US7074758B2 (en) Two gonadotropin releasing hormones and a method to isolate the same
GÄDE Hyperprolinaemia caused by novel members of the adipokinetic hormone/red pigment-concentrating hormone family of peptides isolated from corpora cardiaca of onitine beetles
AU2003202800A1 (en) Two Gonadotropin releasing hormones and a method to isolate the same
Smart et al. Peptide tyrosine phenylalanine: a novel neuropeptide F-related nonapeptide from the brain of the squid, Loligo vulgaris
US5470826A (en) Single chain proteins with FHS suppressing activity isolated from follicular fluid and uses thereof
Mongiat et al. Experimental data supporting the expression of the highly conserved GnRH-II in the brain and pituitary gland of rats
Dores et al. Immunological evidence for multiple forms of α-melanotropin (α-MSH) in the pars intermedia of the Australian lungfish, Neoceratodus forsteri
Nozaki et al. Distribution of immunoreactive adenohypophysial cell types in the pituitaries of the Atlantic and the Pacific hagfish, Myxine glutinosa and Eptatretus burgeri
Hatfield et al. Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development: II. β-lipotropin (β-LPH)-related peptides
Gelman et al. Ecdysiotropic activity in the lepidopteran hindgut—an update
Nagle et al. Delta-bag cell peptide from the egg-laying hormone precursor of Aplysia. Processing, primary structure, and biological activity.
Wilson et al. Chromogranin from normal human adrenal glands: purification by monoclonal antibody affinity chromatography and partial N-terminal amino acid sequence
Moszkowska et al. Some new aspects of a sheep pineal gonadotropic inhibiting activity in in vitro experiments
Kawauchi Evolutionary aspects of pituitary hormones
Gröneveld et al. Identification, cellular localization and in vitro release of a novel teleost melanin-concentrating hormone gene-related peptide
EP0996457A1 (en) Fsh-releasing peptides

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i

Free format text: IN PCT GAZETTE 32/2003 UNDER (22) REPLACE "10 JANUARY 2003 (10.01.2003)" BY "30 JANUARY 2003 (30.01.2003)"

WWE Wipo information: entry into national phase

Ref document number: 2003564080

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2003202800

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2003701712

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2003701712

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 1691/DELNP/2005

Country of ref document: IN

WWG Wipo information: grant in national office

Ref document number: 2003701712

Country of ref document: EP