Abstract:
The invention provides novel methods for IVM of bovine oocytes. These methods can be used in efficient propagation of superior animals at low cost, which is very helpful in improving ecological and economic performance in the cattle industry.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a Continuation in Part of U.S. patent application Ser. No. 14/429,065, filed Jul. 16, 2015, which is a National Phase Application of PCT International Application No. PCT/CN2012/001633, International Filing Date Dec. 6, 2012, which are hereby incorporated by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The invention relates to a novel method for in vitro maturation (IVM) of bovine oocytes. Specifically, the invention relates to the application of C-type natriuretic peptide (CNP) in method for IVM of bovine oocytes. The invention provides a method for producing high-quality IVM oocytes, which can be used in livestock propagation through in vitro fertilization (IVF), as well as production of cloned or transgenic animals. The invention herein belongs to the field of embryo biotechnology. 
       BACKGROUND OF THE INVENTION 
       [0003]    In vitro embryo production (IVP), which comprises in vitro oocyte maturation (IVM), in vitro fertilization (IVF) and in vitro embryo culture (IVC), holds many benefits in agricultural livestock settings, facilitating the mass production of embryos at low cost. From 2010 to 2012, the amount of transferred IVP bovine embryos has been raised over eight times, reaching 350,000 annually. Moreover, commercial bovine IVP embryo transfer has become a large international business to reduce costs of genetic export and import (2013). However, the existing methods for IVM of bovine oocytes, always lead to nuclear-cytoplasmic asynchrony, which would compromise cytoplasmic maturation of oocytes and subsequent embryonic development. Therefore, nuclear-cytoplasmic asynchrony of IVM oocyte is the primary bottleneck for the large-scale application of IVP process in enhancing bovine reproductive efficiency. 
         [0004]    Efficient IVP process is largely dependent on the fully matured oocytes. The oocytes used for IVM were mainly collected from ovaries by ovum pick-up (OPU) or from slaughtered animals. Distinct with naturally (in vivo) matured oocytes, the oocytes used for IVM are recovered from 3-8 mm follicles. In these follicles, immature oocytes are maintained at meiotic arrest, which is also referred as germinal vesicle (GV) stage. The isolation of GV oocyte from its follicular environment triggers meiotic resumption (also referred to as “spontaneous nuclear maturation”). During the process of conventional IVM, spontaneous nuclear maturation causes a premature breakdown of oocyte-cumulus cell gap junctions (Thomas et al., 2004), which impairs the intercellular transport of beneficial cumulus cell metabolites to oocytes (Gilchrist and Thompson, 2007). Therefore, the conventional IVM systems always lead to an insufficient cytoplasmic maturation in large part of oocytes that nuclear maturation are fully achieved. This is referred as “nuclear-cytoplasmic asynchrony”. 
         [0005]    Numerous efforts have been made to overcome the nuclear-cytoplasmic asynchrony of IVM oocytes. Many chemical agents that could inhibit oocyte meiotic resumption, were used to inhibit spontaneous maturation temporarily and thus improve the synchronization between nuclear and cytoplasmic maturation, which could enhance developmental competence of oocytes. These chemicals, such as roscovitine, butyrolactone (Mermillod et al., 2000), cycloheximide (Kastrop et al., 1991), 6-DMAP (Saeki et al., 1997), forskolin (Albuz et al., 2010), etc., always showed evident inhibitory effect on nuclear maturation. Furthermore, some of those meiotic inhibitors could improve subsequent developmental rate of preimplantation embryos. However, the safety of these agents for oocytes and embryos has not been fully evaluated and certain of agents have been identified to have detrimental effects on oocyte ultrastructure and function (Fair et al., 2002; Lonergan et al., 2003). Furthermore, inappropriate usage of chemical agents during oocyte IVM could subsequently impair implantation and fetal development after embryo transfer (Albuz et al., 2010). 
         [0006]    Therefore, safe, efficient and reversible oocyte meiotic inhibition during IVM is critical for oocyte cytoplasmic maturation and subsequent embryonic development. It would be desirable if endogenous physiological factors responsible for oocyte meiotic arrest could be used for inhibiting spontaneous meiotic resumption, thereby improving nuclear-cytoplasmic synchronization and subsequent developmental competence of IVM oocytes. 
         [0007]    C-type natriuretic peptide (CNP), encoded by the natriuretic peptide precursor C (Nppc) gene, is an essential factor for maintaining oocyte meiotic arrest. In bovines, we have demonstrated an efficient and essential role of CNP in maintaining oocyte meiotic arrest. CNP derived from MGCs targets directly the NPR2 localized on both cumulus cells (CCs) and oocytes, and thus increase intra-oocyte cGMP levels via both CC-dependent and independent pathways. After that, increased intra-oocyte cGMP levels leads to an elevation of intra-oocyte cAMP levels by inhibiting PDE3A, and thus maintains oocyte meiotic arrest. This mechanism is very distinct from that reported in mice and porcines, in which only CC-dependent pathway functions via gap junctions (Zhang et al., 2010; Hiradate et al., 2014). 
         [0008]    Based on the direct and efficient inhibitory effect on meiotic resumption in bovine oocytes, CNP could be a potential candidate for improving IVM method. Until the application of the invention, CNP, as a naturally presented factor in follicle fluid, has not been used for improving oocyte IVM methods. 
       SUMMARY OF THE INVENTION 
       [0009]    It is therefore an object of the present invention to develop a novel method for IVM of bovine oocytes. 
         [0010]    It is another object of the present invention to apply CNP in improving IVM culture medium of bovine oocytes. 
         [0011]    It is further an object of the present invention to develop an IVM culture medium which could enhance developmental competence of IVM bovine oocytes and subsequent embryonic development. 
         [0012]    The present invention is directed to a novel biphasic IVM system, which comprises a CNP-based pretreatment (pre-IVM) phase, followed by an extended-IVM phase. The development of this novel system is based on our study of mechanism for CNP-induced meiotic arrest in bovine oocytes, which is very distinct to that in mice and porcine. 
         [0013]    The use of CNP in IVM culture medium is superior to other chemical meiosis inhibitor, such as roscovitine, butyrolactone, cycloheximide, 6-DMAP, etc. As a molecule that is naturally present in the follicular fluid, CNP doesn&#39;t show any evident adverse effect on bovine oocytes and subsequent preimplantation embryos. Further more, by monitoring fetal development after embryo transfer and offspring growth, we could also excluded the possible long-term detrimental effects of CNP. Meanwhile, its efficient and reversible inhibitory effect on spontaneous meiotic resumption provides a alternative for improving existing IVM system of bovine oocytes. 
         [0014]    The present invention specifically provides a novel IVM method for bovine oocytes, which comprises two phases: the first phase directs a pre-IVM culture medium containing CNP. Furthermore, our study indicates that the inhibitory effect of CNP on bovine oocyte meiotic resumptuon is dose-dependent. Preferably, oocytes resumption during IVM could significantly inhibited by pretreatment with CNP (pre-IVM) at the concentration in the range of 100-400 nM, more especially at 200nM. While with higher concentration than 400 nM or lower than 100 nM, no evident inhibitory effect could be observed. It should also be noted that the preferable concentration of CNP in maintaining oocyte meiotic arrest is variable between different species. In mice, the preferable concentration of CNP for meiotic inhibition is 100 nM, and the inhibitory effect shows a increased trend with the elevated concentrations (Zhang et al., 2010). 
         [0015]    Moreover, the invention comprises a pre-IVM culture medium for bovine oocytes. Preferably, the pre-IVM culture medium used herein is TCM-199 (tissue culture medium-199) that contains CNP at concentration of 100-400 nM, and BSA at concentration of 2.5-3.5 mg/ml. More especially, CNP at concentration of 200 nM, and BSA at concentration of 3 mg/ml would be more preferable. 
         [0016]    The novel IVM method for bovine oocyte provided in the invention comprises two phases (biphasic IVM system): CNP-based pretreatment phase, followed by the extended IVM phase. Preferably, the extended IVM culture medium used herein is TCM-199 that contains 8-12 μg/ml FSH, 0.8-1.2 μg/ml LH, 0.8-1.2 μg/ml E2, 8-1.2 ng/ml EGF, 8-12% FBS. 
         [0017]    Further, the preferable duration of pre-IVM is 5-7 hours, while the preferable duration of extended IVM phase is 24-28 hours. It must be noted that the extended IVM is required for enhancing developmental competence of matured oocytes. In Franciosi&#39;s study, CNP pretreatment (100 nM) alone could not lead to an evidently satisfying results of oocyte maturation and embryonic development (Franciosi et al., 2014). 
         [0018]    Preferably, the oocytes used for IVM are collected from ovaries by OPU or from slaughtered animals. 
         [0019]    Specially, the term “CNP” is also applicable for other type of peptides or derivates that have inhibitory effect on oocyte meiotic resumption. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0020]    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. 
       1. Detailed Methods 
     (1) Collection of Oocytes 
       [0021]    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. 
         [0000]    (2) Pretreatment (Pre-IVM) of Oocytes with CNP 
         [0022]    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 μl pre-IVM medium and 50 COCs in each well) and cultured for 6 hours at 38.5° C., in humidified air with 5% CO 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. 
       (3) Extended-IVM of Oocytes. 
       [0023]    After cultured in pre-IVM medium containing 200 nM CNP, COCs are further cultured in extended-IVM medium (TCM199 supplemented with 10 μg/ml FSH, μg/ml LH1, 1 μg/ml E2, 10 ng/ml EGF, 10% FBS) at 38.5° C., in humidified air with 5% CO 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. 
       (4) In Vitro Fertilization and Culture 
       [0024]    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 μl 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. 
       2. Results 
     (1) Effect of CNP at Different Concentrations on Spontaneous Meiotic Resumption of IVM Oocytes 
       [0025]    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&lt;0.01) (Table 1). However, higher CNP concentrations do not show increased inhibitory effect. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 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 c ) 
                 40 (44 ± 2.6 c ) 
               
               
                 CNP (50 nM) 
                 101 
                 64 (63.4 ± 3.1 bc ) 
                 37 (21.3 ± 3.1 bc ) 
               
               
                 CNP (100 nM) 
                 98 
                 68 (69.4 ± 2.6 abc ) 
                 30 (30.6 ± 2.6 abc ) 
               
               
                 CNP (200 nM) 
                 97 
                 79 (81.4 ± 2.1 a ) 
                 18 (19.6 ± 2.1 a ) 
               
               
                 CNP (400 nM) 
                 106 
                 79 (74.5 ± 2.8 ab ) 
                 27 (25.5 ± 2.8 ab ) 
               
               
                   
               
               
                 Table shows the mean ± SEM values of at least three independent experiments. 
               
               
                 Data with different letters within same column are significantly different (p &lt; 0.01) 
               
             
          
         
       
     
       (2) Effect of CNP Pre-IVM and Extended-IVM on Developmental Competence of Bovine Oocytes 
       [0026]    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. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 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 b)   
                 58 (23.1 ± 2.3 b ) 
               
               
                   
                 26 h 
                 216 
                 168 (77.8 ± 3.0 b ) 
                 52 (24.7 ± 1.9 b ) 
               
               
                   
                 28 h 
                 198 
                 143 (72.2 ± 3.4 b ) 
                 42 (21.2 ± 2.4 b ) 
               
               
                 6 h 
                 24 h 
                 193 
                 157 (81.3 ± 2.7 ab ) 
                 53 (27.5 ± 1.3 bc ) 
               
               
                   
                 26 h 
                 217 
                  89 (87.1 ± 0.75 a ) 
                 62 (28.6 ± 0.7 c ) 
               
               
                   
                 28 h 
                 214 
                 186 (88.3 ± 3.0 a ) 
                 80 (37.4 ± 2.5 a ) 
               
               
                   
               
               
                 Table shows the mean ± SEM values of at least three independent experiments. 
               
               
                 Data with different letters within same column are significantly different (p &lt; 0.01)