KR100995382B1 - Method for Screening Endocrine Disrupter Using Sperm - Google Patents

Abstract

The present invention relates to a method for selecting an environmental hormone estimation material by culturing the environmental hormone estimation material with the sperm of a pig and then measuring the ratio of sperm showing fertility gain or acrosome reaction. According to the method of the present invention, the environmental hormone estimating material can be screened at high speed / in bulk in a simple and economic manner.

Environmental hormones, sperm, mammal, fertility gain, acrosome reaction, screening method

Description

Screening method for environmental hormone using sperm {Method for Screening Endocrine Disrupter Using Sperm}

The present invention relates to a method for screening environmental hormones using sperm of an animal and to a composition for screening environmental hormones comprising sperm of an animal.

The basic concept of classical toxicity assessments to date has been to observe biotoxicological changes with high exposures. In the future, however, toxicological assessments in real life, such as trace and residual and long-term exposures, will be the focus. An example would be an example of an environmental hormone represented by a chemical that causes endocrine dysfunction following prolonged exposure.

Originally, the World Wildlife Fund (WWF), which began to point out the dangers of environmental hormones in earnest from the 1990s, selected 68 types of environmental hormones exposed to nature. In addition, about 80,000 chemicals used commercially in the United States have not yet been tested for environmental hormone activity, and are currently searching for environmental hormones known to date. However, because hundreds of thousands of chemicals are synthesized each year, it is not yet known how many environmental hormones exist in nature.

It has been known that these environmental hormones have a surprisingly large impact on our ecosystem and humans. In the 1990s, it was revealed that the reproductive and immune functions are weakened, abnormal behaviors and increased cancer incidence. In addition, the hormonal distinction of sex itself goes beyond mere weakening of the reproductive function by environmental hormones.

Observing these phenomena, the US Environmental Protection Agency has formed a committee called the Endocrine Disrupters Screening and Testing Advisory Committee (EDSTAC) to screen environmental hormones, as well as studies on the types, levels, sources, and pathways of environmental hormones, as well as their impact on wildlife populations. Research is being conducted to examine the effects of hormones.

In Korea, research on environmental hormones has begun, and research on developing test methods and search programs to identify specific environmental hormones is underway, and the Environmental Hormone Countermeasure Council, which is organized by the Ministry of Environment, has also begun activities.

Currently, more than 80,000 chemicals suspected of environmental hormones are in the process for screening for their effects as environmental hormones. One substance is tested for toxicity and identified as an environmental hormone. About 600-1200 experimental animals are used. Screening for one environmental hormone costs between $ 2 and $ 4 million. Currently, a budget of 320 billion won is needed to conduct screening tests for environmental hormones. Also, considering the chemicals to be produced in the future, huge economic costs must be paid.

Since most of the environmental hormones are chemicals, their impact on the industry that handles, produces or consumes them can be an immediate economic impact. Therefore, the establishment of environmental hormone detection screening system will minimize the economic loss.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have conducted research to establish a system capable of mass / fast screening of environmental hormone substances at low cost and high efficiency, and found that the sperm of an animal exhibits fertility or acrosome reaction when exposed to environmental hormones. Based on the findings, the present inventors completed the present invention by experimentally confirming that the environmental hormone estimation substance can be effectively selected by exposing the environmental hormone estimation substance to the sperm of the animal and then measuring the ratio of sperm showing fertility or acrosome reaction. .

It is therefore an object of the present invention to provide a method for screening environmental hormone estimating compounds using animal sperm.

Another object of the present invention is to provide a composition for screening an environmental hormone estimating compound comprising an animal sperm as an active ingredient.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a method for screening an environmental hormone estimating compound comprising the following steps:

(a) incubating the sperm of the animal with a compound suspected of an environmental hormone; (b) measuring the change in fertility gain or acrosome reaction of the sperm cultured in step (a); And (c) selecting a compound that increases the fertility gain or the ratio of the acrosome reaction sperm.

The present inventors have conducted research to establish a system capable of mass / fast screening of environmental hormone substances at low cost and high efficiency, and found that the sperm of an animal exhibits fertility gain or acrosome reaction when exposed to environmental hormone substances. Based on the findings, the present invention was completed by experimentally confirming that the environmental hormone estimating substance is exposed to the sperm of an animal and then measuring the ratio of sperm showing fertility or acrosome reaction to effectively select the environmental hormone estimating substance. It was.

Hereinafter, the method of the present invention will be described in detail according to the steps.

(a) culturing the animal's sperm with a compound suspected of an environmental hormone

In the present invention, the sperm is used as a sperm separated from an animal. The sperm used in the present invention can be used from any species of animal. It is preferably a sperm of a mammal, which includes but is not limited to bovine, equine, porcine, ovine, elk and bison. Most preferably, sperm derived from pigs is used. Among the sperm of the animal, the pig's sperm responds most sensitively to environmental hormones, so it shows a change in fertility gain or acrosome reaction.

Sperm used in the present invention are normally viable sperm. Thus, at least 50%, preferably at least 75%, of sperm used in the present invention are morphologically normal. In addition, at least 40%, preferably at least 60%, of sperm used in the present invention exhibit progressive motility.

As used herein, the term "environmental hormone" refers to a substance whose chemical structure is similar to that of a biological hormone, which disrupts or affects the function of a normal hormone when introduced into an organism. More specifically, the term "exogenous endocrine disruptor" Or "endocrine obstruction."

The expression "compound presumed to be an environmental hormone" used in the present invention means a compound which is predicted to act as an environmental hormone, and is a compound to be selected by the method of the present invention.

According to a preferred embodiment of the present invention, the compound that is assumed to be an environmental hormone is a compound that exhibits estrogen or progesterone-like activity. Preferably, a compound which is predicted to exhibit a similar activity to estrogen or progesterone and thus acts as a sex hormone upon in vivo is a compound to be screened by the screening method of the present invention.

In the method of the present invention, the method of culturing the sperm of the animal together with the compound estimated to be an environmental hormone includes an sperm compound after adding the environmental hormone estimating compound to the culture medium in which the sperm of the animal can survive and remain. It is carried out by culturing to expose for a certain time.

The time to incubate the sperm and environmental hormones of the animal together is sufficient time for the environmental hormones to affect and affect the sperm is not limited to a specific time. Preferably 1 minute-1 hour, more preferably 10-40 minutes, most preferably 15-30 minutes.

(b) measuring the fertility acquisition or acrosome reaction of the sperm cultured in step (a)

As used herein, the term "capacitation" refers to certain changes, such as maturation or activity and processes that the sperm undergo to develop the ability to fertilize the egg. Sperm for which fertility has not been obtained significantly decreases the ability to fertilize an egg. Fertility gain can be confirmed through changes occurring inside the sperm, such as changes in mitochondria tissue in the tail of the sperm or changes occurring on the cell surface. Fertility gains include physical characteristics (total athleticism, forward athleticism, rapid athleticism), kinematic parameters (linear velocity, average path velocity, curve velocity, and average displacement of the lateral head) and channel activator, A23187 ionophore It can be measured by the conversion to acrosome reaction ability caused by the stimulation by ionophore and the like.

In the present invention, the term "acrosome" refers to a cap portion that occupies the front half of the head of a sperm and includes enzymes necessary for permeation of eggs. .

As used herein, the term "acrosome reaction" refers to a reaction that occurs in the acrosome while the sperm approaches the zona pellucida of the egg. When the sperm approaches the egg, the membrane surrounding the sperm's acrosome is fused with the plasma membrane of the sperm to secrete the contents containing enzymes inside the acrosome so that the sperm can fuse with the egg.

In the method of the present invention, the rate of increase in sperm that causes fertilization-acquired sperm or acrosome after exposure to an environmental hormone predicting substance using animal sperm is measured.

According to a preferred embodiment of the present invention, the sperm acquisition in step (b) or the measurement of the change in acrosome reaction is carried out by a chlortetracycline (CTC) staining method. In the method of the present invention, the determination of fertilization-acquired sperm or acrosome reaction sperm can be performed using a chlortetracycline fluorescence assay known in the art. Sperm chlortetracycline (CTC) staining is a method used to measure various physiological changes in sperm, including sperm motility, viability, capacitation, and acrosome reaction. (Journal of Andrology, Vol 21, Issue 6 938-943; Zygote. 2006 Aug; 14 (3): 259-73; Hum Reprod. 2006 Jun; 21 (6): 1555-63).

(c) screening for compounds that acquire fertility or increase the proportion of acrosome reaction sperm

The environmental hormone estimating compound to be screened in the method of the present invention is a potential environmental hormone compound as a compound having the ability to promote fertilization or acrosome reaction of animal sperm. The ability of sperm to acquire fertility or acrosomal reaction is the ability of the biological hormone, typically estrogen or progesterone, to be expected to function similarly to biohormones when introduced into the body. . Therefore, in the method of the present invention, a compound that increases the number of sperm that exhibits this reaction by measuring fertility-acquired sperm or acrosome reaction sperm is judged and selected as a potential environmental hormone.

According to another aspect of the invention, there is provided a composition for screening environmental hormone estimation compound comprising sperm of the animal as an active ingredient.

According to a preferred embodiment of the present invention, the animal in the present invention is a mammal, more preferably bovine, equine, porcine, sheep, elk and bison (bison) any one animal selected from the group consisting of, most preferably pigs.

According to another preferred embodiment of the present invention, the environmental hormone estimating compound is a compound exhibiting estrogen or progesterone-like activity.

The present invention relates to a method for selecting an environmental hormone estimating substance by culturing the environmental hormone estimating substance with an animal sperm and measuring a ratio of sperm showing fertility gain or acrosome reaction. According to the method of the present invention, a screening method of environmental hormone substances in a simple and economical manner at a high speed and in large quantities, studies on the mechanism of action of environmental hormone substances, development of environmental hormone blocking material and alternative substances, and prevention of environmental pollution Can be used effectively in research.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

1. Preparation of pig and cow semen

In the method of the present invention, pig liquid semen and bovine frozen semen were used.

2. Treatment of environmental hormones

In the method of the present invention, estrogen (E2), progesterone (P4), Genistein (GEN), and 4-tertocylphenol (0P), which are used hormones or environmental hormone substances, are adjusted to a concentration of 0.001 μl / L-100 μl / L in the culture medium. Was used. The liquid semen from pigs was divided into 1 ml each, diluted to an appropriate concentration, and then hormone or environmental hormone estimating substances were added thereto. Hormone-added semen-containing solutions were incubated for 15 or 30 minutes in a 5% carbon dioxide gas incubator.

The frozen frozen semen of the cow was thawed and diluted to more than about 50 million birds per sample with diluent, and then treated in the same manner as the liquid liquid pig.

3. Acquisition of fertilization ability or measurement of additive reaction sperm using CTC method

To the semen (2 × 10 ⁸ cell / ml) solution was added 135 μl of PBS and 15 μl of H258 solution. This solution was then incubated at 37 ° C. for 10 minutes, after which 250 μl of 2% v polyvinylpyrolidone (polyvinylpyrrolidone) was added and centrifuged at 400 × g for 10 minutes. The supernatant was removed from the centrifuged sample and 500 μl of CTC (chlortetracycline) solution and 500 μl PBS were added. After 20 seconds, 10 μl of glutaraldehyde solution was added thereto, and the mixture was left in a dark place at 4 ° C. and confirmed by fluorescence microscopy.

Observed sperm are: i) dead sperm (the head of the D-sperm is blue), ii) live noncapacitated; Fluorescence staining is not strongly expressed in the equatorial region of iv), i) live capacitated; B-the acrosome of the sperm head is expressed in light green and the aterior acrosome is dark. Iv) live acrosome (AR-where green expressing material appears like spots on the head and the green expressing material is only in the posterior arcrosome or no fluorescence is present in the head) The evaluation was based on four distinct pattern criteria.

4. Experimental Results

(1) Results of experiments in which swine sperm were exposed to estrogen

Porcine sperm was exposed to estrogen (E2) at 0.001-100 μmol / L for 15 minutes and 30 minutes, respectively, and sperm gain and acrosome responses were measured. The results are shown in panels A and C of FIG.

In the graphs of all the following figures, F represents intact sperm, B represents capacitated sperm, and AR represents acrosome reacted sperm.

In panel A of FIG. 1, after sperm exposure to estrogen (E2) for 15 minutes, sperm showing fertilization gain and acrosome reaction were measured. According to this result, it was confirmed that the ratio of fertilization gain and acrosome reaction sperm was increased from the concentration of 0.001 μmol / L. This indicates that pig sperm tofu is being changed by estrogen.

Panel C of Figure 1 shows the results of measuring the sperm showing the fertilization gain and acrosome reaction after the pig sperm exposed to estrogen (E2) for 30 minutes. As in the case of the 15-minute exposure, it was confirmed that the ratio of acquiring fertilization and acrosome reaction sperm was increased from the concentration of 0.001 μmol / L.

(2) Results of experiment that exposed bovine sperm to estrogen

After bovine sperm was exposed to estrogen (E2) at 0.001-100 μmol / L for 15 minutes and 30 minutes, respectively, sperm gain and measurement of acrosome response were measured. It is shown in D.

Panel B of FIG. 1 shows the results of measuring sperm showing fertility gain and acrosome reaction after 15 minutes exposure of small sperm to estrogen (E2). The results showed that the ratio of sperm gaining sperm did not increase, but the sperm showing acrosome reaction increased at some concentrations.

Panel D of FIG. 1 shows the results of measuring sperm showing fertility gain and acrosome reaction after 30 minutes exposure of small sperm to estrogen (E2). As in the case of 15-minute exposure, it was confirmed that the sperm showing the acrosome reaction increased at some concentrations.

(3) Results of experiments in which swine sperm were exposed to progesterone

The results of observing sperm fertilization gain and acrosome response after exposing pig sperm to progesterone (P4) at 0.001-100 μmol / L concentration for 15 minutes and 30 minutes, respectively, are shown in panel A and panel C of FIG. 2. .

Panel A of FIG. 2 shows the results of measuring sperm showing fertilization gain and acrosome response after 15 minutes of exposure to pig sperm to progesterone (P4). According to this result, it was confirmed that the ratio of fertilization gain and acrosome reaction sperm was increased from the concentration of 0.001 μmol / L. In particular, the ratio of acrosome reaction sperm increase was greater than that of fertility sperm.

Panel C of Figure 2 shows the results of measuring the sperm showing the fertilization gain and acrosome reaction after 30 minutes exposure to pig sperm to progesterone (P4). As in the case of 15-minute exposure, the ratio of fertilization gain and acrosome reaction sperm was increased from 0.001 μmol / L concentration. It appeared greatly.

(4) Results of experiments in which bovine sperm were exposed to progesterone

The results of measuring the ratio of sperm showing fertility gain and acrosome reaction after 15 min and 30 min exposure of bovine sperm to 0.001-100 μmol / L concentration of progesterone (P4) are shown in panel B and panel D of FIG. 2, respectively. It was.

Panel B of FIG. 2 shows the results of measuring sperm showing fertility gain and acrosome reaction after 15 minutes exposure of small sperm to progesterone (P4). According to this result, the ratio of sperm obtained from fertilization did not increase significantly, but it was observed that the ratio of sperm showing acrosome reaction at some concentrations increased.

Panel D of FIG. 2 shows the results of measuring sperm showing fertility gain and acrosome reaction after 30 minutes exposure of small sperm to progesterone (P4). As with the 15 minute exposure, the percentage of fertilization gained sperm did not increase significantly, but at some concentrations it was observed that the percentage of sperm showing acrosome reactions increased.

(5) Pig sperm to environmental hormone Exposed  Results of the experiment

Fig. 3 shows the results of measuring the changes in sperm capacities and acrosome reactions after the sperm were exposed to representative environmental hormones Genistein and 4-tertocylphenol for 15 minutes and 30 minutes, respectively, for 15 and 30 minutes.

Panel A of FIG. 3 shows a result of measuring sperm showing fertilization gain and acrosome reaction after exposing pig sperm to Genistein for 15 minutes. According to this result, fertility gain was increased at most concentrations, and the ratio of acrosome reaction sperm increased from 0.001 μmol / L concentration.

Panel B of Figure 3 shows the results of measuring the sperm showing the fertilization gain and acrosome reaction after the pig sperm exposed to Genistein 30 minutes. According to these results, fertility gain was slightly increased at several concentrations and the ratio of acrosome reaction sperm increased significantly from 0.001 μmol / L concentration.

Panel C of FIG. 3 shows the results of measuring sperm showing fertilization gain and acrosome reaction after exposing pig sperm to 4-tertocylphenol for 15 minutes. The results showed that the proportion of fertilization sperm did not increase, but the proportion of acrosome reaction sperm increased at all concentrations.

Panel D of Figure 3 shows the results of measuring the sperm showing the fertilization gain and acrosome reaction after the pig sperm exposed to 4-tertocylphenol for 30 minutes. The results showed that fertility gain was slightly increased at some concentrations, and the ratio of acrosome reaction sperm increased significantly from 0.001 μmol / L.

According to the above experimental results, it was confirmed experimentally that the environmental hormone estimation material can be selected very efficiently using a sperm of an animal.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Figure 1 shows porcine sperm and bovine sperm exposure to estrogen (E2) concentration of 0.001-100 μmol / L for 15 minutes and 30 minutes, respectively, to obtain sperm fertility and change in acrosome response. A graph showing one result. F stands for intact sperm, B stands for capacitated sperm, and AR stands for acrosome reacted sperm.

Figure 2 is a graph of the results of sperm fertilization gain and acrosome reaction after sperm and bovine sperm exposed to progesterone (P4) concentration of 0.001-100 μmol / L for 15 minutes and 30 minutes, respectively. F stands for intact sperm, B stands for capacitated sperm, and AR stands for acrosome reacted sperm.

Figure 3 shows the results of measuring the changes in the sperm capacities and acrosome reaction after the pig sperm exposed to representative environmental hormones Genistein and 4-tertocylphenol for 15 minutes and 30 minutes respectively. F stands for intact sperm, B stands for capacitated sperm, and AR stands for acrosome reacted sperm.

Claims (9)

Screening method of environmental hormone estimating compound comprising the following steps: (a) incubating the sperm of the animal with a compound suspected of an environmental hormone; (b) measuring the change in fertility gain or acrosome reaction of the sperm cultured in step (a); And (c) screening for compounds that increase fertility gain or ratio of acrosome reaction sperm. The method of claim 1 wherein the animal of step (a) is a mammal. The method of claim 2, wherein the mammal is any one selected from the group consisting of bovine, equine, porcine, sheep, elk and bison. How to. The method of claim 1, wherein the environmental hormone estimating compound is an estrogen or progesterone-like compound. The method according to claim 1, wherein the acquisition of sperm fertilization or change in acrosome reaction of the sperm of step (b) is performed by a chlortetracycline (CTC) staining assay. Environmental hormone estimation compound screening composition comprising the sperm of the animal as an active ingredient. 7. The composition of claim 6, wherein said animal is a mammal. The method of claim 7, wherein the mammal is any one selected from the group consisting of bovine, equine, porcine, sheep, elk and bison. Composition. 7. The composition according to claim 6, wherein the environmental hormone estimating compound is a compound showing estrogen or progesterone-like activity.

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