The production of mature oocytes from adult ovaries following primary follicle culture in a marsupial

Abstract

A model marsupial culture system has been developed whereby individual primary follicles, obtained from adult ovaries, can be grown in vitro to the antral stage and oocytes retrieved from these follicles can achieve nuclear maturation (metaphase II) in the presence of LH. Primary follicles isolated from adult Sminthopsis macroura ovaries were cultured individually in one of four systems: microdrops under oil, upright, inverted, or roller culture. After 6 days of culture, cumulus-oocyte complexes (COCs) were excised from early antral follicles and incubated for an additional 24 h to assess meiotic competence and the effects of LH and lithium on oocyte maturation. Histology and transmission electron microscopy established normal in vivo standards and verified oocyte and follicular integrity following culture. On day 6 of culture, follicle viability was significantly greater in the inverted system (73%) than in the other three systems (10-46%). The inverted system was the most effective in supporting development with follicles demonstrating progressive growth during culture and showing antral signs by day 4. Meiotic resumption during COC culture was facilitated by LH, but hindered by lithium. The ability to resume meiosis and progress to metaphase II was equivalent in oocytes retrieved following follicle culture and those matured in vivo. This study highlights the importance of oxygen and nutrient availability during marsupial follicle culture, and demonstrates for the first time that primary follicles isolated from adult mammalian ovaries can undergo normal growth and development in vitro, to produce mature, meiotically competent oocytes.

A novel marsupial pri-miRNA transcript has a putative role in gamete maintenance and defines a vertebrate miRNA cluster paralogous to the miR-15a/miR-16-1 cluster

“…Oocytes were measured along the longest axis, and the axis perpendicular in relation to the scale bar. The measurements were within the expected range for the identified follicles (Kress et al 2001, Nation & Selwood 2009). …”

Induced ovulation mimics the time-table of natural development in the stripe-faced dunnart, Sminthopsis macroura and results in the birth of fertile young

“…1). Following induction, developing follicles showed normal morphology, and follicle diameter measurements recorded were within the range expected for naturally cycling animals (Kress et al 2001, Nation & Selwood 2009). Primordial, primary, and early secondary follicles were present in all examined ovaries.…”

“…Mean follicle diameters were calculated by measuring the longest axis, and the axis perpendicular, using a calibrated ocular micrometer. Follicles were determined to be primary, secondary, tertiary, or antral based on size and morphology (Kress et al 2001, Nation & Selwood 2009). Antral follicles were carefully ruptured using 29 G needles to assess oocyte maturity (Merry et al 1995).…”

Towards a 3D culture of mouse ovarian follicles

“…Recent attempts have been made to maintain the 3D organization by culturing follicles in inverted microdrop suspension (Wycherley et al, 2004;Nation and Selwood 2009), rotating wall vessels (Rowghani et al, 2004), orbiting test tubes (Rowghani et al, 2004;Heise et al, 2005Heise et al, , 2009 or in roller bottle systems (Nation and Selwood 2009). In an inverted microdrop suspension system, mouse follicles were cultured for up to 6 days in 96-well round-bottomed plates that allowed the maximum oxygen access and nutrients supply at the media/gas interface and supported growth better than upright cultures (Wycherley et al, 2004).…”