JP6725942B2 - Follicle-stimulating hormone secretagogue - Google Patents

Description

The present invention relates to a bovine follicle-stimulating hormone secretagogue, which contains plasmalogen extracted from bovine as an active ingredient.

A cow is a single animal, and normally only one ovulates in one sexual cycle, so basically one cow is born in one pregnancy. Therefore, in the breeding of cattle, a method of inducing superovulation, recovering a large number of eggs from a cow and transplanting them to another cow, and obtaining a large number of offspring at one time is widely used.

Further, in the case of cattle breeding management, one cow is ideally produced once a year, and if the number of empty days before reconception after calving exceeds a predetermined number of days, an economic loss will occur. Therefore, a method of inducing ovulation to control the number of days of gestation is also widely used.

It is known that ovulatory induction involves gonadotropins (LH) and follicle stimulating hormones (FSH), which are extremely important hormones for reproduction, and regulate the secretion of these LH and FSH. This is important in cattle breeding. Such LH and FSH are secreted from the gonadotroph cells present in the anterior pituitary. The gonadotroph cells play a central role in the regulation of sexual function, and in recent years, the present inventors have been mainly conducting the analysis of gonadotroph cells.

For example, the present inventors observed gonadotroph cells in the anterior lobe of the bovine pituitary by a fluorescent immunostaining method, and the gonadotrophin-releasing hormone receptor (GnRHR) was localized to a lipid icada (Lipid Raft), which is a special structure on the cell membrane of gonadotrophs. It was clarified (see Non-Patent Document 1). Next, the present inventors analyzed all genes expressed in bovine anterior pituitary gland using a next-generation sequencer, and discovered GPR61 as a novel receptor expressed in bovine anterior pituitary gland (non- See Patent Document 2).

Furthermore, the present inventors also discovered that GPR61 coexists with GnRHR in lipid saccharids on the cell surface of gonadotrophins (see Non-Patent Document 3). Although GPR61 was an orphan (orphan) receptor whose ligand was unknown, it was recently reported that plasmalogen may be the ligand (see Non-Patent Document 4). However, it is the result of plasmalogen derived from avian skin cells, and it is only the result of knocking down not only GPR61 but also GPR1, GPR19, GPR21, and GPR27. What is the relationship between GPR61-mediated FSH secretion and plasmalogen? Not shown.

On the other hand, studies have been conducted on LH and FSH secretion in cattle, and some methods have been proposed. For example, the present inventors have presented a method for stimulating LH secretion from bovine gonadotroph cells using kisspeptin (see Patent Document 1).

In addition, anti-inhibin antibody was injected into cows for passive immunization to suppress the function of the dominant follicle secreting inhibin and to increase the number of small follicles by endogenous gonadotropins, followed by exogenous A method of inducing superovulation of cows by gonadotropin (see Patent Document 2), or a method of inducing superovulation of cows by active immunization with inhibin and then administering follicle stimulating hormone to ovulate a large number of developed follicles. (See Patent Document 3). With this method, the blood FSH concentration can be increased, but its reliability is poor despite its complexity and high cost.

Further, a method of inducing superovulation in cattle by one injection as an aqueous solution containing porcine FSH containing polyvinylpyrrolidone or carboxymethylcellulose (see Patent Document 4) has been proposed. In such a method, secretion of FSH can be stimulated, but since FSH, which is a heterologous porcine-derived protein, is administered to cattle, there is a problem that antibodies to the above-mentioned porcine-derived FSH can be formed in the administered cattle. there were.

JP, 2005-145351, A Japanese Patent Laid-Open No. 10-072370 JP, 08-154957, A Japanese Patent Laid-Open No. 05-038344

Gonadotropin-releasing hormone (GnRH) receptors of cattle aggregate on the surface of gonadotrophs and are increased by elevated GnRH concentrations. Anim. Reprod. Sci. Kadokawa H, Pandey K, NaharA, Nakamura U, Rudolf FO. Deep sequencing of the transcriptome in the anterior pituitary of heifers before and after ovulation.J. Vet. Med. Sci. Pandey K, Mizukami Y, Watanabe K, Sakaguti S, Kadokawa H. Heifers express G-protein coupled receptor 61 in anterior pituitary gonadotrophs in stage-dependent manner.Anim.Reprod.Sci. Pandey K, Kereilwea O, Borromeo V, Kadokawa H. Neuronal Orphan G-Protein CoupledReceptor Proteins Mediate Plasmalogens-Induced Activation of ERK and AktSignaling. PLoS One. Hossain MS, Mineno K, Katafuchi T.

An object of the present invention is to provide an agent that promotes secretion of follicle-stimulating hormone from bovine gonadotroph cells.

The present inventors conducted an experiment in which plasmalogen extracted from commercially available bovine brain was added to a culture system of bovine anterior pituitary. As a result, they have found that addition of plasmalogen promotes FSH secretion from bovine anterior pituitary cells. Further, it was confirmed that GnRH is not necessary for promoting FSH secretion, and the present invention was completed.

That is, the present invention is as follows.
(1) A bovine follicle-stimulating hormone secretagogue containing plasmalogen extracted from bovine as an active ingredient.
(2) The bovine follicle-stimulating hormone secretagogue according to (1) above, wherein the plasmalogen is extracted from bovine brain.
(3) The follicle-stimulating hormone secretagogue according to the above (1) or (2), wherein the plasmalogen is ethanolamine plasmalogen.
(4) The bovine according to any one of (1) to (3) above, which has a hexadecyl group or a hexadecenyl group through a vinyl ether bond at the 1-position (sn-1 position) of the glycerol skeleton in plasmalogen. Follicle-stimulating hormone secretagogue.

The plasmalogen used in the present invention is a phospholipid having a small molecular weight, and is derived from a bovine of the same species and has a small molecular weight, so that the antigenicity is extremely low, and FSH can be produced in the administered cow without causing an antibody against the plasmalogen. It can be secreted.

It is a graph which shows the result of having investigated the FSH secretion amount from the bovine anterior pituitary cell after the culture sexual maturation when the plasmalogen derived from bovine brain of various concentrations was administered. In the figure, "b" indicates that there is a significant difference (P<0.05) from the control (no plasmalogen and GnRH).

Plasmalogen is one of the glycerophospholipids represented by the following general formula (I), and has an alkyl group or an alkenyl group via a vinyl ether bond at the 1-position (sn-1 position) of the glycerol skeleton.

In the formula, R ¹ is an alkyl group or an alkenyl group having 1 to 20 carbon atoms, and specifically, it has 12 to 20 carbon atoms such as dodecyl group, tetradecyl group, hexadecyl group (hexadecanyl group), octadecyl group, and icosanyl group. Examples thereof include an alkyl group and an alkenyl group having 14 to 18 carbon atoms such as a tetradecenyl group, a hexadecenyl group, and octadecenyl. On the other hand, R ² is an alkyl group or alkenyl group having 1 to 20 carbon atoms or a polyunsaturated fatty acid residue having 6 to 26 carbon atoms. As the alkyl group or alkenyl group having 1 to 20 carbon atoms, similar to the case of R ¹ , an alkyl group having 12 to 20 carbon atoms such as a dodecyl group, a tetradecyl group, a hexadecyl group, an octadecyl group, and an eicosanyl group, or tetradecenyl group Group, a hexadecenyl group, octadecenyl, and other alkenyl groups having 14 to 18 carbon atoms. Further, as the polyunsaturated fatty acid residue having 6 to 26 carbon atoms, octadecadienoyl group, octadecatrienoyl group, icosatetraenoyl group, icosapentaenoyl group, docosatetraenoyl group, docosa Examples thereof include a pentaenoyl group and a docosahexaenoyl group. Further, X can be a polar group such as an ethanolamine group, a choline group, an inositol group, a glycerol group, or a serine group, and is preferably an ethanolamine group.

Examples of the bovine plasmalogen include those extracted from living tissues such as brain, skin, intestine, stomach, reproductive organs, heart, and the like, and those extracted from brain are preferred. In addition, in the present invention, “extracted from bovine” includes “extracted from microorganisms growing in bovine stomach or intestine”.

The method for extracting plasmalogen from a living tissue or a gastrointestinal microorganism is not particularly limited, and examples thereof include a method using a solvent such as hexane, acetone, ether, or ethanol. If necessary, an acetone precipitation method, a column chromatography method, or high performance liquid chromatography may be used to remove triglycerides and partial glycerides, and only the phospholipid fraction containing plasmalogen may be separated and purified. Alternatively, the lysoplasmalogen may be removed by distribution of an aqueous solution.

The ratio of plasmalogen-type phospholipid to total phospholipid is preferably 50% or more, more preferably 55% or more, and further preferably 60% or more. The bovine plasmalogen of the present invention may contain glycerophospholipids, sphingophospholipids, etc. other than bovine plasmalogen.

The bovine FSH promoter of the present invention can be formulated by a method known to those skilled in the art. For example, a pharmaceutically acceptable carrier or medium, specifically, sterile water or saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, preservative, binding agent. It may be a composition formulated by admixing it in an appropriate combination with an agent and the like in a unit dose form required for generally accepted pharmaceutical practice.

In the composition, for oral administration, the bovine FSH promoter of the present invention is mixed with a pharmaceutically acceptable carrier well known in the art to give tablets, pills, dragees, capsules, liquids. , Gels, syrups, slurries, suspensions and the like. As the carrier, those widely known in the art can be widely used, for example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid and the like; Water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone and other binders, dried starch, sodium alginate, agar powder, laminaran powder, hydrogen carbonate Disintegrators such as sodium, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, starch and lactose; disintegration inhibitors such as sucrose, stearin, cocoa butter and hydrogenated oil; quaternary ammonium salts, Absorption enhancers such as sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid; enriched talc, stearate, boric acid powder, polyethylene glycol, etc. Agents and the like can be used. Further, the tablet may be a tablet coated with a usual coating, for example, a sugar-coated tablet, a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, or a double tablet or a multilayer tablet, if necessary.

In the above composition, for parenteral administration, the bovine FSH promoter of the present invention can be formulated according to the usual formulation practice using pharmaceutically acceptable excipients well known in the art. ..

Examples of the water-soluble excipient for injection include physiological saline, isotonic solution containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and the like. You may use together with a solubilizing agent, for example, alcohol, specifically ethanol, polyalcohol, for example, propylene glycol, polyethylene glycol, a nonionic surfactant, for example, Polysorbate 80(TM), HCO-50.

Oily excipients include sesame oil and soybean oil, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. Further, a buffering agent such as a phosphate buffer solution, a sodium acetate buffer solution, a soothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol, phenol, and an antioxidant may be added. The prepared injection solution is usually filled in a suitable ampoule.

For the purpose of controlling the dispersion of the bovine FSH promoter of the present invention, a carrier such as collagen, hydroxyapatite, hyaluronic acid, fibrin, polysaccharide, aluminum hydroxide gel or liposome may be added. The prepared preparation can be used as an oral preparation or a parenteral preparation.

The administration route of the bovine FSH promoter of the present invention is not particularly limited, and may be oral, rectal, transmucosal, intestinal, intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, vitreous. Includes body, intraperitoneal, intranasal, or intraocular injection. The administration route can be appropriately selected in consideration of the age and medical condition of the sick animal, other drugs used in combination, and the like.

The dose of the follicle-stimulating hormone secretagogue of the present invention may be any amount capable of promoting FSH secretion, and can be appropriately determined depending on the administration method, age, symptoms and the like. For example, based on the results of this example, it is preferable that the plasmalogen concentration in bovine blood is 1 to 2000 ng/L, preferably 3 to 1000 ng/L, and more preferably 5 to 500 ng/L.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the technical scope of the present invention is not limited to these exemplifications.

1. Preparation of Anterior Pituitary Cells Anterior pituitary cells were obtained from 26-month-old mid-luteal phase Japanese Black calves (n=10) after sexual maturation. The obtained anterior pituitary cells were enzymatically dispersed using collagenase, hyaluronidase and deoxyribonuclease according to the method of Hashizume et al. (Biology of Reproduction, Volume 64, Issue 3, 1 March 2001, Pages 898-903). The viable cell ratio of the dispersed cells was confirmed to be 90% or more by trypan blue staining.

Next, the anterior pituitary cells dispersed above were treated with 10% horse serum (produced by Thermo Fisher Scientific), 2.5% FBS, 1% non-essential amino acid (produced by Thermo Fisher Scientific), 100 IU/mL penicillin, 100 μg/ The cells were suspended in Dulbecco's modified Eagle medium (DMEM) medium (Thermo Fisher Scientific) containing mL streptomycin and 2.5% fetal bovine serum.

The suspended cells (2.5×10 ⁵ cells/mL, 0.3 mL in total) were seeded in a 48-well plate (Sumitomo Bakelite Co., Ltd.), and a microscope observation chamber (μ-Slide VI 0.4, Ibidi Co., Ltd.) was used. It was cultured for 82 hours under the conditions of 5% CO ₂ and 37°C. When using cultured cells, recombinant human activin A (final concentration 10 ng/mL: manufactured by R&D systems) was added 24 hours before the addition of plasmalogen to stimulate FSH synthesis.

2. Secretion of FSH In order to evaluate the effect of plasmalogen on the secretion of FSH in the absence of GnRH, the DMEM medium after the 82-hour culture was replaced with 295 μL of DMEM medium containing 0.1% BSA and 10 ng/mL activin A. And cultured for 2 hours. Next, 5 μL of DMEM alone or various concentrations of bovine brain-derived plasmalogen (hexadecanyl group derived from stearic acid or hexadecenyl group derived from oleic acid via a vinyl ether bond at the 1-position of the glycerol skeleton (of hexadecanyl group and hexadecenyl group The ratio is about 1:1.27) and has a hexadecanyl group derived from stearic acid or a hexadecenyl group derived from oleic acid via an ester bond at the 2-position of the glycerol skeleton: final concentration 0, 5, 50, 500, 5 μL of DMEM containing 5,000, 50,000 pg/mL: Product no 39-0200: manufactured by Larodan) was added, and the mixture was further cultured for 2 hours. After culturing, the medium in each well was collected for radioimmunoassay (RIA) of FSH levels.

<RIA analysis>
The FSH concentration in the medium recovered above was ¹²⁵ I-labeled bFSH and anti-oFSH antiserum (AFP5318C, AFP5346D, and AFPC5288113, National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases: NIDDK). Double analysis was carried out by the double antibody RIA. The detection limit value was 0.20 ng/mL.

FIG. 1 shows the results of examining the amount of FSH secretion from cultured bovine anterior pituitary cells after sexual maturation, when various concentrations of plasmalogen derived from bovine brain were administered. In the figure, the X-axis is the added plasmalogen concentration, and the Y-axis is the plasma derived from bovine brain at each concentration with respect to the FSH concentration in the culture solution without plasmalogen (0 pg/mL: Control). The ratio of the FSH concentration in the culture solution when a logen is added is shown. As shown in FIG. 1, plasmalogen increased FSH secretion at 5,50,500,5000 pg/mL and particularly significantly increased FSH secretion at 50,500 pg/mL (P<0. 05). Although not shown in the figure, even in the presence of GnRH, FSH secretion was increased at 5,50 pg/mL of plasmalogen compared with the control, and particularly FPS secretion was significantly increased at 50 pg/mL (P< 0.05).

From the above results, it was revealed that addition of plasmalogen extracted from bovine brain stimulates FSH secretion from bovine anterior pituitary cells. Further, it was revealed that such FSH secretion is not dependent on GnRH, that is, the secretion of FSH can be stimulated without GnRH. Considering the analysis of gonadotroph cells by the present inventors, it is considered that this FSH secretion was caused by the activation of intracellular transduction pathway of GnRHR by plasmalogen. Therefore, even immediately after parturition in which GnRH is hardly secreted in vivo, it is possible to activate the intracellular transduction pathway of GnRHR and secrete FSH by adding plasmalogen extracted from bovine brain. It has been suggested.

Further, since it works with a very small amount of 5 pg/mL and 50 pg/mL, it is highly practical from the viewpoint that the dosage is small and various administration methods can be selected at low cost.

FSH is an important hormone that regulates postpartum recovery of the ovary and uterus, estrus clarification, and conception promotion. Since FSH secretion from bovine gonadotroph cells can be stimulated by plasmalogen extracted from bovine, the present invention can be used for treatment of superovulation and reproductive disorders in cattle.

Claims (4)

A bovine follicle-stimulating hormone secretagogue containing plasmalogen extracted from bovine as an active ingredient. The bovine follicle-stimulating hormone secretagogue according to claim 1, wherein the plasmalogen is extracted from bovine brain. Plasmalogen is ethanolamine plasmalogen, The follicle-stimulating hormone secretagogue according to claim 1 or 2, characterized in that the plasmalogen is ethanolamine plasmalogen. The bovine follicle-stimulating hormone secretagogue according to any one of claims 1 to 3, which has a hexadecyl group or a hexadecenyl group through a vinyl ether bond at the 1-position (sn-1 position) of the glycerol skeleton in plasmalogen. ..