Developmental Competence of Intrafollicular Oocytes Derived from Preantral Follicle Culture with Different Protocols after Parthenogenetic Activation

This study was conducted to improve efficiency of a follicle culture system without reducing developmental competence of intrafollicular oocytes. Preantral follicles (100 to 125 μm in diameter) of F1 hybrid (B6CBAF1) mice were cultured singly for 216 h in modified α-MEM-glutamax medium, to which 2.5 IU/ml hCG and epidermal growth factor was added 16 h prior to the end of culture. Medium change was either performed three times (54 h interval), twice (72 h interval), once (108 h interval), or not at all (216 h interval). Maturation (progression to the metaphase II stage) of intrafollicular oocytes was detected from 4 days after culture in the three-times change treatment, while all treatments yielded mature oocytes from day 5 of culture. Compared with the three-times change, decreasing the change frequency to once did not reduce the capacity to begin maturation (germinal vesicle breakdown of 82 to 86%), to mature (78 to 79%) and to develop into blastocysts after parthenogenetic activation (29 to 32%). Morphological parameters were similar among these treatments. Except for the no medium change treatment, similar colony-forming activity of inner cell mass cells after culturing of blastocysts in leukemia inhibitory factor-containing medium was detected, while the morphology of the colonyforming cells deteriorated in the change-once treatment compared with the change twice or three-times. In conclusion, the efficiency of the preantral follicle culture system could be improved by reducing frequency of medium change up to a 72 h interval (three times in total 216 h culture) without decreasing developmental competence of oocytes. (


INTRODUCTION
We recently succeeded to establish homozygous embryonic stem (ES) cell-like cells derived from parthenogenesis of intrafollicular oocytes matured in in vitro-cultured preantral follicles. This achievement can contribute not only to securing autologous, immune-specific ES cells without undertaking somatic cell nuclear transfer, but also to retrieving developmentally-competent oocytes from numerous preantral follicles being destined to degenerated. Our effort was subsequently focused to further optimize the culture system and as the series of research, we attempted to increase culture efficiency by decreasing the frequency of medium change. This was because of using single follicle culture system for follicle development, which might have insufficient autocrine or paracrine factors for oocyte maturation. This study was consequently designed to evaluate whether increasing medium change interval (decreasing the frequency of medium change) could influence developmental competence of intrafollicular oocytes. Morphological parameters including oocyte maturation, development after parthenogenetic activation and colony-formation of inner cell mass (ICM) cells of blastocysts were employed for this attempt. management, breeding and surgery followed the standard operation protocols of Seoul National University. Appropriate management of experimental samples, quality control of the laboratory facility and equipment were also conducted.

Culture of secondary follicles
Secondary follicle of 100 to 125 µm in diameter with multiple layers of granulosa cells and an intrafollicular oocyte were collected with an ocular micrometer of an inverted microscope (TE-2000; Nikon, Tokyo, Japan) at 40× magnification. The secondary follicles isolated mechanically from the ovaries were washed three times in 10 µl droplets of L-15 medium and subsequently cultured at 37°C, 5% CO 2 in air atmosphere.

In vitro growth of secondary follicles
The secondary follicles were placed singly in 10 µl culture droplets overlaid with washed-mineral oil (Sigma-Aldrich Corp.) in 60×15 mm Falcon plastic Petri-dishes (Becton Dickinson, Franklin Lakes, NJ). The medium used for the culture of secondary follicle is ribonucleoside and deoxyribonucleoside-containing α-MEM-glutamax medium (Gibco Invitrogen), to which 1% (v/v) heat-inactivated fetal bovine serum (FBS; HyClone), 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 100 mIU/ml recombinant human FSH (Organon, Oss, The Netherlands), 10 mIU/ml LH (cat. no. L-5259, Sigma-Aldrich Corp) and 1% (v/v) penicillin and streptomycin were added. On day 1 of culture, 10 µl of fresh medium was added to each droplet and, from the third day after the replacement, half of a medium was changed. The interval was different according to experimental design.

The maturation of follicular oocytes and parthenogenetic activation
To induce final maturation of intrafollicular oocytes, 2.5 IU/ml hCG (Pregnyl™; Organon) and 5 ng/ml epidermal growth factor (EGF; cat. no E-4127, Sigma-Aldrich Corp) were added to the culture medium 16 hours prior to the end of culture. Retrieved oocytes were freed from cumulus cells by mechanical pipetting and subsequently placed in M2 medium, consisting of 94.66 mM NaCl, 4.78 mM KCl, 1.71 mM CaCl 2 ⋅2H 2 O, 1.19 mM KH 2 PO 4 , 1.19 mM MgSO 4 ⋅7H 2 0, 4.15 mM NaHCO 3 , 20.85 mM HEPES, 23.28 mM sodium lactate, 0.33 mM sodium pyruvate, 5.56 mM vglucose, 1% (v/v) penicillin/streptomycin, and 4 mg bovine serum albumin (BSA)/ml, supplemented with 200 IU/ml hyaluronidase. Initiation of meiotic maturation determined by germinal vesicle breakdown (GVB) and completeness of the maturation to reach the metaphase II stage (having metaphase II plate with polar body) was monitored under a phase-contrast microscope after staining with Lacmoid solution. Size (diameter) of mature oocytes derived from each treatment was also measured by soft imaging system GmbH (Version3.0, Biocompare, Münster, Germany).

Culture of ICM cells for colony-formation
For culturing ICM cells of blastocysts, zona pellucida of the blastocysts obtained from the parthenogenetic oocytes were removed with acid Tyrode solution and the zone-free blastocysts were cultured on mouse embryonic fibroblast (MEF) feeder layer that were mitotically inactivated with mitomycin C. Knock-out Dulbecco's minimal essential medium (KDMEM; Gibco Invitrogen) supplemented with 0.1 mM β-mercaptoethanol (Gibco Invitrogen), 1% (v/v) nonessential amino acids (Gibco Invitrogen), 2 mM Lglutamine, 1% (v/v) lyophilized mixture of penicillin and streptomycin and 2,000 units/ml mouse leukemia inhibitory factor (LIF; Chemicon, California, US) and 3:1 mixture of FBS and knock-out serum replacement (KSR,Sigma-Aldrich, St Louis, MO) were used for culturing the zonafree blastocysts. Colony-formation of ICM cells was monitored daily under an inverted microscope.

Experimental design
Prospective, randomized study was conducted and all secondary follicles retrieved were equally distributed into each experimental treatment. In general, secondary follicles were cultured with medium change three-times (54 h interval; considered as control treatment), twice (72 h interval), once (108 h intervals) throughout the culture (9 days) or not (216 h interval). In experiment 1, hCG and EGF, the final maturation inducers, were added to 16 h prior to the end of day 1 to 9 of culture and meiotic maturation of the oocytes to the metaphase II stage was monitored. In experiment 2, intrafollicular oocytes derived from 9-day cultured preantral follicles were retrieved 16 hours after the treatment with hCG and EGF. Number of follicles grown into the pseudoantral stage, initiation of maturation to reach the germinal vesicle breakdown and metaphase II stages, meiotic maturation, the morphology of mature oocytes, and cleavage and blastocyst formation after parthenogenetic activation were subsequently monitored. In addition, colony-forming activity of ICM cells of parthenogenetic blastocysts was evaluated after culturing in LIF-containing medium.

Statistical analysis
A generalized linear model (PROC-GLM) in a Statistical Analysis System (SAS) program was employed for statistical analysis. One-way ANOVA followed by the least-square method was conducted when the parameter had  model effect. Significant differences among treatments were determined where the p value was less then 0.05.

Experiment 1
Completion of meiotic maturation was first detected in hCG-and EGF-treated intrafollicular oocytes that were retrieved from preantral follicles cultured for 4 days ( Figure  1). Regardless of the treatments, number of mature oocytes were gradually increased and subsequently peaked on day 9 of culture. A significant (p<0.05) model effect among treatments was detected from day 6 to day 9 of culture. Regardless of observation time, more oocytes reached to the metaphase II stage in the medium change three-times, twice or once ( 54 to 108 h intervals) than no change (216 h intervals).

Experiment 2
In total, 680 preantral follicles were evenly distributed into 4 groups (170 each). As shown in Table 1, more (p<0.05) follicles developed into the pseudoantral stage in the change three-times, twice or once than no medium change (78 to 79% vs. 55%). Similar pattern was detected in the proportion of oocytes begun maturation (82 to 86% vs. 64%; p<0.05), matured (47 to 52% vs. 22%; p<0.05) and developed into blastocysts (29 to 32% vs. 10%; p<0.05). There was no difference in oocyte morphology among treatments, which were observed by light microscope. Diameters of mature oocytes derived from different treatments were within the range of 68.2±1.6 to 68.8±1.8 µm and no significant (p>0.05) difference was detected among the treatments (Figure 2). In the case of colonyformation of ICM cells, 11 to 13% of ICM cells formed colonies in any groups of medium change, while no colonyformation was detected in no medium change. No prominent difference in the morphology of the colonyforming cells was detected between the cells derived from the change three-times and the change twice (Figure 3). Morphological deterioration, however, was detected in the colony-forming ICM cells derived from the medium change once.

DISCUSSION
The results of this study demonstrated that decreased frequency of medium change to certain extend (increased the change interval up to 72 h) was not detrimental to support developmental competence of intrafollicular oocytes in in vitro-cultured preantral follicles. Similar, even insignificantly increased rates of follicle development, initiation and completeness of oocyte maturation, blastocyst formation after parthenogenesis was obtained and there was no change in oocyte morphology by the increased interval of medium change. This modification contributes to improving the efficiency of follicle culture system by reducing labor and maintenance cost without decreasing developmental competence and morphological integrity of intrafollicular oocytes.
In vitro-culture system for preantral follicles is a useful tool for retrieving sufficient number of oocytes for providing various clinical and industrial purposes. Moreover, it also contributes to understanding the underlying mechanisms of oocyte growth and differentiation. Several culture systems for culturing different stages preantral follicles have been suggested in different species (Katska et al., 1998;Cecconi et al., 1999;Wu et al., 2001;Gupta et al., 2002). Live births were derived from in vitro-fertilization of intrafollicular oocytes derived from in vitro-cultured preantral follicles followed by embryo transfer (Liu et al., 2001;Delapena et al., 2002). On the other hand, homozygous (cloned) animals by transfer of parthenogenetic embryos have been attempted for a long time for efficient undertaking artificial reproduction in domestic animals (Kono, 2006). Recently, homozygous ES cells were derived from parthenogenetic activation of ovulated oocytes in primates including human (Cibelli et al., 2002;Vrana et al., 2004;Sanchez-pernaute et al., 2005). We further combined these two technologies for preantral follicle culture and parthenogenetic ES cell establishment, which resulted establishment of homozygous ES cell-like cells (Lee et al., 2006).
Oocytes in preantral follicles cultured in vitro were tolerable against reduced frequency of medium change and morphological integrity remained after increasing the change intervals to some extend. In our supplementary data, there was no difference in cortical granule distribution and the formation of chromosome-spindle complex in mature oocytes among treatments (data not shown). Probably, paracrine role of follicular cells such as granulosa and theca cells or autocrine function of intrafollicular oocyte is enough to acquire developmental competence during in vitro-culture. In other words, essential substrates for acquiring developmental competence to the blastocyst stage, which were secreted from follicles and/or oocytes, may not be diluted by at least the 108 h interval change.
Data on ICM morphology suggested the deleterious effect of excessive extension of the change interval up to 108 h or 192 h (medium change once or no change during the 9-day culture) on the colony-formation of ICM cells. These results showed that the 72 h interval (medium change twice during the culture) might be the optimal. Further increased interval might accumulate embryo-toxic metabolites or dried up essential factors for ICM colony formation in medium substrates. On the other hand, it has been reported that the size of intrafollicular oocytes matured in vitro was smaller than that of in vivo-ovulated oocytes (Eppig and O'Brien, 1996). In this study, we failed to increase the diameter similar to in vivo-derived oocytes (around 75 to 80 µm). Additional attempt was subsequently conducted to further improve developmental competence of oocytes, such as applying extracellular matrix gel for threedimensional culture of the follicles and modifying culture medium composition.
In conclusion, the results obtained from our presented studies contribute to developing alternative technology of immune-specific stem cell establishment (colony-formation rate of more than 10%) and to optimizing reproductive performance of domestic animals having desired genotype. Also, our follicle culture technique could be applies for securing large number of developmentally-competent oocytes from infertile patients with ovarian factor.