JP5401736B2 - Sea cucumber egg release and fertilization inducer, and sea cucumber production method using the same - Google Patents

Description

  The present invention relates to a peptide that induces ovulation or fertilization of echinoderms, particularly sea cucumbers, and a method for efficiently performing ovulation or fertilization. The present invention is useful in sea cucumber production and sea cucumber seedling breeding.

  In recent years, in response to increasing demand for dried sea cucumbers in China, the export of dried sea cucumbers from Japan to China and Hong Kong has increased rapidly. The amount of sea cucumber landing in Japan has only increased slightly, and there is a concern that the resources will be reduced.

Manamako ( Stichopus japonicus ) is produced by seedlings at farming and fishery centers in each prefecture, and seedlings are released through fishery cooperatives. However, the actual number of seedlings supplied is significantly lower than the desired number of seedlings to be released, and there is an urgent need to increase seedling production efficiency. In order to increase the efficiency of seedling production, it is necessary to increase the production efficiency in each of the two stages of fertilized egg production and juvenile sea cucumber breeding.

  For efficient production of fertilized eggs, a technique that ensures reliable egg collection and collection is desired. However, the method currently used at the seed and seedling production site is to obtain a good parent individual during the spawning season, collect mature eggs and sperm, and artificially inseminate the fertilized egg until it reaches the release size. Is. In order to obtain mature eggs and sperm, it is common to hold the parent individual in seawater irradiated with ultraviolet rays or warmed seawater, and to stimulate egg release and fertilization by stimulation. However, this method requires seawater treatment facilities and is an indirect stimulation method, so the induction efficiency of ovulation and fertilization is low and the induction rate (number of individuals) cannot be predicted. Significant limitations are imposed on the establishment of seedling plans and the efficiency of seedling operations.

And since there is no reliable egg collection method from a parent individual with excellent traits, it is difficult to carry out selective breeding.
On the other hand, various peptides were isolated and purified from invertebrate nerve tissue, and consisted of 5 amino acids of asparagine (N) -glycine (G) -isoleucine (I) -tryptophan (W) -tyrosine (Y), carboxyl It has been reported that a peptide in which the terminal carboxyl group is amidated has a contracting action on muscles and the like (Non-patent Document 1). As for this peptide, there are also reports on the action of regulating the contraction of starfish tube feet, sea cucumber muscles, and sea cucumber connective tissue hardening (Non-Patent Documents 2 to 4).

For the echinodermatid starfish, gonad-stimulating hormone purified from radiation nerves has been studied. This starfish hormone is a peptide having a molecular weight of 4737 and a number of amino acids of 43, and it has been reported that eggs and sperm are released when it is given to the ovary or testis (Patent Document 1).
WO2006 / 022343 Muneoka, Y., Morishita, F., Furukawa, Matsushima, O., Kobayashi, M., Ohtani, M., Takahashi, T., Iwakoshi, E., Fujisawa, Yuko. And Minakata, H .: Acta Biologica Hungarica 51 (2-4), 111-132 (2000). Saha AK, Tamori M, Inoue M, Nakajima Y, Motokawa T .: NGIWYamide-induced contraction of tube feet and distribution of NGIWYamide-like immunoreactivity in nerves of the starfish Asterinapectinifera., Zoological Science, 23, 627-32 (2006). Inoue, M .: Localization of the neuropeptide NGIWYamide in the holothurian nervous system and its effects on muscular contraction .: Proceedings of the Royal Society of London series B., 266, 993-1000 (1999) Birenheide R, Tamori M, Motokawa T, Ohtani M, Iwakoshi E, Muneoka Y, Fujita T, Minakata H, Nomoto K .: Peptides controlling stiffness of connective tissue in sea cucumbers., Biological Bulletin, 194, 253-9 (1998)

The present inventors have found an active ingredient that acts on a parent individual having a developed ovary / testis from the body tissue of the sea cucumber and induces ovulation and fertilization. A method for judging and selecting the maturity level of the sea cucumber parent individual, a method for judging the maturity level of the ovary and testis, and a method for inducing ovulation and fertilization of the mature parent individual were established, and the present invention was completed. The present invention provides the following:
1) A compound represented by the following formula and having an ovulation activity and / or a fertilization activity
A ₁ -A ₂ -A ₃ -WA ₄ -R
Or an ovarian oviposition / fertilization inducer comprising a labeled product thereof.
(Where:
R is OH or NH ₂ ;
A ₁ is an asparagine residue, a pyroglutamic acid residue or a glutamine residue, preferably an asparagine residue;
A ₂ is a glycine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, preferably a glycine residue or an alanine residue, more preferably a glycine residue,
A ₃ is a leucine residue or an isoleucine residue;
W is a tryptophan residue;
A ₄ is a tyrosine residue or a phenylalanine residue, preferably a tyrosine residue. )
2) wherein R is NH ₂ ; A ₁ is an asparagine residue; A ₂ is a glycine residue; A ₃ is an isoleucine or leucine residue, and A ₄ is Or a fertilization inducer according to 1), which comprises a compound that is a tyrosine residue or a labeled product thereof.
3) A method for producing echinoderms, comprising the step of administering the compound defined in 1) or 2) or a labeled product thereof to an echinoderm male parent and / or female parent.
4) A male parent for the production of echinoderms comprising the step of providing the compound or label thereof defined in 1) or 2) to all or part of the testis and / or ovary of echinoderms A method of selecting and selecting individuals and / or female parents.
5) The production method according to 3), wherein the male parent individual and / or the female parent individual of the echinoderm are selected by the method described in 4). 6) The gonad is an ovary, a female parent individual is selected, and the selected female parent individual is administered the compound defined in 1) or 2) or a labeled product thereof in 3) The production method described.
7) A method of inducing fertilization or ovulation of echinoderms, comprising the step of administering the compound defined in 1) or 2) or a labeled product thereof to a male or female parent of echinoderms.
8) A method for producing a compound defined in 1) or 2) or a labeled product thereof, comprising a step of isolating from an invertebrate extract belonging to the sea cucumber class.
9) A peptide represented by the following formula or a labeled product thereof.

NGLWY-R
Or a labeled product thereof.
(Wherein R is OH or NH ₂ )
In the present specification, the term “echinoderm” includes invertebrates of sea cucumber, sea urchin, starfish, spider starfish, or sea urchin, unless otherwise specified. Examples of invertebrates included in the sea cucumber are Manamako, Kinko, Gummodoki, Murasakiki Gumodomoki, Hamanakiko, Kuriiroko, Janomeko, Tetsuiroko, Ryukyu Fujinamiko, Fujinamiko, Nisekuronamiko, Yonaco , Sea cucumber sea cucumber, sea cucumber sea cucumber, sea bream sea cucumber, sea urchin sea cucumber, sea urchin sea cucumber, sea cucumber sea cucumber, sea cucumber sea cucumber, sea cucumber sea cucumber, sea apple. The present invention is preferably applied to sea cucumber ( Stichopus japonicus ) or silkworm ( Cucumaria frondosa ), and more preferably applied to sea cucumber. The sea cucumbers are mainly oceanic and inhabit many reefs and gravel bottoms, with red sea cucumbers with mottled brown and brown spots, and mainly in the sandy mud bottom of the inner bay, from dark blue green to black. Including close blue sea cucumber and black black sea cucumber.

I. Ovulation and fertilization inducer [Structure]
The egg release / fertility inducing agent of the present invention is a compound represented by the following formula and having an egg releasing activity and / or fertilizing activity:
A ₁ -A ₂ -A ₃ -WA ₄ -R
Or a labeled product thereof (hereinafter sometimes referred to as “ovulation / fertilization-inducing substance of the present invention”). The definition of each character in the formula is as described above.

  In the present invention, the term “residue” in relation to amino acids is derived by removing H (hydrogen atom) from the N (amino) terminus of the amino acid and removing OH (hydroxyl group) from the C (carboxyl) terminus, The group which can be connected with the amino acid residue of or other group.

In the oviposition / fertilization-inducing substance of the present invention, a preferred example of the A ₁ -A ₂ -A ₃ -WA ₄ portion is NGIWY (SEQ ID NO: 1) or NGLWY (SEQ ID NO: 2). In natural products extracted from sea cucumbers, A ₃ is isoleucine (I). According to the study by the present inventors, however, what was changed to leucine (L) was surprisingly 100% more than natural products. It can have double or more activity.

In the egg-laying / fertilization-inducing substance of the present invention, a preferred example of the R moiety is NH ₂ . The C-terminus of the polypeptide chain is preferably amidated (R is NH ₂ ). Those that are not amidated (where R is OH) may be less active than those that are amidated.

Particularly preferred examples of the ovulation / fertility-inducing substance of the present invention are NGIWY-NH ₂ (sometimes described as NGIWYamide) and NGLWY-NH ₂ (sometimes described as NGLWYamide). The present inventors named the natural type former as Cubifrin-I and the latter as Cubifrin-L.

  In the present specification, the oviposition / fertilization-inducing substance of the present invention may be described using Cubifrin-I and Cubifrin-L as examples. This also applies to the ovulation / fertilization-inducing substance of the present invention.

  As a method of labeling in the oviposition / fertility-inducing substance of the present invention, a compound that selectively acts on the amino group at the N-terminus (for example, a carbodiimide-based condensing agent such as dicyclohexylcarbodiimide) or the like is used. The method of combining is mentioned. By using such a labeled product, fluorescence is imparted to the egg-laying / fertilization-inducing substance of the present invention, and optical observation and measurement of the amount of binding become possible. Examples of fluorescent compounds include fluorescein isothiocyanate (FITC), Texas Red (TR; sulforhodamine), dansyl (Dns; (5-dimethylamino) naphthalene-1-sulfonyl), carbocyanine (Cy3, Cy5, PE- Cy5), DOXYL (N-oxy-4,4-dimethyloxazolidine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6- Tetramethylpiperidine), umbelliferone, dinitrophenyl, acridine, coumarin, erythrosine, rhodamine, tetramethylrhodamine, or 7-nitrobenzo-2-oxa-1-diazole (NBD), or groups derived therefrom.

As the group for labeling, various groups can be used as long as the group does not lose the activity of the egg-laying / fertilization-inducing substance of the present invention by binding. If appropriate, a group for thermochemically and photochemically inactive linkage formation, that is, a spacer (for example,-(CH ₂ ) _n- (n is about 2 to 6) in order to maintain activity. An integer), a group for labeling may be linked via polyethylene oxide (PEO) _n ). The type of linker to be used can be appropriately selected by those skilled in the art in consideration of various factors such as length, molecular flexibility, and ease of reaction for linking. In addition, as a group for labeling, a reactive reagent itself having a fluorescence property by binding to an amino group may be used. An example of this is NBD-F (4-fluoro-7-nitrobennzofurazan). Such a thing is also included in the scope of the present invention.

  The egg-releasing / fertilizing inducer of the present invention induces egg-releasing / fertilizing behavior in about 1 hour by administration to a parent of Manamako. The released eggs / sperms are fertilized mature eggs / mature sperm, and fertilized eggs can be obtained by mixing and fertilizing them. Moreover, the oviposition / fertilization inducer of the present invention induces the release of fertilized eggs / sperm by administration to a part or the whole of the ovary / testis tissue taken out in vitro (see Examples).

  As used herein, “having ovulation activity and / or fertilization activity” refers to at least one of ovulation activity or fertilization activity against at least one kind of echinoderm, for example, manama, except in special cases. It means having. There may be both activities. Whether or not a certain compound has an ovulation activity or a fertilization activity can be determined, for example, according to the description of the present specification, by removing a candidate compound from an animal or an animal body at an appropriate concentration. Can be determined by whether or not egg or sperm release is observed.

The oviposition / fertilization-inducing substance of the present invention can be synthesized, and can be isolated and purified from sea cucumber by the following procedure.
[Isolation / Purification]
1. Extraction Cut and collect a portion of the echinoderm that contains the nerve tissue (eg, the tip of the head, including up to the rear end of the lime ring at the base of the sea cucumber mouth). After the fine powder, an ice-cold extract (for example, containing 4 M acetic acid, 0.2 mM β-aminoethyl benzensulfonyl fluoride, 5 μg / ml leupeptin, 2 μM pepstatin A) is added and homogenized. The homogenized sample is centrifuged under cooling to obtain a supernatant. From the supernatant, a filtered fraction having a molecular weight of 10,000 or less is obtained using an ultrafiltration device or the like (neural extract).

2. Chromatographic purification Dissolve the nerve extract, obtain the supernatant, and desalinate using a Develosil C8-UG-5 column. For elution from the column, 80% acetonitrile aqueous solution containing 0.1% TFA is used. The acetonitrile elution fraction is freeze-dried to obtain a freeze-dried product, which is used as a chromatographic sample.

  The chromatographic sample is fractionated by elution with a linear concentration gradient (10-40%) of acetonitrile containing, for example, 0.1% trifluoroacetic acid. The presence or absence of egg release from the ovary is confirmed, and the fraction having the ovulation activity is identified. If necessary, it is further subjected to chromatography, and further purified fractions having an ovulation activity are identified.

II. Method for producing echinoderms The present invention provides a method for producing echinoderms comprising the step of administering the oviposition / fertilization-inducing substance of the present invention to male and / or female parents of echinoderms. To do. In this method, the oviposition / fertilization-inducing substance of the present invention is provided to all or a part of the testis and / or ovary of echinoderms, and the male parent individual and / or excellent in production. You may further include the process of selecting a female parent individual.

The administration of the ovulation / fertility-inducing substance of the present invention to the male parent and / or female individual of echinoderms,
After a certain amount is dissolved in seawater, artificial seawater, physiological salt solution, or other solutions that do not have an inappropriate effect on the living body, and adjusted to the target concentration, it does not give a large load to the living body such as a syringe or injector. It may be administered by a method. Usually, the final concentration in the sea cucumber after administration of the ovulation / fertility-inducing substance should be maintained at about 10 ⁻⁸ M (10 nM) at most.

Selection of parent individuals that are excellent for production can be performed by determining the maturity of the gonad. This can be done by a method comprising the following steps:
(1) removing part of the gonad from an invertebrate belonging to the sea cucumber class; and
(2) including a step of supplying to the excised piece the ovulation / fertilization-inducing substance of the present invention at a concentration effective for ovulation / fertilization, for example, a concentration of 10 nM or less, When the egg is released, the maturity of the gonad is judged to be high.

  As for the degree of maturation of the ovary, when the ovulation / fertilization-inducing substance of the present invention is provided at a concentration effective for ovulation / fertilization, for example, a concentration of 10 nM or less, a fertilization effective ratio (for example, 50% or more Preferably ovary when maturation is observed in eggs that are 70% or more, more preferably 80% or more, and more preferably 90% or more (the germinal vesicles observed in immature eggs collapse and disappear) You may judge that the maturity level of is high.

A parent individual from which a gonad determined to have a high maturity can be selected as a superior parent individual for production.
Regarding the determination of ovarian maturity in the present invention, “mostly” when releasing most eggs means that echinoderms, for example sea cucumbers, lay eggs multiple times within a single spawning period, and therefore the degree of growth ( There may be a plurality of egg groups with different diameters), and in such a case, when the egg release / fertilization inducer of the present invention is administered, substantially all mature eggs can be released. It is meant that unmature eggs in the ovary are not released. The same applies to determination of testicular maturity in the present invention when most sperm are released.

In the production method of the present invention, ovulation can be performed by removing the ovary from the parent individual and performing an in vitro ovulation / fertilization inducer action to obtain a mature egg, regardless of the method of injection into the parent individual. More specifically, the egg released from the ovary after ovulation and fertilization-inducing substance is added to the ovary taken out in seawater etc. so that the final concentration is about 10 ^-8 M at the most and cultured for a certain period of time. Can be recovered and used for fertilization.

  As for sperm, the testis extracted from the parent individual can be fragmented in a container using a scissors or the like to obtain sperm that oozes from the testis strip, which is appropriately diluted to give Can be used. In this case, an oviposition / fertilization-inducing substance is unnecessary.

  By using the sea cucumber in the spawning season, the method according to the present invention can surely cause the egg laying and fertilization at any time, so that planned and efficient fertilization at the seedling production site Egg production can be carried out inexpensively, simply and reliably.

[Example 1: Isolation and purification of peptide]
The active peptide was extracted and purified by the following procedure.
1. Extraction
1) The head part of the head was cut and sampled from 150 Manamako so as to include the rear end of the lime ring at the base of the mouth. After removing the body wall part, it was frozen with liquid nitrogen and stored frozen at −80 degrees (frozen sea cucumber head 219.3 g).

2) The frozen sea cucumber head was finely ground using a grinder under freezing.
3) Add an equal amount of ice-cold extract (containing 4M acetic acid, 0.2mM β-aminoethyl benzensulfonyl fluoride, 5μg / ml leupeptin, 2μM pepstatin A) to the finely ground frozen sample, and immediately use a hiscotron homogenizer. Homogenized.

  4) The homogenized sample was centrifuged under cooling (15,200 × g, 15 minutes, 4 ° C.) to obtain supernatant 1. An equal amount of ice-cold extract was added to the precipitate and homogenization / centrifugation was repeated again to obtain supernatant 2. Supernatant 1 and supernatant 2 were combined and then centrifuged under cooling (45,000 × g, 2 hours, 4 ° C.) to obtain supernatant 3.

5) The supernatant 3 was filtered with a pressure ultrafiltration apparatus equipped with Amicon YM30 to obtain a filtered fraction having a molecular weight of 30,000 or less (30K fraction).
6) The 30K fraction was filtered with a pressurized ultrafiltration device equipped with Amicon YM10 to obtain a filtered fraction with a molecular weight of 10,000 or less (10K fraction)
7) The 10K fraction was freeze-dried to obtain a freeze-dried product (nerve extract 24.27 g).

2. Chromatographic purification
1) Desalination of nerve extracts:
60 ml of purified deionized water was added to the nerve extract (24.27 g), dissolved, and then centrifuged (6,000 × g, 10 minutes, 4 ° C.) to obtain supernatant 4. The precipitate was resuspended by adding 5 ml of purified deionized water, centrifuged again to obtain a supernatant, and combined with supernatant 4 (supernatant 5).

The supernatant 5 was filtered through a particle removal filter, and then desalted using a Develosil C8-UG-5 column (20 × 50 mm). For elution from the column, 80% acetonitrile aqueous solution containing 0.1% TFA was used.
The acetonitrile elution fraction was freeze-dried to obtain a freeze-dried product, which was used as a chromatographic sample (123 mg freeze-dried product).

2) First stage chromatography:
Chromatographic samples were fractionated using a Develosil RPAQUEOUS-AR-5 column (10 × 250 mm) by linear concentration gradient elution (10-40%) of acetonitrile containing 0.1% trifluoroacetic acid. Each fraction collected was lyophilized individually.

  Dissolve each freeze-dried fraction in 0.5 ml of purified water, dilute and dissolve a portion of it in seawater, add a piece of sea cucumber ovary, incubate at room temperature for 1.5 hours, and then check for egg release from the ovary Then, a fraction having an ovulation activity was identified (ovulation assay method).

The fractions containing the egg release activity were combined into the first-stage active fraction and subjected to the second-stage chromatography.
3) Second and third stage chromatography:
The first-stage active fraction was fractionated using a Develosil RPAQUEOUS-AR-5 column (10 × 250 mm) by performing a linear concentration gradient (10-30%) of acetonitrile containing 20 mM ammonium acetate (pH 6.0). Each fraction collected was lyophilized individually.

  Dissolve each lyophilized fraction in 0.5 ml of purified water, dilute a portion of it in seawater, add a piece of octopus ovary and incubate at room temperature for 1.5 hours, then check for the release of eggs from the ovaries. Then, the fraction having the ovulation activity was identified by the ovulation assay (second stage active fraction).

 The second stage active fraction was fractionated using a Develosil RPAQUEOUS-AR-3 column (2 × 250 mm) with a linear concentration gradient (15-22%) of acetonitrile containing 0.1% trifluoroacetic acid. Each fraction collected was lyophilized individually.

  Dissolve each freeze-dried fraction in 0.5 ml of purified water, dilute and dissolve a portion of it in seawater, add a piece of sea cucumber ovary, incubate at room temperature for 1.5 hours, and then check for egg release from the ovary Then, the fraction having the ovulation activity was identified by the ovulation assay (third stage active fraction).

3. Structure analysis of the active ingredient After confirming the purity of the third-stage active fraction using a matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer, the amino acid sequence was determined using a protein sequencer (Procise 494HT, manufactured by ABI). When analyzed, the amino acid sequence NGIWY (SEQ ID NO: 1) was obtained. Subsequently, when the molecular weight was confirmed by a liquid chromatograph-tandem mass spectrometer (nanoFrontier LD, manufactured by Hitachi High-Technologies; Quattro Premier, manufactured by Nihon Waters), the measured molecular weight was 1 less than the expected molecular weight, and the inside of the peptide When the structure was analyzed, data that seemed to indicate a protein sequencer was obtained, but amidation was estimated together with the observation that the measured value was shifted by 1 (FIG. 4).

[Example 2: Activity comparison test of NGIWYamide, NGLWYamide and NGIWY-OH types for ovary pieces]
1. Method The title peptides were synthesized by FMoc method using a peptide synthesizer (ABI433A type, manufactured by ABI). After synthesis, the molecular weight was confirmed by analysis with a mass spectrometer. Each was dissolved in filtered seawater and adjusted so that each peptide concentration would be 1 μM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, 100 fM.

  Of the small ovary pieces extracted from the sea cucumber, the eggs are densely packed inside, and the small pieces of eggs with the same diameter are immersed in seawater in which the peptide is dissolved. The number of eggs released was counted. Filtered seawater was used as a negative control.

2. Results The results are shown in the table below.

In the table, wells in which 50% or more of the eggs showed a maturation (disappearance of nuclei) reaction were shaded.
NGIWYamide induced egg maturation and ovulation at concentrations from 1 nM to 10 pM. NGLWYamide induced egg maturation and ovulation at concentrations of 10pM to 100fM and higher. NGIWY-OH induced egg maturation and ovulation at concentrations of 100 nM and above.

NGLWYamide was 100 to 1000 times stronger than NGIWYamide. On the other hand, the activity of NGIWY-OH type was very weak from 1/100 to 1/10000 compared to natural type (NGIWYamide).
[Example 3: Activity comparison test of NGIWYamide and NGLWYamide for mature individuals]
1. Method The body weight of sea cucumber was measured, and the body volume for each individual was calculated with the value as 100% water. The peptide was dissolved in seawater and the peptide was injected intraperitoneally.

2. Results The results are shown in FIG. In the NGIWYamide administration group, both males and females did not ovulate or fertilize at 1 nM or less. On the other hand, in the NGLWYamide administration group, males did not fertilize below 100 pM. Females did not ovulate below 10 pM. NGLWYamide showed the same effect at a concentration of 1/10 or less of natural NGIWYamide.

[Example 4: Example of seed production]
1. Method Manamako (average weight 142 g) caught in Ise Bay and bred for about 80 days at Mie Prefectural Cultivation Fisheries Center were used. For females, a part of the ovary (about 1 cm in length) is cut out and left in seawater containing 100 nM NGIWYamide for 1 hour and 30 minutes. Judged. Manamako was labeled with individual identification and housed in a 100 L water tank for both males and females (water temperature 19 ° C.), and NGIWYamide was administered intraperitoneally to a final concentration of 10 nM to induce ovulation and fertilization. After confirming the start of fertilization and ovulation (about 1 hour after peptide administration), fertilization was established by adding seawater from the male aquarium containing sperm to the female aquarium during ovulation slightly cloudy.

2. Results Female individuals with reactivity in the previous maturity assessment showed egg-laying behavior at a high rate and resulted in egg-laying (5/7 individuals), but non-responsive individuals did not lay eggs (0 / 6 individuals). Male individuals also fertilized at a high rate (13/15 individuals). Finally, 3.45 million eggs could be collected, and the hatching rate was 94%.

  6-8, after fertilization using mature eggs and spermatozoa obtained by peptide administration, late auriclaria larvae on day 13, dorioraria larvae on day 13 after insemination, juveniles after transformation on day 20 after fertilization A picture of a sea cucumber is shown (the lower right scale is 0.1 mm).

FIG. 1 shows the results of the first stage chromatography. Physiological activity was detected in the part indicated by the bar graph. FIG. 2 shows the results of the second stage chromatography. Physiological activity was detected in the part indicated by the bar graph. FIG. 3 shows the results of the third stage chromatography. Physiological activity was detected in the part indicated by the bar graph. FIG. 4 is a chart showing the measurement results of the active ingredient by a liquid chromatograph-tandem mass spectrometer. Samples are introduced by the ElectroSpray method. FIG. 5 shows the results of an activity comparison test of NGIWYamide (upper) or NGLWYamide (lower) on mature individuals. It is a photograph of the late Aurichralia larvae on day 13 after fertilization using mature eggs and sperm obtained by peptide administration (lower right scale is 0.1 mm). This is a photograph of Dorioralaria larvae on the 13th day after insemination (lower right scale is 0.1 mm). It is a photograph of a sea cucumber after transformation (20th day after fertilization) (lower right scale is 0.1 mm).

Claims (8)

A compound represented by the following formula and having an ovulatory activity and / or fertilizing activity
A ₁ -A ₂ -A ₃ -WA ₄ -R
(Where:
R is OH or NH ₂ ;
A ₁ is an asparagine residue;
A ₂ is a glycine residue;
A ₃ is a leucine residue or an isoleucine residue;
W is a tryptophan residue;
A ₄ is a tyrosine residue. )
Or an invertebrate ovulation / fertilization inducer belonging to the sea cucumber class, comprising a labeled product thereof. A method for producing an invertebrate belonging to the sea cucumber class, comprising the step of administering the compound defined in claim 1 or a labeled product thereof to a male parent and / or a female parent of an invertebrate belonging to the sea cucumber class. Including the step of providing the testis and / or ovary of an invertebrate belonging to the sea cucumber class with all or part of the test substance and the labeled product thereof as defined in claim 1 A method of selecting and selecting male and / or female parents for production. The production method according to claim 2, wherein the invertebrate male parent and / or female parent belonging to the sea cucumber class is selected by the method according to claim 3. The production according to claim 4, wherein the gonad is an ovary, wherein a female parent individual is selected, and the selected female parent individual is administered the compound defined in claim 1 or a labeled product thereof. Method. Insemination or release of an invertebrate belonging to the sea cucumber class, comprising the step of administering to the male parent or female parent of an invertebrate belonging to the sea cucumber class, the compound defined in claim 1 or a labeled product thereof. How to induce an egg.   A method for producing a compound as defined in claim 1 or a labeled product thereof, comprising the step of isolating from an invertebrate extract belonging to the sea cucumber class. Peptide represented by the following formula
NGLWY-R
(Wherein R is NH ₂ )
Or a labeled product thereof.