Method for in vitro oocyte maturation

Abstract

Methods are provided for in vitro maturation (IVM) of bovine oocytes that include steps of (a) pre-culturing bovine germinal vesicle (GV) oocytes in the presence of C-type natriuretic peptide (CNP) and (b) subsequently culturing the oocytes of (a) for an extended duration in medium containing follicle stimulating hormone (FSH), luteinizing hormone (LH), 17-estradiol (E2), epidermal growth factor (EGF), and fetal bovine serum (FBS).

Claims

1. A method for in vitro maturation (IVM) of bovine oocytes, comprising: (a) pre-culturing one or more bovine germinal vesicle (GV) oocytes in a pre-IVM culture medium that comprises a meiosis-inhibiting concentration of C-type natriuretic peptide (CNP), under conditions and for a time sufficient to obtain bovine oocytes in meiotic arrest; wherein the pre-IVM culture medium comprises 200 nM CNP and 3 mg/ml BSA; wherein the step of pre-culturing is for 6 hours at 38.5 C., in humidified air with 5% CO.sub.2; and (b) subsequently culturing the bovine oocytes in meiotic arrest obtained in step (a) for 28 hours in an extended-IVM culture medium containing follicle stimulating hormone (FSH), luteinizing hormone (LH), 17-estradiol (E2), epidermal growth factor (EGF), and fetal bovine serum (FBS) to obtain matured bovine oocytes having enhanced developmental competence relative to bovine GV oocytes that have not been pre-cultured with CNP present.

2. The method of claim 1, wherein the extended-IVM culture medium comprises 8-12 g/ml FSH, 0.8-1.2 g/ml LH, 0.8-1.2 g/ml E2, 8-12 ng/ml EGF, and 8-12% FBS.

3. The method of claim 2, wherein the extended-IVM culture medium comprises TCM-199.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The following embodiment should be considered as a detailed description of the invention, but not the limiting description. Any changes or modifications based on the invention, belongs to the definition of the invention.

(2) 1. Detailed Methods

(3) (1) Collection of Oocytes

(4) Bovine ovaries were obtained from a local slaughterhouse, transported at 30-35 C. to the laboratory within 1 hour, and washed in warmed normal saline containing 2% Penicillin G-Streptomycin. Ovarian antral follicles (3-8 mm) were aspirated using an 18-gauge needle. cumulus-oocyte complexes (COCs) with homogenous oocyte cytoplasm and complete, compacted, and multilayered CCs (Grade-1) or complete but fewer than three layers of CCs (Grade-2), were collected using micropipettes.

(5) (2) Pretreatment (Pre-IVM) of Oocytes with CNP

(6) Grade-1 and -2 COCs were washed in the washing buffer (HEPES-TCM199) for three times, and then washed in pre-IVM medium (TCM199 containing 3 mg/ml BSA and CNP at different concentrations (50, 100, 200 and 400 nM) for two times. After that, COCs were transfer into pre-IVM medium (pre-warmed for 2 hours) in 4-well plate (500 l pre-IVM medium and 50 COCs in each well) and cultured for 6 hours at 38.5 C., in humidified air with 5% CO.sub.2. After pre-IVM phase, a proportion of COCs are digested with hyaluronidase to remove cumulus cells. To determine the most preferable concentration of CNP for pre-VIM, meiosis kinetics of oocytes was evaluated according to nuclear morphology using DAPI staining under a fluorescence microscope. The oocytes without pre-IVM phase are used as control.

(7) (3) Extended-IVM of Oocytes.

(8) After cultured in pre-IVM medium containing 200 nM CNP, COCs are further cultured in extended-IVM medium (TCM199 supplemented with 10 g/ml FSH, g/ml LH1, 1 g/ml E2, 10 ng/ml EGF, 10% FBS) at 38.5 C., in humidified air with 5% CO.sub.2. To determine the most preferable duration for extended-IVM, COCs were cultured for 24, 26 or 28 hours. In addition, COCs without pre-IVM phase, are also cultured in pre-IVM medium for 24, 26 or 28 hours as controls.

(9) (4) In Vitro Fertilization and Culture

(10) After maturation, the COCs were fertilized in BO medium for 8 hours at 39 C. under 5% CO2 in a humidified atmosphere. After fertilization, the presumptive zygotes were transferred to 100 l CR1 culture medium droplets (15 zygotes in each droplets) at 39 C. in a humidified atmosphere. The culture medium was changed every 2 days during the culture period. The percentage cleavage and rates of subsequent embryo development to the blastocyst stage were recorded on day 2 and 7, respectively.

(11) 2. Results

(12) (1) Effect of CNP at Different Concentrations on Spontaneous Meiotic Resumption of IVM Oocytes

(13) The inhibitory effect of CNP on meiotic resumption of bovine oocytes shows dose-depended patterns, with maximal effect at 200 nM. The rate of oocytes maintaining at GV stage is significant higher than that in control group (P<0.01) (Table 1). However, higher CNP concentrations do not show increased inhibitory effect.

(14) TABLE-US-00001 TABLE 1 The effect of pretreatment with CNP at different concentrations on the meiotic resumption of bovine oocytes No. of No. of GV oocytes No. of GVBD oocytes Treatments oocytes (%) (%) Control 91 51 (56 2.6.sup.c) 40 (44 2.6.sup.c) CNP (50 nM) 101 64 (63.4 3.1.sup.bc) 37 (21.3 3.1.sup.bc) CNP (100 nM) 98 68 (69.4 2.6.sup.abc) 30 (30.6 2.6.sup.abc) CNP (200 nM) 97 79 (81.4 2.1.sup.a) 18 (19.6 2.1.sup.a) CNP (400 nM) 106 79 (74.5 2.8.sup.ab) 27 (25.5 2.8.sup.ab) Table shows the mean SEM values of at least three independent experiments. Data with different letters within same column are significantly different (p < 0.01)
(2) Effect of CNP Pre-IVM and Extended-IVM on Developmental Competence of Bovine Oocytes

(15) CNP pre-IVM shows a beneficial effect on developmental competence of IVM oocytes. After IVF, the cleavage rates of oocytes pre-treated with CNP and followed by a extended IVM phase are significantly higher that of their counterparts. In addition, our novel method significantly enhances blastocyst rates.

(16) TABLE-US-00002 TABLE 2 Effect of CNP pre-treatment and extended IVM on developmental rate of bovine oocytes after IVF CNP based Extended-IVM Pre- or IVM standard-IVM No. of No. of cleavage No. of blastocysts phase phase oocytes (%) (%) 0 h 24 h 251 190 (75.7 2.7.sup.b) 58 (23.1 2.3.sup.b) 26 h 216 168 (77.8 3.0.sup.b) 52 (24.7 1.9.sup.b) 28 h 198 143 (72.2 3.4.sup.b) 42 (21.2 2.4.sup.b) 6 h 24 h 193 157 (81.3 2.7.sup.ab) 53 (27.5 1.3.sup.bc) 26 h 217 89 (87.1 0.75.sup.a) 62 (28.6 0.7.sup.c) 28 h 214 186 (88.3 3.0.sup.a) 80 (37.4 2.5.sup.a) Table shows the mean SEM values of at least three independent experiments. Data with different letters within same column are significantly different (p < 0.01)