Two alternative methods for the retrieval of somatic cell populations from the mouse ovary

Abstract

Many modern techniques employed to uncover the molecular fundamentals underlying biological processes require dissociated cells as their starting point/substrate. Investigations into ovarian endocrinology or folliculogenesis therefore necessitate robust protocols for dissociating the ovary into its constituent cell populations. While in the mouse, methods to obtain individual, mature follicles are well-established, the separation and isolation of single cells of all types from early mouse follicles, including somatic cells, has been more challenging. Herein we present two methods for the isolation of somatic cells in the ovary. These methods are suitable for a range of applications relating to the study of folliculogenesis and mouse ovarian development. First, an enzymatic dissociation utilising collagenase and a temporary, primary cell culture step using neonatal mouse ovaries which yields large quantities of granulosa cells from primordial, activating, and primary follicles. Second, a rapid papain dissociation resulting in a high viability single cell suspension of ovarian somatic cells in less than an hour, which can be applied from embryonic to adult ovarian samples. Collectively these protocols can be applied to a broad array of investigations with unique advantages and benefits pertaining to both.

Pregranulosa cells upregulate cardiac troponin I Tnni3 upon cell cycle resumption during primordial follicle activation.

“…We focused on three critical timepoints representative of ovary development before, during and after follicle activation, namely: embryonic day 18.5 (E18.5), when germ cell nest breakdown is initiated to form primordial follicles; post-natal day 4 (PND4), when the initial wave of primordial follicle activation is occurring; and PND7, when populations of activated follicles (termed primary follicles) are present within the ovary (Figure 1A). A previously described and validated dissociation protocol using the enzyme papain was utilised to achieve a single cell suspension of the ovarian tissue at each stage (Frost et al, 2021b). In total, our dataset contained 24,810 cells, with 8,210 cells from one E18.5 ovary, 7,587 cells from one PND4 ovary and 9,013 cells from one PND7 ovary (Table S1).…”

“…Notably we observed a decrease in the number of cells in the Oo_1 cluster, particularly at PND7 (Figure 2A). The dissociation protocol used to prepare the ovarian samples for single cell RNA-sequencing in this study enriches the suspension for somatic cells, rather than germ cells, via size selection, as oocytes increase in size as activation occurs and primordial follicles transition to primary follicles (Frost et al, 2021b). This may explain the decrease in oocyte numbers in the postnatal timepoints (Zhang et al, 2014).…”

“…Female ovaries at E18.5, PND1 and PND7 were harvested and the ovarian bursa and fat were removed, leaving only ovary tissue. Ovarian tissue was dissociated as previously described (Frost et al, 2021b). Briefly, two ovaries from each timepoint were incubated separately in a solution of papain (10108014001, Sigma-Aldrich, 1mg/ml in HBSS) for 15min at 37°C in presence of 10µg/ml of both DNAse I (10104159001, Thermo Scientific) and Rock Inhibitor (M1817, Abmole Bioscience) (Moncho-Amor et al, 2020).…”

“…Protocols have recently been developed for the dissociation of the neonatal mouse ovary, generating single-cell suspensions for sequencing, that can be used during this initial wave of activation (Y.-Y. Chen et al, 2022; Frost et al, 2021a). To date, most studies have utilised genetically modified mouse models and microarray experiments to explore the expression of gene-driven signals in stimulating or inhibiting primordial follicle activation (reviewed in (Y.…”

Transcriptomic profiling of neonatal mouse granulosa cells reveals new insights into primordial follicle activation

“…Ovaries were collected immediately after euthanasia and either snap-frozen, fixed in 10% neutral buffered formalin for ≤10 h, or placed into 37°C Leibovitz L-15 media supplemented with 1% fetal bovine serum (FBS) for immediate granulosa cell collection. For a detailed description of the methodology and rationale behind time point selection and granulosa cell collection, see [ 17 ].…”