Abstract
The mammalian ovarian follicle is comprised of the germ cell or oocyte surrounded by the somatic cells, the granulosa and theca cells. The ovarian stroma, including the collagen-rich matrix that supports the three-dimensional disk-like follicular structure, impacts the integrity of the ovarian follicle and is essential for follicular development. Maintaining follicular integrity during cryopreservation has remained a limiting factor in preserving ovarian tissues for transplantation because a significant proportion of developed follicles in the frozen-thawed ovaries undergo atresia after transplantation. In this study, we show for the first time that during vitrification of the mouse ovary, the attachment of the oocyte to the granulosa cells was impaired by the loss of the cadherin adhesion molecules. Importantly, exposure to a high osmotic solution greatly decreased the ratio of oocyte diameter to the diameter of its follicle but did not alter the collagen-rich matrix surrounding the follicles. By treating ovaries briefly with collagenase before exposure to the hyper-osmotic solution the ratio of oocyte diameter to follicle diameter was maintained, and cadherin adhesion junctions were preserved. When frozen-thawed ovaries were transplanted to the bursa of recipient hosts, pretreatment with collagenase significantly increased serum levels of AMH, the number of intact follicles and the total number of viable offspring compared to frozen-thawed ovaries without collagenase pretreatment, even 6 months after transplantation. Thus, the collagenase pretreatment could provide a beneficial approach for maintaining the functions and viability of cryopreserved ovaries in other species and clinically relevant situations.
Highlights
The mammalian ovarian follicle is comprised of the germ cell or oocyte surrounded by the somatic cells, the granulosa and theca cells
The physical fusion/attachment of the granulosa cell membrane projections with those of the oocyte is observed in secondary follicles[24], indicating that this specific contact communication mechanism between the oocyte to granulosa cells is essential for early stages of follicular development
In particular the presence of stromal collagen impacts the rigidity of the tissue surrounding each follicle and this in turn impacts follicular function and development involving the maintenance of cell-cell communication between the oocyte and the granulosa cells
Summary
The mammalian ovarian follicle is comprised of the germ cell or oocyte surrounded by the somatic cells, the granulosa and theca cells. Maintaining follicular integrity during cryopreservation has remained a limiting factor in preserving ovarian tissues for transplantation because a significant proportion of developed follicles in the frozen-thawed ovaries undergo atresia after transplantation. Cryopreservation of ovarian tissue containing immature oocytes in preantral follicles is an experimental option for patients before the start of chemical anti-cancer therapy[6]. The limitation of the xenograft technique for delivering frozen-thawed www.nature.com/scientificreports human ovarian tissue to immunodeficient mice has been reported; the morphology of the human frozen ovaries looks normal just after thawing, but a significant portion of the developed follicles undergo atresia within 6 days after in vitro culture[12]. After transplantation the function of frozen-thawed ovarian tissue preserved by vitrification did not improve compared to that of the slowly-frozen ovaries[18]. If the elegant follicular structure is disrupted by a hyperosmotic solution due to the different shrinking speeds among the cell types follicular integrity is compromised severely
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