Effects of ovarian disaggregation on adult murine follicle yield and viability

Abstract

Follicles are isolated from ovaries for numerous reasons, including IVM, but adult murine yields are <2 folliclesmg. The aim of the present study was to optimise ovarian disaggregation and develop methods applicable to the rapid screening of follicle viability. Ovaries from adult mice (n=7) were halved and disaggregated mechanically, or by using collagenase IV (Col-IV; 590UmL) or animal origin-free collagenase IV (AOF) at 590 or 1180UmL. Isolated follicles were stained with 4',6'-diamidino-2-phenylindole (DAPI; nuclei), chloromethyl-X-rosamine (CMXRos; mitochondria) or fluorescein isothiocyanate-conjugated anti-α-tubulin antibody. Follicle diameters and staining were measured and analysed using ImageJ, and data analysed using GraphPad Prism. Col-IV disaggregation yielded the highest number of follicles (17±10 folliclesmg ovarian tissue). All disaggregation methods released more secondary follicles (86±20 per ovary; P<0.05) than any other size cohort. Mechanical and Col-IV disaggregation yielded similar numbers of morphologically intact follicles, whereas AOF disaggregation caused more damage (P<0.01). As the morphological disruption increased, DAPI and CMXRos staining decreased (P<0.05), and tubulin localisation became more heterogeneous. Col-IV disaggregation gave the best yield of morphologically intact follicles containing viable granulosa cells. In conclusion, we improved adult murine follicle yields and applied molecular markers to assess follicle morphology, cellular cytoskeleton and mitochondrial function.

Three Dimensional &lt;i&gt;In Vitro&lt;/i&gt; Culture of Murine Secondary Follicles in a Defined Synthetic Matrix

“…The morphology of each Live-Dead stained follicle was classified as being intact (M1, the follicle appeared completely enclosed by the basal lamina), or largely intact but of lower morphological integrity (M2, the basal lamina appeared irregular or broken in places, but without any loss or displacement of GC), or slightly (M3) or completely disrupted (M4) [46] [47].…”

Establishment of a protocol for the isolation of ovarian preantral follicles derived from collared peccaries (Pecari tajacu)

“…The collagenase used in the present study was a partially purified bacterial enzyme (Clostridium histolyticum) that could attack native collagen without affecting related proteins and damaging the epithelial tissue (Mandl et al, 1958). The efficiency of type IV collagenase enzyme has previously been proven in bovine (Figueiredo et al, 1993), caprine (Machado et al, 2002), porcine (Choi et al, 2008), human (Lierman et al, 2014, and murine (Young et al, 2017) tissues. In addition, the crucial factors of this enzyme are its concentration and duration of digestion (Shuttleworth et al, 2002).…”

Interaction between oocytes, cortical germ cells and granulosa cells of the mouse and bat, following the dissociation–re-aggregation of adult ovaries

“…Ovarian reconstruction in vitro from cells dissociated from postnatal ovaries of mammals has been documented previously in the rat (Zenzes and Engel, 1981), pig (Ol et al, 2015), mouse (Young et al, 2017) and in re-aggregated chimeric mice and rats (Eppig and Wigglesworth, 2000). However, among re-aggregated ovaries, the expression of markers that promote the histogenetic recapitulation of organs such as Cx43 and Laminin has not been reported, including the viability of the oocytes (Ddx4) and their proliferation (pH3).…”