In Vitro Culture of Mouse Preantral Follicles Using Membrane Inserts and Developmental Competence of In Vitro Ovulated Oocytes

Abstract

Abstract. To develop a reliable follicle culture system, mouse preantral follicles 150-200 µm in diameter were cultured individually for 5 or 6 days in membrane inserts or in droplets, and then induced to ovulate with hCG (Experiment 1). The nuclear maturation and developmental competence of the oocytes that ovulated from the follicles cultured in inserts were determined (Experiment 2). There was no significant difference between the two culture systems in the survival rate (83 and 77%). However, follicles cultured in inserts showed a higher ovulation rate (63%) than those cultured in droplets (39%, P<0.05). About 80% of the oocytes that ovulated from the follicles cultured in inserts were at the metaphase II stage. After in vitro fertilization, 75 and 48% of in vitro ovulated oocytes cleaved and developed into blastocysts, respectively. These results demonstrate that the insert culture system is superior to the droplet culture system in terms of follicular growth and ovulation, and can be used to investigate the growth and ovulation of follicles in vitro. Key words: Culture system, In vitro ovulation, Mouse preantral follicle, Oocyte maturation (J. Reprod. Dev. 50: [579][580][581][582][583][584][585][586] 2004) he culture of preantral follicles provides the means not only of producing a uniform population of developmentally competent oocytes but also of investigating the physiology of follicular development and ovulation in the absence of systemic influences. Different culture systems have been developed for mouse preantral follicles [1][2][3]. Some culture syste ms maintain the threedimensional structure of follicles, while others allow adherence to an underlying substrate causing the collapse of the follicle, resulting in a twod i m e n s i o n a l s t r u c t u r e . T h e c u l t u r e o f enzymatically isolated preantral follicles produces many fertile oocytes, but the enzymatic treatment removes the thecal cells and perforates the basal membrane so that an antrum will not form [4]. A culture system that allows the attachment of follicles yields a large number of oocytes but can not retain the follicle structure [5]. A threedimensional culture has many advantages over a two-dimensional culture for investigating the mechanism of folliculogenesis, oocyte growth, ovulation and ovarian dysfunctions. Maintenance of the three-dimensional structure of isolated intact follicles has been achieved by various approaches such as culturing in membrane inserts [6], or simply by the daily transfer of follicles to new wells [7], or new droplets [8]. However, the efficiencies of these three-dimensional culture systems have not been evaluated by direct comparison under the same culture conditions. Moreover, only a few

A simple and efficient method for preparation of isolated ovarian follicles for transmission electron microscopy

“…The polyester membranes, transfixed to the transwell inserts, are available in 3 pore sizes; 0.4 µm, 0.8 µm and 3 µm. Transwell inserts have been used for in vitro culture and maturation of ovarian follicles [24][25][26]. The polyester membrane is fixed to the bottom of the insert, onto which ovarian follicles are cultured.…”

“…In contrast, the polyester membrane in the transwell insert is translucent, allowing excellent cell visualization under phase contrast microscopy without the need for staining. The transwell insert with polyester membrane is a well documented system for in vitro culture and maturation of ovarian follicles [24][25][26]. The major limitation with this technique is that the original three-dimensional structure of the ovarian follicles is lost by attachment to the transwell membrane.…”

A feasibility study of prepubertal and over mature aged local goat in relation to results of In Vitro growth culture to obtain additional M-II oocyte resources

“…In this culture system, we used supplementation of follicular fluid in a fixed presentation (5%) rather than hypoxanthine. Down et al 9 reported that hypoxanthine in follicular fluid has been identified as one such meiosis arresting substance.…”

“…There is quite a variation in the oocyte calcium intensity. 9 Results reported from IVG culture systems from different species of the mammalian oocyte 1,10 , from 1989 to 2015 show a different potential of different species including mouse, pig, and cow with matrix collagen, resulting in a positive result for IVM or IFV methods and obtaining offspring. In domestic species, the first successful IVG culture system that supports the growth of oocytes from mid-growth phase in preantral (late secondary) follicles to the final size was reported by Hirao et al 11 (Table 3).…”

A New In Vitro Ovulation Model for Medaka Based on Whole Ovary Culture

“…In view of clinical and agricultural interests, efforts have been made to develop culture systems using primordial follicles, because the follicles at this stage potentially represent a large source of oocytes in humans and large mammals (Eppig and O'Brien, 1996;Abir et al, 1999;Abir et al, 2001;Demeestere et al, 2002). While in vitro experimental systems suitable for studying follicular growth are available, only a handful of reports describing in vitro ovulation models in mammals have been published (Baranczuk and Fainstat, 1976;Brännström et al, 1987;Rose et al, 1999;Adam et al, 2004). This is probably due to the difficulty of setting stable conditions to obtain reproducible results.…”