Follicle development is accompanied by proliferation of granulosa cells and increasing
oocyte size. To obtain high-quality oocytes in vitro, it is important to
understand the processes that occur in oocytes and granulosa cells during follicle
development and the differences between in vivo and in
vitro follicle development. In the present study, oocytes and granulosa cells
were collected from early antral follicles (EAFs, 0.5–0.7 mm in diameter), small antral
follicles (SAFs, 1–3 mm in diameter), large antral follicles (LAFs, 3–7 mm in diameter),
and in vitro grown oocyte-and-granulosa cell complexes (OGCs), which were
cultured for 14 days after collection from EAFs. Gene expression was analyzed
comprehensively using the next-generation sequencing technology. We found top upstream
regulators during the in vivo follicle development and compared them with
those in in vitro developed OGCs. The comparison revealed that
HIF1 is among the top regulators during both in vivo
and in vitro development of OGCs. In addition, we found that
HIF1-mediated upregulation of glycolysis in granulosa cells is important for the growth of
OGCs, but the cellular metabolism differs between in vitro and in
vivo grown OGCs. Furthermore, on the basis of comparison of upstream regulators
between in vivo and in vitro development of OGCs, we
believe that low expression levels of FLT1 (VEGFA receptor),
SPP1, and PCSK6 can be considered causal factors of
the suboptimal development under in vitro culture conditions.
This study examined the concentration of cell-free mitochondrial DNA (cf-mtDNA) in porcine follicular fluid (FF) and explored whether the cfDNA level in the culture medium could reflect
mitochondrial dysfunction in cumulus cell-oocyte complexes (COCs). cfDNA concentration was higher in the fluid of small-sized follicles, compared to that in larger follicles. The length of
cfDNA ranged from short (152 bp) to long (1,914 bp) mtDNA in FF, detected by polymerase chain reaction (PCR). cfDNA concentration in FF significantly correlated with the mtDNA copy number in
FF but not with the number of one-copy gene (nuclear DNA) in FF. When the COCs were treated with the mitochondrial uncoupler, namely carbonyl cyanide m-chlorophenyl hydrazone (CCCP), for 2 h
and incubated for 42 h, subsequent real-time PCR detected significantly higher amount of cf-mtDNA, compared to nuclear cfDNA, in the spent culture medium. The mtDNA number and viability of
cumulus cells and oocytes remained unchanged. When the oocytes were denuded from the cumulus cells following CCCP treatment, PCR detected very low levels of cfDNA in the spent culture medium
of the denuded oocytes. In contrast, CCCP treatment of granulosa cells significantly increased the amount of cf-mtDNA in the spent culture medium, without any effect on other markers,
including survival rate, apoptosis of cumulus cells, and lactate dehydrogenase levels. Thus, cf-mtDNA was present in FF in a wide range of length, and mitochondrial dysfunction in COCs
increased the active secretion of cf-mtDNA in the cultural milieu.
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