Long-term in vitro culture of bovine preantral follicles: Effect of base medium and medium replacement methods

Abstract

Two culture media and replacement methods were compared during long-term in vitro culture of secondary follicles of cattle using α-MEM⁺ or TCM-199⁺ as base media. The medium replacement methods were: Conventional – removal and subsequent addition of the same amount (60 μl) in a 100 μl aliquot (MEM-C and TCM-C), and Small Supplementation – addition of 5 μl of fresh medium to an initial small aliquot (50 μl), resulting in a final volume of 125 μl on the last day of culture (MEM-S and TCM-S). A total of 207 secondary follicles were cultured individually for 32 days at 38.5 °C in 5% CO₂ and medium replacement was performed every other day. The MEM-S treatment resulted in a larger (P < 0.01) follicular diameter, greater (P < 0.02) growth rate, greater (P < 0.02) antrum formation, as well as greater (P < 0.0001) estradiol concentrations when compared with the MEM-C treatment. The medium change methods did not affect (P > 0.05) the follicular and estradiol end points for TCM-199⁺. The expression of the FSHR gene was greater (P < 0.03) with the TCM-C than TCM-S treatment, while the relative amounts of mRNA for IGF1 was greater (P < 0.02) with MEM-S than TCM-S treatments and for VEGF was greater (P < 0.02) with MEM-C than TCM-C treatment. In conclusion, the type of base medium and the effect of periodic addition of medium differentially affected follicle development, estradiol production, and gene expression. Furthermore, α-MEM⁺ can be used to replace TCM-199⁺ for culture of preantral follicles of cattle if progressive addition of medium is used for medium change.

Introduction

Development of preantral follicles to a stage where the oocyte can mature in vitro requires a long-term culture period and involves several follicular changes during its development (Thomas et al., 2007). However, a long-term culture period can impair the steroidogenic capability of the follicle and affect oocyte competency (McLaughlin and Telfer, 2010). Moreover, the pattern of gene expression can also be affected by in vitro culture systems. During preantral follicle development the oocyte is growing and synthetizing mRNA to allow further development of the embryo after fertilization. Furthermore, some genes encoding for enzymes involved in steroidogenesis (P450 aromatase; Drummond, 2006), follicle stimulating hormone receptor (FSHR; Xu et al., 1995), vascular endothelial growth factor (VEGF; Greenaway et al., 2004), and insulin-like growth factor-1 (IGF-1; Sudo et al., 2007) may be affected during follicular growth. Therefore, to successfully culture preantral follicles of cattle it is necessary to appropriately define in vitro culture conditions which would allow the oocyte to survive, grow, and induce the differentiation of the somatic cell compartment for a prolonged period.

Development of an ideal culture system for in vitro culture of preantral follicles has been the focus of different research groups. However, to our knowledge, in vitro experiments with preantral follicles of cattle are few and with limited success, compared to other species such as goats (Saraiva et al., 2010, Magalhães et al., 2011), sheep (Arunakumari et al., 2010, Luz et al., 2012), and mice (Eppig and O’Brien, 1996, O’Brien et al., 2003). The in vitro follicular culture performance may be affected by different factors such as follicle size (Katska and Rynska, 1998), type of base culture medium (Rossetto et al., 2012), addition of supplements (Figueiredo et al., 1994), gas concentration (Gigli et al., 2006), method of medium replacement (i.e., interval, removal, and addition of culture medium; Araújo et al., 2011), and presence or absence of mineral oil (Fukui et al., 1996).

Several studies have evaluated the developmental characteristics of large (≥150 μm) preantral follicles of cattle in culture media, which have a larger growth rate than small preantral follicles in culture (Katska and Rynska, 1998). In this regard, culture media such as α-MEM (Braw-Tal and Yossefi, 1997, Rossetto et al., 2012, Araújo et al., 2014), TCM-199 (Katska and Rynska, 1998, Itoh and Hoshi, 2000, Saha et al., 2000, Saha et al., 2002, Rossetto et al., 2012), and McCoy (Gutierrez et al., 2000, McCaffery et al., 2000, Thomas et al., 2001, Thomas et al., 2007, McLaughlin et al., 2010, McLaughlin and Telfer, 2010, Rossetto et al., 2012) have been used to maintain viability and improve in vitro development of cattle follicles. The use of TCM-199 has been found to be the most desirable medium to culture isolated secondary follicles of cattle, based on the high percentage of viable follicles after 16 days of in vitro culture (Rossetto et al., 2012). With regard to the most desirable medium replacement method, in a study with isolated secondary follicles of goats, it was found that periodic addition of medium maintained survival and promoted in vitro follicular development, and was the only treatment that resulted in production of a competent oocyte which resumed meiosis (Araújo et al., 2011). However, it is not known if this medium addition method could also be effective in the culture of isolated preantral follicles of cattle. Furthermore, there is a lack of information concerning the effects of type of base medium, as well as medium replacement method on estradiol production and gene expression for the key factors related to folliculogenesis.

Therefore, the aim of this study was to evaluate two different culture media using two distinct methods for media supplementation every other day during long-term in vitro culture of large secondary follicles of cattle, such as: (i) partial removal and replacement of medium, and (ii) addition of a small amount of medium. The following end points were evaluated: follicle viability and development, antrum formation, estradiol production, and mRNA for FSHR, IGF1, VEGF, and P450AROM.