A Reproducible Two-Step Culture System for Isolated Primary Mouse Ovarian Follicles as Single Functional Units1

Abstract

A reproducible two-step culture system for isolated mouse ovarian follicles smaller than 100 microm (type 3a follicles) was designed. First, isolated follicles were grown in single droplets of alpha-minimal essential medium (MEM) without (deoxy)ribonucleosides at a lower concentration of fetal bovine serum (FBS; 1%) for 6 days with mechanical prohibition of thecal cell attachment. Growing follicles reaching at least 100 microm were transferred to alpha-MEM medium enriched with a higher concentration (5%) of FBS to allow attachment and were cultured subsequently for an additional 12 days. Overall, more than 85% of the follicles survived the first culture step, and oocyte growth and granulosa cell proliferation had increased by 25% (P < 0.05). Follicle survival at Day 18 was related to initial follicle diameters at isolation. Average meiotic maturation rates and estrogen secretion were lower compared to those of cultures starting with early preantral follicles of 100-130 microm. Although reverse transcription-polymerase chain reaction analysis revealed the presence of LH-receptor mRNA in thecal cells, an exogenous androstenedione replacement resulted in an increase of estrogen production, suggesting substrate insufficiency. The time needed to grow from early preantral stages to in vitro ovulation is strongly dependent on the initial follicle diameter at isolation. Morphological characteristics of cultured follicles were suggestive for combined transforming growth factor beta deficiencies during in vitro culture.

In vitro development of mouse fetal germ cells into mature oocytes

“…During the last few years, the formation and development of mouse primordial follicles have been investigated in vitro , Klinger & de Felici 2002, Obata et al 2002a, O'Brien et al 2003, Lenie et al 2004, Niwa et al 2004, Shen et al 2006a, 2006b). The primordial follicles within the newborn mouse ovaries were able to achieve complete development and maturation in vitro, although the frequency of preimplantation development was low , O'Brien et al 2003.…”

siRNA-mediated depletion of stable proteins in mouse oocytes

“…(ii) in vitro growth (IVG) medium without PVP (Eppig and O'Brien, 1996;Hartshorne, 1997;Lenie et al, 2004;Smitz and Cortvrindt, 2002) Take 10 ml of !-MEM with GlutaMax (Life technologies, #32571-036) that is opened within 3 months. Add 12.5 "l insulin (f. 5 "g/ml), 10 "l transferrin (f. 5 "g/ml), 10 "l sodium selenite (f. 5 ng/ml), and 10 "l Penicillin & Streptomycin.…”

“…Further information on selection criteria can also be found elsewhere (Hartshorne, 1997;Lenie et al, 2004;Pesty et al, 2006). Usually, 10-20 follicles per female can be collected.…”

“…Representative pictures of follicles cultured in vitro for 12 days can be found elsewhere (Inoue and Zhang, 2014;Lenie et al, 2004;Smitz and Cortvrindt, 2002). After 12 days, 80-100% of oocytes normally reach the fully grown GV-stage.…”

Preservação de fertilidade

“…De qualquer maneira não haveria a exposição ao risco de reimplantar células malignas. Outra possibilidade é o isolamento de folículos primordiais a partir do tecido congelado-descongelado, utilizando-se técnicas de digestão enzimática 37,38 ou por dissecção mecânica 39,40 , seguido de cultivo para maturação in vitro. As téc-nicas atualmente propostas para maturação in vitro (MIV) de folículos, e que vêm obtendo sucesso nos centros de reprodução assistida, partem de folículos imaturos, mas já em estádio de desenvolvimento mais avançado, ou de folículos antrais visualizados ao ultra-som e captados sem estimulação de ovulação prévia ou com baixas doses de gonadotrofi nas 41,42 .…”