Applications of Ackermania muciniphila and its metabolite butyric acid in preparation of drugs for relieving HPA stress axis and HPG reproductive axis disorder

Abstract

The invention discloses Akkermansia muciniphila and application of a metabolite butyric acid of the Akkermansia muciniphila in preparation of drugs for relieving reproductive disorder of hypothalamus-pituitary-adrenal axis (HPA axis) and hypothalamus-pituitary-gonad axis (HPG axis), and develops a new function of the strain for relieving reproductive injury. The strain inhibits the HPA axis activity and reduces the stress hormone level by secreting butyric acid, so that the HPG axis function is activated, the reproductive system injury of ICR mice caused by high-density crowding stress is remarkably improved, and the sperm density is increased, the reproductive hormone level is increased, and the testis structure is improved. In addition, as a core metabolite of the Akkermansia muciniphila, the butyric acid can independently mediate the regulation and control process. The invention provides a new mechanism of animal population density-dependent reproductive homeostasis mediated by butyric acid, provides a potential micro-ecological intervention strategy for coping with reproductive capacity decline caused by social stress, and provides a new thought and method for treating animal and human reproductive injury.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and more particularly, to the application of Akkermansia muciniphila ( Akkermansia muciniphila ) and its metabolite butyric acid in the preparation of drugs for alleviating the disorders of the HPA stress axis and the HPG reproductive axis. Background Art

[0002] In natural ecosystems, animal population densities exhibit dynamic fluctuation characteristics. Many internal factors (such as intraspecific competition) and external factors (such as food resources, interspecific competition, climate, and predation) can regulate population abundance. Density dependence is a key mechanism in population ecology for regulating and stabilizing population density (size). Due to resource scarcity, high-density crowding leads to increased social stress in animals, which in turn causes a decline in immune function, reproductive performance, and survival rate through accelerated aging, thus inducing a decline in animal population numbers.

[0003] The inhibitory effect of high-density populations on the reproduction of small rodents has been widely studied. For example, in wild small rodents, during high-density periods, changes such as increased adrenal gland weight, decreased testis weight, and reduced sex hormone secretion have been observed in Brandt's voles and red-backed voles; in livestock farming, intensive high-density livestock farming leads to a decline in their reproduction and survival rate. In addition, in recent years, as urban population density has continued to rise, the social stress and mental illness risks faced by humans have increased, and the decline in male semen quality and infertility may be closely related to psychological disorders such as anxiety and depression induced by high density. Therefore, the development of new intervention measures to effectively alleviate reproductive damage caused by high-density crowding stress not only has important ecological value for animal population regulation but also provides a new research perspective for maintaining human reproductive health and has broad application prospects.

[0004] Previous studies have mostly emphasized the inhibitory effect of social stress induced by high-density crowding on the hypothalamic-pituitary-gonadal axis (HPG axis) through the activation of the hypothalamic-pituitary-adrenal axis (HPA axis). Specifically, when faced with social stress, the hypothalamus secretes corticotropin-releasing hormone (CRH), which stimulates the pituitary gland to release adrenocorticotropic hormone (ACTH), resulting in the adrenal gland secreting glucocorticoids (GC; including cortisol in primates or corticosterone [CORT] in birds and rodents). The secretion of CRH inhibits the pulsatile release of gonadotropin-releasing hormone (GnRH) from the hypothalamus and reduces the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary gland. In addition, GC inhibits the release of various components of the HPG reproductive axis by directly acting on GnRH neurons, FSH, LH, and sex hormones (such as testosterone [T]).

[0005] With the rapid development of high-throughput sequencing technology, microbiome research has shown that the gut microbiota plays an important role in regulating the reproductive process of animals, such as affecting testicular morphological development, spermatogenesis, and the secretion of reproductive-related hormones. Among them, Akkermansia muciniphila ( Akkermansia muciniphila ) as the dominant bacterium in the intestinal mucus layer plays a core role in maintaining the function of the intestinal mucosal barrier by decomposing mucin and promoting its mucus regeneration. Existing studies have shown that this bacterium and its key metabolite butyric acid (a typical representative of short-chain fatty acids) can not only effectively relieve anxiety-like behaviors and reduce the levels of pro-inflammatory factors, but also enhance the intestinal barrier function and regulate neurotransmitter balance, and are regarded as potential microbial and metabolite targets for anti-stress intervention. However, Akkermansia muciniphila the roles and molecular mechanisms of and butyric acid in density-dependent reproduction regulation have not been elucidated.

[0006] The purpose of the present invention is to provide an application of Akkermansia muciniphila ( Akkermansia muciniphila ) and its metabolite butyric acid in the preparation of hormone drugs for regulating the HPA stress axis.

[0007] The present invention discloses an application of Akkermansia muciniphila ( Akkermansia muciniphila ) and its metabolite butyric acid in the preparation of drugs for relieving the HPA stress axis.

[0008] Furthermore, the stress-related hormones are corticotropin-releasing hormone, adrenocorticotropic hormone, and corticosterone.

[0009] Another purpose of the present invention is to disclose an application of Akkermansia muciniphila ( Akkermansia muciniphila ) and its metabolite butyric acid in the preparation of drugs for relieving the dysfunction of the HPG reproductive axis.

[0010] Furthermore, the reproductive-related hormones are gonadotropin-releasing hormone, follicle-stimulating hormone, luteinizing hormone, and testosterone.

[0011] Furthermore, the Akkermansia muciniphila is preserved in the American Type Culture Collection (ATCC) with the preservation number BAA-835, and the concentration of Akkermansia muciniphila in the drug is at least 1×10 9 [CFU] / 0.2 mL PBS. The active ingredients of the drug include an effective amount of the viable or dead form of Akkermansia muciniphila, its isolates, and / or secretions.

[0012] Furthermore, the metabolite butyric acid includes sodium butyrate and excipients. The dose of butyric acid is 500 mg / kg, and the excipient is physiological saline with a dosage of 0.2 mL.

[0013] Furthermore, the steps for preparing the butyric acid solution are as follows: Weigh 16.875 g of butyric acid and dissolve it in 150 mL of physiological saline. Stir with a glass rod until it is completely dissolved to prepare the butyric acid solution.

[0014] Furthermore, the Akkermansia muciniphila and the butyric acid solution are administered orally.

[0015] Taking social stress as the starting point, the present invention systematically explores for the first time the Akkermansia muciniphila Akkermansia muciniphila and the improvement effect of its metabolite butyric acid on reproductive damage caused by social stress, and through simultaneously investigating the synergistic effect of viable bacteria colonization and butyric acid supplementation, conducts research from multiple dimensions such as sperm density, testicular tissue morphology, and hormone levels. Specifically, the Akkermansia muciniphila Akkermansia muciniphila inhibits the excessive activation of the HPA axis in ICR mice induced by high-density social stress by producing butyric acid, reduces the levels of stress-related hormones (CRH, ACTH, CORT), and then activates the HPG axis, promotes the secretion of reproductive hormones such as (GnRH, FSH, LH, T), and finally increases sperm density and improves testicular structural abnormalities. This study not only fills the blank in the application under social stress Akkermansia muciniphila but also provides a theoretical basis for the development of precision microbiotherapy through the targeted research on the metabolite butyric acid, and has the potential of translational medicine.

[0016] Beneficial effects of the present invention: (1) Inhibiting stress and promoting reproduction: The present invention discovers that Akkermansia muciniphila and its metabolite butyric acid can significantly inhibit the activation of the HPA axis, reduce the levels of stress-related hormones (CRH, ACTH, CORT), and significantly up-regulate the secretion of reproductive-related hormones (GnRH, FSH, LH, T), increase sperm density, and improve the morphology of seminiferous tubules, thereby effectively alleviating the anxiety behavior and social stress of ICR mice under high-density crowding stress and improving their overall reproductive health.

[0017] (2) Mechanism innovation: The present invention systematically reveals for the first time the Akkermansia muciniphila improvement effect and mechanism of and its metabolite butyric acid on reproductive damage induced by social stress, and through simultaneously investigating the synergistic effect of viable bacteria colonization and butyric acid supplementation, reveals from multiple dimensions such as sperm density, testicular tissue morphology, and hormone levels the Akkermansia muciniphila new mechanism of inhibiting the HPA axis and activating the HPG axis by producing butyric acid, providing a new intervention target for animal and human social stress-related reproductive disorders.

[0018] (3) Application value: The present invention clearly defines for the first time the Akkermansia muciniphila –butyric acid–HPA / HPG axis cascade regulation mechanism, for the development based onAkkermansia muciniphila Provide a theoretical basis for microecological preparations with butyric acid, the core component of metabolites, and it is expected to be applied in the future to alleviate reproductive disorders in livestock and humans caused by social stress. Description of the Drawings

[0019] Figure 1 For orally administering to high-density stressed mice Akkermansia muciniphila Changes in the butyric acid content in the colon feces after 4 weeks. The results are expressed as the mean ± standard error of the mean (SEM), (* P <0.05, ** P <0.01, *** P <0.001) indicates that the data is statistically significant (the same below); Figure 2 For orally administering to high-density stressed mice Akkermansia muciniphila And the change in the time to enter the central area of the open field after 4 weeks of butyric acid. Among them, A and B are respectively Akkermansia muciniphila The time for high-density stressed mice in the colonization experiment and the butyric acid oral administration experiment to enter the central area of the open field; Figure 3 For orally administering to high-density stressed mice Akkermansia muciniphila And the change in sperm density after 4 weeks of butyric acid. Among them, A and B are respectively Akkermansia muciniphila The sperm density of high-density stressed mice in the colonization experiment and the butyric acid oral administration experiment; Figure 4 For orally administering to high-density stressed mice Akkermansia muciniphila And the results of hematoxylin & eosin (H&E) staining of testicular tissues after 4 weeks of butyric acid. A and B are respectively the H&E staining results of group AB mice magnified 10 times and 20 times, C and D are respectively the H&E staining results of group AKK mice magnified 10 times and 20 times, E and F are respectively the H&E staining results of group SA mice magnified 10 times and 20 times, G and H are respectively the H&E staining results of group BA mice magnified 10 times and 20 times; Figure 5 For orally administering to high-density stressed mice Akkermansia muciniphila And the results of the area, perimeter, diameter of seminiferous tubules and the thickness of the germinal epithelium in the testis after 4 weeks of butyric acid. Among them, A, B, C, D are respectively Akkermansia muciniphila The area, perimeter, thickness of the germinal epithelium and diameter of seminiferous tubules in the testis of mice in the colonization experiment, and E, F, G, H are respectively the area, perimeter, thickness of the germinal epithelium and diameter of seminiferous tubules in the testis of mice in the butyric acid oral administration experiment; Figure 6 For orally administering to high-density stressed mice Akkermansia muciniphila And the stress-related hormone results after 4 weeks of butyric acid. Among them, A, B, C are respectively Akkermansia muciniphilaResults of corticotropin-releasing hormone, adrenocorticotropic hormone, and corticosterone in colonized experimental mice. D, E, and F are the results of corticotropin-releasing hormone, adrenocorticotropic hormone, and corticosterone in experimental mice orally administered butyric acid, respectively; Figure 7 For orally administered to high-density stress mice Akkermansia muciniphila and the results of reproductive-related hormones after 4 weeks of butyric acid administration. Among them, A, B, C, and D are Akkermansia muciniphila the results of gonadotropin-releasing hormone, follicle-stimulating hormone, luteinizing hormone, and testosterone in colonized experimental mice. E, F, G, and H are the results of gonadotropin-releasing hormone, follicle-stimulating hormone, luteinizing hormone, and testosterone in experimental mice orally administered butyric acid, respectively. Specific embodiments

[0020] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Unless otherwise specified, the experimental methods, reagents, and equipment used in the present invention are conventional methods, reagents, and equipment in the technical field.

[0021] Example 1 Establishment of animal model: Sixty male SPF-grade ICR mice weighing 35–40 g were purchased from Henan Skobes Biotechnology Co., Ltd. The mice were placed in polycarbonate ventilated cages (6 mice / cage) and raised for 8 weeks at a room temperature of 23 ± 1°C, a relative humidity of 55 ± 5%, and a light cycle of 14 h light: 10 h dark cycle (hereinafter referred to as "high-density stress mice") to establish a high-density crowded stress mouse model. During the high-density treatment period, the mice could freely ingest standard mouse food (Beijing Keao Biotechnology Co., Ltd.) and pure water.

[0022] Example 2 Akkermansia muciniphila Colonization experiment Thirty-six high-density stress mice were selected for Akkermansia muciniphila the colonization experiment. To provide Akkermansia muciniphila space for colonization and release, 1 week before gavage Akkermansia muciniphila , part of the original intestinal microorganisms in 36 high-density stress mice were cleared by continuously transplanting a fresh broad-spectrum antibiotic mixture (streptomycin 50 μg / mL, neomycin 100 μg / mL, penicillin 100 μg / mL; 200 μL / day for 7 days). Subsequently, the 36 high-density stress mice were randomly divided into two groups: the antibiotic transplantation group (AB group) and Akkermansia muciniphila the colonization group (AKK group) (18 mice in each group). The AB group was gavaged with 200 μL of PBS (once a day for 4 weeks), and the AKK group was gavaged with 200 μL Akkermansia muciniphila (1 × 10 9[CFU] / 0.2 mL PBS; once a day for 4 weeks). On the last day of gavage treatment, the open-field behavior of the experimental mice was recorded using a camera from 8:00 to 10:00 in the morning; fresh feces of the experimental mice were collected from 10:00 to 12:00 and stored at -80 °C for further determination of butyric acid content. After the gavage treatment, the experimental mice were anesthetized with isoflurane and decapitated at 8:00 to 10:00 in the morning, and then the following experimental operations were carried out: (1) Fresh blood of the experimental mice was immediately collected, left to stand at 4 °C for 2 h, centrifuged at 3000 rpm for 20 min to collect serum, and stored at -80 °C for further serum hormone detection and analysis; (2) The left testis was removed, fixed in 4% paraformaldehyde pre-cooled at 4 °C, and stored at 4 °C for further testicular histological detection and analysis; (3) The left epididymis was removed, the cauda epididymidis was minced and placed in 1 mL of 0.9% sterile physiological saline, and placed in an incubator at 37 °C for 20 min to completely release sperm, and a sperm suspension was prepared for further sperm density detection.

[0023] Example 3 Akkermansia muciniphila Butyric acid content in the feces of colonized mice After the fresh feces of the experimental mice in the AB group and the AKK group were sent to Novogene Co., Ltd., the butyric acid content in the colon feces of the two groups of experimental mice was measured using an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) system (Vanquish™ Flex UHPLC-TSQ Altis™, Thermo Fisher Scientific).

[0024] The results showed that, compared with the antibiotic transplantation group, Akkermansia muciniphila colonization significantly increased the content of its metabolite butyric acid ( P = 0.02, Figure 1 ). Therefore, to further confirm whether Akkermansia muciniphila alleviates mouse reproductive damage caused by high-density stress by producing butyric acid (i.e., whether the two act on the same molecular pathway), we conducted an oral butyric acid experiment with the same experimental conditions as Akkermansia muciniphila colonization in another 24 high-density stress mice.

[0025] Example 4 Oral butyric acid experiment Another 24 high-density stressed mice were used for oral butyric acid experiments. The 24 high-density stressed mice were randomly divided into two groups: a normal saline control group (SA group) and a butyric acid transplantation group (BA group) (12 mice in each group). The SA group was gavaged with 200 μL of 0.9% sterile normal saline (once a day for 4 weeks), and the BA group was gavaged with 200 μL of 500 mg / kg butyric acid solution (once a day for 4 weeks). The open-field behavior of the experimental mice was recorded using a camera from 8:00 to 10:00 am on the last day of the gavage treatment. After the gavage treatment, the experimental mice were anesthetized with isoflurane and decapitated at 8:00 - 10:00 am, and then the following experimental operations were carried out: (1) Fresh blood of the experimental mice was immediately collected, allowed to stand at 4 °C for 2 h, centrifuged at 3000 rpm for 20 min to collect serum, and stored at -80 °C for further serum hormone detection and analysis; (2) The left testis was removed and fixed in 4% paraformaldehyde pre-cooled at 4 °C, and stored at 4 °C for further testicular histological detection and analysis; (3) The left epididymis was dissected, the cauda epididymis was minced and placed in 1 mL of 0.9% sterile normal saline, and placed in an incubator at 37 °C (for 20 min) to completely release sperm, and a sperm suspension was prepared for further sperm density detection.

[0026] Example 5 Akkermansia muciniphila And the effect of butyric acid on the anxiety behavior of high-density stressed mice The open field consisted of a black PVC floor and four opaque acrylic panel walls (50 cm × 50 cm × 50 cm). The open field was divided into a central area (30 cm × 30 cm) and a peripheral area. The high-density stressed mice were placed in the central area and allowed to freely explore the open field. The time the mice entered the central area within 5 minutes was recorded using a Canon LEGRIA HF-806 camera. Before detecting each mouse, the open area was wiped with 75% alcohol.

[0027] The results showed that compared with the AB group and the SA group, the AKK group ( P = 0.01, Figure 2 A) and the BA group ( P = 0.03, Figure 2 B) The time of high-density stressed mice entering the central area increased significantly. Oral Akkermansia muciniphila and butyric acid could significantly relieve the anxiety behavior of high-density stressed mice.

[0028] Example 6 Akkermansia muciniphila And the effect of butyric acid on the sperm density of high-density stressed mice The sperm suspension was diluted 10-fold with 0.9% sterile normal saline. The number of sperm in the central (1) and diagonal (4) small grids was counted using a hemocytometer under a microscope (Olympus BX63), and the sum was accumulated. The sperm density was the total number of sperm × 5 × 10 (1 mm3 (Number of spermatozoa in internal sperm) × 1000 (number of spermatozoa in 1 mL of diluent) × 10 (dilution factor).

[0029] The results showed that the sperm density of mice in the AKK group was significantly increased compared with that in the AB group ( P = 0.02, Figure 3 A); the trend of increased sperm density in mice in the BA group compared with that in the SA group ( P = 0.3, Figure 3 B). These results indicate that oral administration of Akkermansia muciniphila and butyric acid can increase the sperm density of mice under high-density stress.

[0030] Example 7 Akkermansia muciniphila Effect of and butyric acid on the testicular morphology of mice under high-density stress

[0031] The left testicular tissue preserved in 4% paraformaldehyde was transferred to 70% ethanol, and after paraffin embedding, sectioning and H&E staining, the testicular sections were scanned by a single-slide digital panoramic scanner (Pannoramic MIDI, 3DHISTECH, Hungary). The area, perimeter, thickness of the spermatogenic epithelium and diameter of the cross-section of seminiferous tubules approximated to a perfect circle in each testicular section sample (n ≥ 8) were measured using CaseViewer 2.4 software. Figure 4 C-D) and the BA group ( Figure 4 G-H), compared with the AB group ( Figure 4 A-B) and the SA group ( Figure 4 E-F), the arrangement of seminiferous tubules in experimental mice was sparse and severely vacuolated, the spermatogenic cell layer was significantly reduced, the number of central spermatids was small, and the intercellular space was large. Further quantitative analysis of seminiferous tubules showed that: compared with the AB group, the area of seminiferous tubules in mice in the AKK group ( P = 0.005, Figure 5 A), perimeter ( P = 0.006, Figure 5 B), diameter ( P = 0.02, Figure 5 D) were significantly increased, and there was a tendency for the thickness of the spermatogenic epithelium to increase ( P = 0.2, Figure 5 C); compared with the SA group, the area of seminiferous tubules in mice in the BA group ( P <0.001, Figure 5 E), perimeter ( P <0.001, Figure 5 F), thickness of the spermatogenic epithelium ( P <0.001, Figure 5 G), diameter (P = 0.004, Figure 5 H) were all extremely significantly increased. These results indicate that oral administration of Akkermansia muciniphila and butyric acid significantly increased the testicular morphological development of high-density stressed mice.

[0032] Example 8 Akkermansia muciniphila Effects of and butyric acid on stress and reproduction-related hormones in high-density stressed mice

[0033] The contents of reproduction-related hormones (GnRH, FSH, LH and T) and stress-related hormones (CRH, ACTH and CORT) in the serum of experimental mice were detected using an enzyme-linked immunosorbent assay (ELISA) kit (specific for mice, Shanghai Xinfan Biotechnology Co., Ltd.). The specific operation is as follows: Blank wells, standard wells and sample wells to be tested were set up on a 96-well plate. 50 μL of the standard was accurately added to the standard well, and 10 μL of the serum sample and 40 μL of the sample diluent were added to the sample well to be tested. Subsequently, 100 μL of the detection antibody labeled with horseradish peroxidase (HRP) was added to the standard well and the sample wells. After covering the plate with a sealing plate film, it was incubated in a constant temperature incubator at 37 °C for 60 min. The liquid was discarded, and each well was manually washed 5 times with the washing solution, and left standing for 1 min each time. 50 μL of each of the chromogenic substrates A and B solutions was added to each well, and color development was carried out at 37 °C in the dark for 10 min. 50 μL of the stop solution was added to each well to stop the reaction, and the OD value of each well was measured at a wavelength of 450 nm (within 15 min). A standard curve was plotted, with the standard concentration as the abscissa and the corresponding OD value as the ordinate, and the concentration value of the sample to be tested was calculated.

[0033] The results showed that compared with the AB group, the stress-related hormones CRH ( P = 0.009, Figure 6 A), ACTH ( P <0.001, Figure 6 B) and CORT ( P <0.001, Figure 6 C) in the AKK group were significantly decreased; the reproduction-related hormones GnRH ( P <0.001, Figure 7 A), FSH ( P <0.001, Figure 7 B), LH ( P <0.001, Figure 7 C) and T ( P <0.001, Figure 7 D) were extremely significantly increased; compared with the SA group, the stress-related hormones ACTH ( P = 0.002, Figure 6 E) and CORT ( P = 0.002, Figure 6F) showed a significant decrease, however, CRH ( P = 0.1, Figure 6 D) showed no significant change (with a downward trend); the reproductive hormone FSH ( P = 0.02, Figure 7 F), LH ( P = 0.02, Figure 7 G) and T ( P <0.001, Figure 7 H) showed a significant increase, however, GnRH ( P = 0.4, Figure 7 E) showed no significant change (with an upward trend). These results showed that oral Akkermansia muciniphila and butyric acid significantly inhibited the secretion of stress-related hormones on the HPA stress axis, while significantly increasing the release of reproductive hormones on the HPG reproductive axis, thereby significantly improving the reproductive damage induced by social stress.

[0034] In summary, the above results indicated that Akkermansia muciniphila inhibiting the over-activation of the HPA stress axis by producing the metabolite butyric acid, thereby restoring the function of the HPG axis, increasing sperm density and improving gonadal development, and ultimately improving the reproductive disorders of ICR mice caused by high-density crowding stress.

[0035] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of Akkermansia muciniphila ( Akkermansia muciniphila ) and its metabolite butyric acid in the preparation of drugs for relieving disorders of the HPA stress axis or the HPG reproductive axis.

2. The application according to claim 1, characterized in that Alleviating the HPA stress axis means regulating stress-related hormones, and alleviating the HPG axis means regulating reproductive-related hormones.

3. The application according to claim 1, characterized in that The Akkermansia muciniphila is deposited at the American Type Culture Collection (ATCC) with the deposit number BAA-835, and the concentration of Akkermansia muciniphila in the drug is at least 1×10 9 [CFU] / 0.2 mL phosphate buffer solution (PBS). The active ingredient of the drug includes an effective amount of Akkermansia muciniphila the viable or dead form thereof, and its isolates and / or secretions.

4. The application according to claim 1, characterized in that The metabolite butyric acid of Akkermansia muciniphila, including sodium butyrate and excipients. The effective dose concentration of the butyric acid is 500 mg / kg, and the excipient is physiological saline with a dosage of 0.2 mL.

5. The application according to claim 4, characterized in that The steps for preparing the butyric acid solution are as follows: Weigh 16.875 g of butyric acid and dissolve it in 150 mL of physiological saline, and stir with a glass rod until it is completely dissolved to prepare a butyric acid solution with a concentration of 500 mg / kg.

6. The application according to claim 1, characterized in that, The Akkermansia muciniphila and the butyric acid solution are administered orally.

7. The application according to any one of claims 1-6, characterized in that, The stress-related hormones are corticotropin-releasing hormone, adrenocorticotropic hormone, and corticosterone, and the reproductive-related hormones are gonadotropin-releasing hormone, follicle-stimulating hormone, luteinizing hormone, and testosterone.

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