METHOD FOR ACTIVATING FOLLICLES BY MEANS OF USING SMALL-MOLECULE COMPOUND, AND PREPARATION THEREOF

Abstract

Disclosed are a method and culture medium for performing an in vivo treatment on ovaries of mammals including humans or performing in vitro culturing on ex vivo ovaries or ovarian tissue by means of using 4-vinylcyclohexene diepoxide. The method or culture medium provided facilitates the retention and enhancement of the developmental and reproductive potential of mammalian ovaries or ovarian tissue, and is particularly capable of promoting follicle maturation and increasing the number of ovulations.

Claims

1. A culture medium for cultivating mammalian ovary or ovarian tissue, comprising 4-vinylcyclohexene diepoxide or its analogue, wherein the content of 4-vinylcyclohexene diepoxide in the medium is about 0.1 nM-5000 nM.

2. The culture medium according to claim 1, wherein said culture medium is used for culturing isolated ovaries or parts thereof.

3. (canceled)

4. The culture medium according to claim 1, wherein the culture medium further comprises a background medium selected from a minimal medium, a simple medium or a supplemented medium.

5. The culture medium according to claim 1, further comprising one or more of the following other compounds: inorganic salts, energy sources, amino acids, proteins, cytokines, chelating agents, antibiotics, hyaluronic acid, growth factors, hormones or vitamins.

6. The culture medium according to claim 1, wherein the mammals are rodentia, lagomorpha, carnivora, artiodactyla, persistodactyla, or primates and apes.

7. An in vitro culture method of ovary or ovarian tissue of a mammal, said method comprising adding 4-vinylcyclohexene diepoxide or its analog in a culture medium, wherein the 4-vinylcyclohexene diepoxide is about 0.1 nM-5000 nM in the culture medium.

8. The in vitro culture method according to claim 7, wherein the time for the ovary or ovarian tissue being cultured in the culture medium with 4-vinylcyclohexene diepoxide is no more than 6 days.

9. The in vitro culture method according to claim 7, which is used to treat the ovary or ovarian tissue of juvenile or young mammals, and the time for the ovary or ovarian tissue being cultured in the culture medium with 4-vinylcyclohexene diepoxide is not more than 4 days.

10. The in vitro culture method according to claim 7, which is used to treat the ovary or ovarian tissue of middle-aged or old mammals, and the time for the ovary or ovarian tissue being cultured in the culture medium with 4-vinylcyclohexene diepoxide is not more than 6 days.

11. A method for improving ovarian function in a mammal, wherein the mammal is given about 5-200 mg/kg body weight of 4-vinylcyclohexyl diepoxide.

12. The method according to claim 11, wherein the method stimulates follicle maturation.

13. (canceled)

14. The method according to claim 11, wherein 4-vinylcyclohexene diepoxide is administered at intervals of about 12-48 hours.

15. The method according to claim 11, wherein 4-vinylcyclohexene diepoxide is administered to the mammal for not more than 10 days.

16. The method according to claim 11, wherein 4-vinylcyclohexene diepoxide is administered intraperitoneally.

17. The method according to claim 11, wherein the method is for the treatment of an ovary-related reproductive disorder.

18. (canceled)

19. (canceled)

20. (canceled)

21. The culture medium according to claim 1, wherein the 4-vinylcyclohexene diepoxide in the medium is about 1 nM-20 nM.

22. The in vitro culture method according to claim 7, wherein the 4-vinylcyclohexene diepoxide is about 10 nM-200 nM in the culture medium.

23. The in vitro culture method according to claim 8, wherein the time for the ovary or ovarian tissue being cultured in the culture medium with 4-vinylcyclohexene diepoxide is no more than 1 day.

24. The method according to claim 11, wherein the mammal is given about 20-100 mg/kg body weight of 4-vinylcyclohexyl diepoxide.

25. The method according to claim 12, wherein the method promotes activation of primordial follicles, or promotes transformation of primordial follicles to primary follicles, or promotes transformation of primary follicles to secondary follicles.

26. The method according to claim 17, wherein the method is for the treatment of premature ovarian insufficiency (POI) or diminished ovarian reserve (DOR).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1 shows that culturing with 4-vinylcyclohexene diepoxide (VCD) stimulates activation of primordial follicles in in vitro experiments. FIG. 1A shows the staining of ovarian sections from different treatment groups. FIG. 1B shows the results of counting follicles of all levels in ovarian tissues of different treatment groups. FIG. 1C shows Immunoblotting of phosphorylation levels for several proteins associated with follicle activation in ovaries.

[0041] FIG. 2 shows that short VCD culture duration stimulates the transformation of primordial follicles to primary follicles in in vitro experiments. FIG. 2A shows the staining of ovarian sections in different treatment groups. FIG. 2B shows the results of counting secondary follicles in ovarian tissues of different treatment groups.

[0042] FIG. 3 shows that VCD stimulates follicle development in neonatal mice in vivo experiments. FIG. 3A shows the staining of ovarian sections from different treatment groups. FIG. 3B shows the results of follicle counting in ovarian tissues of different treatment groups.

[0043] FIG. 4 shows that VCD stimulates follicle development as well as more oocytes expulsion in the in vivo experiments in old mice. FIG. 4A shows the staining of ovarian sections from different treatment groups. FIG. 4B shows the results of counting different follicles in the ovarian tissue of different treatment groups. FIG. 4C shows the MII oocytes obtained by ovulation stimulation (arrows point to MII oocytes). FIG. 4D shows the results of MU oocytes counts in the experimental and control groups.

[0044] FIG. 5 shows that VCD stimulates aromatase expression and estrogen production in ovarian granulosa cells. FIG. 5 A-B shows that the expression level of aromatase in granulosa cells of mice was significantly increased after VCD treatment. FIG. 5 C-D shows that the expression level of aromatase in KGN was significantly increased after VCD treatment.

DETAILED DESCRIPTION

[0045] The technical details and benefits of the invention provided in the present disclosure are further described in the following examples, which are intended to illustrate the inventions and not to limit the scope of the present disclosure.

Example 1 Experimental Method and Reagents

[0046] 4-vinylcyclohexene diepoxide (VCD) was purchased from Sigma Aldrich (Cat #94956-100 ML). For in vitro maturation, and in vitro fertilization, VCD was dissolved in phosphate-buffered saline (PBS) (Cat #10010-0323, Gibco). For intraperitoneal injection, VCD was dissolved in saline (0.9% sodium chloride).

[0047] In Vitro Culture of Neonatal Mouse Ovary

[0048] Separating Mouse Ovary

[0049] Neonatal 2-day (PD2) or 12-day (PD12) C57bl/6 mice (provided by Shandong University Experimental Animal Center) were executed by decapitation and the ovaries and surrounding tissues were dissected and separated under a dissecting microscope, and the excised ovaries were placed in Leibovitz's L-15 medium (containing 10% fetal bovine serum+0.5% penicillin+0.5% streptomycin). The adipose tissue was dissected and separated. The separated ovaries were washed three times in PBS.

[0050] Culture of Mouse Ovary

[0051] Configure ovarian medium: 1 mg/ml bovine serum albumin (BSA, Cat #A4161-1G, Sigma), 1 mg/ml AbluMAX II Lipid-Rich BSA (Cat #11021029, Gibco), 5% Insulin-Transferrin-Selenium (ITS-G) (Cat #41400-045, Gibco), 0.5% Penicillin+0.5% Streptomycin and 100 nM VCD were added into DMEM/F12 (Cat #11039-021, Gibco) medium. The prepared medium was mixed and added to a six-well plate with 1.5 ml of medium per well. A sieve (Cat #PICMORG50, Millipore) was put on the medium and placed in the incubator to preheat.

[0052] The separated ovaries were transferred to the ovarian medium with a pipette, washed three times and placed on the upper sieve of the pre-warmed medium. A drop of medium was added around the ovaries in the upper sieve to keep the area around the ovaries moist. The six-well plates were placed in a 37° C., 5% CO2 incubator for four consecutive days, and the lower layer of medium was changed daily. For PD2 mice ovaries, VCD was added to the medium on the first day of incubation only. PD12 mice ovaries were continuously exposed to VCD for four days.

[0053] Neonatal Mice were Injected Intravenously and Ovarian Changes were Observed

[0054] PD12 C57bl/6 mice in the same litter were divided into experimental and control groups according to body weight. The experimental mice were weighed and given either 20 mg/kg or 160 mg/kg of VCD by intraperitoneal injection, while the control mice were given the same volume of saline. After ten days of continuous administration, the mice were executed and the ovaries were dissected to separate the adipose tissue under the microscope and fixed in 4% PFA. Dehydrated sections were used to observe and count the development of follicles in the ovaries at different times.

[0055] Ovulation Stimulation Experiment in Old Mice

[0056] 10-12 month old C57bl/6 rats were divided into experimental and control groups according to body weight, and the experimental group was weighed and injected intraperitoneally with 80 mg/kg of VCD, while the control group was given the same volume of saline. After 5 days of continuous administration, 5 units of PMSG were injected to the experimental group and the control group, and 10 IU of hCG was injected 48 hours later to induce secondary ovulation. Mice were executed 16 hours after hCG injection, and both oviducts were collected and placed in M2 dissecting solution (Cat #M7167, Sigma), and the oocyte-granulosa cell complexes were seen when cutting the dilated oviducts under a dissecting microscope. The granulosa cell complexes from each mouse were transferred to a droplet of hyaluronidase (Cat #90101, Fujifilm Irvine Scientific) and the number of eggs per mouse was counted under a microscope after the granulosa cells around the oocytes had been digested.

[0057] Western Blot Assay for Phosphorylation of Proteins Associated with Follicle Activation in the Ovary

[0058] PD2 mice were dissected in autopsy solution (Leibovitz's L-15 medium, Cat #41400-045, Gibco; +10% fetal bovine serum (FBS)) after execution, washed twice in ovarian medium and incubated in ovarian medium. After incubation in medium with or without VCD for 3 min. ovarian tissue was collected and lysed in RIPA lysis solution (Cat #89900, Thermo) for digestion. Proteins were denatured and separated by SDS PAGE and transferred to PVDF membranes (Millipore). The membranes were incubated with primary antibodies followed by incubation with HRP-conjugated secondary antibodies and the bands were examined using an enhanced chemiluminescence detection kit (Bio-Rad).

[0059] Antibody Information

[0060] anti-rabbit aromatase (Cat #NBP1-45360, Novus, 1:2000), anti-rabbit phosphor-AKT (Serine 473) (D9E) (Cat #4060, Cell Signaling, 1:500), anti-rabbit phosphor-FoxO1 (Threonine 24)/FoxO3a (Threonine 32) (Cat #9464, Cell Signaling, 1:500), anti-rabbit phosphor-P-S6 (Serine 235/236) (Cat #4858, Cell Signaling, 1:1000), anti-rabbit phosphor-P-S6 (Serine 240/244) (Cat #2215, Cell Signaling, 1:1000), anti-rabbit phosphor-P70S6k (Threonine 389) (Cat #9205, Cell Signaling, 1:1000), anti-rabbit (Cat #7074S, CST) or anti-mouse (Cat #7076S, CST).

[0061] Detection of Estradiol (E2) Levels in Cell Culture Supernatants (ELISA)

[0062] KGN cell supernatants were collected after 24 hours of cell culture for the detection of E2 levels. The procedure of the assay followed the instructions of the E2 Assay Kit (Human Estradiol (E2) ELISA Kit, Cat #KAQ0621, Invitrogen).

[0063] Statistical Analysis

[0064] Data are expressed as the mean±standard deviation from at least three independent experiments. Statistical comparisons were performed using one-way ANOVA, and statistical differences were set at P<0.05.

Example 2 Short-Time VCD Culture Stimulates Activation of Primordial Follicles in an In Vitro Experiment

[0065] The experimental procedure and results are shown in FIG. 1.

[0066] Two-day-old (PD2) mouse ovaries were isolated and cultured in vitro according to the method described in Example 1. Different concentrations of 4-vinylcyclohexene diepoxide (VCD) (0, 1 nM, 10 nM, 100 nM, 1000 nM) were added to the medium on the first day of culture, and the medium was changed to normal medium from the second day of culture and continue the culture for 3 days, and then the ovaries were collected; the control group was incubated with 50 ng/ml folliculopoietin FSH (Sigma #F4021-2UG). The collected ovaries were sectioned and stained to observe the morphology, and the results are shown in FIG. 1A (arrows point to primary follicles). FIG. 1B shows the results of counting follicles of all levels in the ovarian tissue of different treatment groups. The results demonstrated that the number of primordial follicles and early primary follicles was significantly increased in ovaries treated with VCD compared with those of the control group. FIG. 1C shows that ovaries of PD2 mice were treated with 10 nM VCD for 3 min in vitro before tissue proteins were collected, and the changes in the levels of protein phosphorylation associated with follicle activation in the ovaries were observed by Western blotting assay. The results showed that VCD treatment for 2-3 min could significantly activate the PI3K-Akt pathway, in which the expression of the involved proteins was significantly elevated. It can be seen that low concentration of VCD treatment stimulated the activation of primordial follicles.

Example 3 Short Duration VCD Culture Stimulates the Transformation of Primordial Follicles to Primary Follicles in In Vitro Experiments

[0067] Mouse ovaries at 12 days (PD12) were isolated and cultured in vitro according to the method described in Example 1. Ovaries were collected after 4 days of incubation with different concentrations of 4-vinylcyclohexene diepoxide (VCD) (0, 1 nM, 10 nM, 100 nM, 1000 nM) in the medium, and 50 ng/ml of folliculopoietin FSH (Sigma #F4021-2UG) in the medium for the control group, in each group of mice N=9. The collected ovaries were sectioned and stained to observe the morphology. The results are shown in FIG. 2A (arrows point to normal secondary follicles). FIG. 2B shows the results of counting secondary follicles in ovarian tissues of different treatment groups. The results demonstrate that the number of secondary follicles in the ovaries treated with VCD was significantly increased compared to the control group.

Example 4 VCD Stimulates Follicle Development in Neonatal Mice In Vivo Experiments

[0068] According to the method described in Example 1, the same litter of PD12 C57bl/6 female mice was divided into experimental and control groups according to body weight and given 20 mg/kg or 160 mg/kg of VCD by intraperitoneal injection, while the control mice were given the same volume of saline. Each group (N=6) was administered for 5 consecutive days, and then the mice were executed and the collected ovaries were stained to observe the morphology as shown in FIG. 3A (yellow arrows indicate early sinus follicles and red arrows indicate sinus follicles). FIG. 3B shows the results of follicle counts in ovarian tissues of different treatment groups after 5 days of continuous administration. The results demonstrate that there were more follicles close to the preovulatory stage in the ovaries of the mice in the administered group compared to the control group.

[0069] Together with the phenomena observed in Example 3, it can be concluded that VCD treatment can stimulate the transformation of primary and secondary follicles to mature oocytes.

Example 5 VCD Promotes Follicle Development as Well as More Mature Oocytes Expulsion in an In Vivo Experiment in Old-Aged Mice

[0070] Aged mice of 10-12 months were used as study subjects to observe the effect of VCD on follicle development and eventual oocytes expulsion in their ovaries.

[0071] According to the method described in Example 1, 10-12 month old C57bl/6 rats were divided into experimental and control groups according to their body weight, and the experimental group was weighed and injected intraperitoneally with 80 mg/kg of VCD, while the control group was given the same volume of saline. After 5 days of administration, 5 units of PMSG were injected into the experimental animals, and then 10 IU of hCG was injected 48 hours later to induce secondary ovulation. 16 hours after the hCG injection, the mice were executed, and the MII oocytes were collected from the oviducts of both sides. The collected ovaries were sectioned and stained to observe the morphology, and the results are shown in FIG. 4A (arrows point to preovulatory follicles, CL refers to corpus luteum). FIG. 4B shows the results of counting different follicles in the ovarian tissues of different treatment groups. The results of follicle counts indicate that there were more sinus follicles as well as newly formed corpus luteum in the ovaries of mice given VCD. FIG. 4C shows the MII oocytes obtained by ovulation (arrows point to MII eggs). FIG. 4D shows the results of MII egg counts in the experimental and control groups. The ovulation results showed that the mice in the VCD group could ovulate more oocytes after 5 days of administration. It can be seen that VCD can improve ovarian function and further stimulate oocyte expulsion in aged mice.

Example 6 VCD Promotes the Expression of Aromatase in Ovarian Granulosa Cells and Estrogen Production

[0072] Primary granulosa cells from PD20 mice were extracted and the effect of VCD treatment on the expression of aromatase in granulosa cells was observed.

[0073] Granulosa cells were isolated from the ovaries of PD20 mice according to the method described by Reddy P et al, Science 2008, 319(5863):611-613 and Liu Y X et al Biol Reprod 1986, 35(1):27-36, from which primary granulosa cell lines were obtained. They were treated with different concentrations of VCD (0, 1 nM, 10 nM, 100 nM, 1000 nM) for 24 h, using 50 ng/ml FSH as control. The results showed that the expression level of aromatase in mouse granulosa cells was significantly increased after VCD treatment (FIG. 5 A-B)

[0074] Human-derived granulocyte-like cell line KGN (provided by Prof. Toshihiko Yanase, Fukuoka University, Japan) was cultured in DMEM/F12 medium. KGN were treated with different concentrations of VCD (0, 1 nM, 10 nM, 100 nM, 1000 nM) for 24 h, using 50 ng/ml FSH as control. The results showed that the expression level of aromatase in KGN was significantly increased after VCD treatment (FIG. 5 C-D). The production of estrogen (E2) in KGN culture supernatant was further observed by ELISA, and it was found that the level of estrogen in the cell supernatant of VCD-treated group was significantly higher than that of the control group (FIG. 5E).

[0075] The above data suggest that VCD has a role in promoting the expression of granulocyte aromatase and its downstream estrogen production.

[0076] The inventors of the present invention discover for the first time that VCD apparently exhibits both dose- and duration-dependent opposing, hormone-like effects on the maintenance of primordial follicle pool, follicle development, and ovulation induction. This effect of VCD is due to that low concentration and/or short duration treatment with 4-vinylcyclohexene diepoxide has hormone-like effects on ovary or cultured ovarian tissue, and can transiently activate PI3K-akt and mTOR pathways; in addition, low concentration and/or short-term 4-vinylcyclohexene diepoxide treatment also increases expression of aromatase in ovarian granulosa cells stimulates estrogen production. The present invention thus provides a method and a culture medium for treating mammalian ovaries including human ovaries in vivo, or culturing isolated ovaries or ovarian tissues in vitro with 4-vinylcyclohexene diepoxide.

[0077] The foregoing is a description of the present invention and is not to be regarded as a limitation of the invention. Unless otherwise indicated, the present invention will be practiced using conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc., and it is clear that the invention can be achieved in other ways than those described specifically in the above description and embodiments. Other aspects and improvements within the scope of the present invention will be apparent to those skilled in the art to which the present invention belongs. Many changes and variations are feasible in accordance with the teachings of the present invention and are therefore within the scope of the present invention.