Computational protocol: RHAMM deficiency disrupts folliculogenesis resulting in female hypofertility

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Protocol publication

[…] Mouse ovaries and uterus were fixed with 4% paraformaldehyde for 16–24 h and embedded in paraffin. 4 µm thick tissue sections were stained with hematoxylin and eosin according to standard protocol. The sections were analyzed in an Olympus BX41 light microscope and images were acquired using the Cell* software (Olympus).For ovarian follicle quantification, 4 µm thick serial sections of the whole ovary were prepared and one of every five sections was stained with hematoxylin and eosin. The sections were examined with bright field microscopy, the follicles, at different stages of folliculogenesis, were counted and the total number of follicles in the sections examined were plotted (see and ) (; ).For the morphological classification of the follicles, the criteria of Pedersen and Peters () were applied. Briefly: follicles type 1,2 and 3 were classified as primordial; type 4 and 5 were classified as primary; type 6 as secondary; type 7 as antral and type 8 as pre-ovulatory.For analysis of the orientation of granulosa cell division, ovary sections, prepared as described above, were scanned with 40× objective in a VS110 virtual microscope (Olympus). From the scanned images, follicles with one or two layers of granulosa cells were selected and the cell division axis of granulosa cells at metaphase or anaphase, was determined: A line dissecting the center of the oocyte, the centre of the mitotic granulosa cell and the basal lamina was defined as the oocyte-basal membrane axis (). For cells at anaphase, the spindle axis was defined as the line parallel to the direction of separating chromosomes (). For cells at metaphase, a line along the metaphase plane was drawn () and a second line perpendicular to the metaphase plate () was used to define the spindle axis. The angle θ between oocyte-basal membrane axis and spindle axis was determined using ImageJ (NIH). The follicles of 4 wild type mice ovaries (n = 41 mitotic cells) and 8 mutant mice ovaries (n = 31 mitotic cells) were thus analyzed. [...] Oocyte in vitro maturation assays were performed as previously described (). Briefly, oocytes were collected from ovaries of 10- or 26-week-old hmmr+/+ and hmmrm/m mice and placed in M2 medium pre-warmed to 37°C and supplemented with 4 mg/ml BSA and 1 mM milrinone. For video microscopy of oocyte meiotic maturation, oocytes were transferred to a Ludin Chamber containing M2 medium with 4 mg/ml BSA. Time-lapse images were acquired using a Photometrics CCD camera (CoolSnap HQ2) mounted on a Leica HC PL APO 20×/0.7 NA objective enclosed in a thermostatic chamber (Life Imaging Service). Images were taken every 15 min for 18–20 h at 20× magnification. Metamorph 7.0 (Universal Imaging) and ImageJ (NIH) software were used for image analysis. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Applications Bright-field microscopy, Microscopic phenotype analysis
Organisms Homo sapiens, Mus musculus
Diseases Amino Acid Metabolism, Inborn Errors, Ocular Motility Disorders