Imager a1

A CCD camera (AxioCam HRm, Carl Zeiss Microscopy, LLC, Thornwood, NY) was used to record the fluorescence intensity of the cells through a microscope (Imager. A1, Carl Zeiss Microscopy, LLC, Thornwood, NY) with a motorized x-y stage (Mechanical stage 75 × 50 R, Carl Zeiss Microscopy, LLC, Thornwood, NY) in a dark room. Images were taken every 30 minutes with an exposure time of 300 ms controlled by a shutter (VS25S2ZM1R1-21, Vincent Associates, Rochester, NY). The excitation light from a mercury lamp (X-Cite 120Q, Dynamics Group Inc., Ramsey, MN) was guided through a filter set (Filter Set 43 HE, Carl Zeiss Microscopy, LLC, Thornwood, NY). A MATLAB routine was developed to sort droplets by the number of cells in them and to track the fluorescence intensity of each cell automatically. A quality control filter code was used to filter out those cells that are improperly tracked. Dark current corrections, bias corrections and flat field corrections were done to all images before tracking the fluorescence intensity of cells.

In order to detect the cell apoptosis in rat ovaries, In Situ Cell Death Detection Kit (KeyGEN BioTech) was used to detect the fragmented DNA histochemically by TUNEL. Fluorescein-labeled nucleotides were incorporated in situ onto 3′ ends of DNA strand breaks of the apoptotic cells. According to the instruction, the paraffin sections were dewaxed at 60°C for 60 min and rehydrated with ethanol (100%, 90%, 80%, and 75%) in step by step manner. The slides were washed thrice with PBS for 5 min and incubated with proteinase K at 37°C for 30 min. Fifty-microlitre TdT enzyme reaction mixture was added to the samples, and the whole setting was incubated for 30 min at 37°C in a humidified atmosphere in the dark and washed thrice in PBS for 5 min. Streptavidin-fluorescein reagent was added to the sections and incubated at 37°C in a humidified atmosphere for 30 min in the dark. After washing thrice with PBS, the cell nucleus was dyed with DAPI for 10 min. Apoptotic cells in the ovary were stained green. The sections were observed with fluorescence microscopy (ZEISS Imager A1, Germany).

Sperm chromatin dispersion (SCD) assay was performed in fresh ejaculate, frozen thawed ejaculate as well in washed spermatozoa from fresh and frozen thawed group at different intervals as described by Fernandez et al. [19 (link)] with minor modifications [20 (link)]. Briefly, the spermatozoa were mixed with 1% low melting point agarose maintained at 37°C. About 150μL of this mixture was layered on a glass slide pre-coated with 0.65% of normal melting point agarose and the gel was allowed to solidify. The slides were immersed in freshly prepared acid denaturation solution (0.08 N HCl) for 7 min at room temperature in dark. Proteins were removed by incubating the slides in lysing solution 1 containing 0.4M Tris, 20mM DTT, 1% SDS, 50mM EDTA, at pH 7.5 for 20 min followed by incubation in lysing solution 2 containing 0.4M Tris, 2M NaCl, at pH 7.5 for 15 min at room temperature. Neutralization was done in Tris buffer (0.4M Tris, pH 7.5) for 2 min, serially dehydrated in graded ethanol, and air dried. Cells were stained with Ethidium Bromide (2μg/mL) and sperm chromatin integrity was assessed in a minimum of 400 spermatozoa under fluorescent microscope (Imager- A1 Carl Zeiss, Germany). The percentage of sperm with chromatin damage was determined by counting the spermatozoa having fragmented nuclei and spermatozoa with no halo and small halo.

Differential interference microscopy (DIC) and fluorescent images were visualized with a Zeiss Axio Imager.A1 fluorescence microscope (60X or 100X objectives). Images were taken with an AxioCam MRm digital camera with ZEN Pro software (Zeiss). High-resolution fluorescent images were taken using a DeltaVision Elite deconvolution microscope equipped with a CoolSnap HQ2 high-resolution charge-coupled-device (CCD) camera. Images were processed using softWoRx software (GE). Images taken on both microscopes were additionally analyzed using ImageJ/Fiji software [94 (link),95 ].

Embryos of different periods were fixed in 4% paraformaldehyde (PFA) solution overnight at 4 °C. After fixation and gradient dehydration, embryos can store at −20 °C for long time. Linearized plasmids were employed as templates to synthesize digoxigenin-labeled antisense probes using the DIG RNA labeling mix (Roche) and T7 RNA Polymerase (TaKaRa). After synthesis, these probes were purified by the RNeasy^® FFPE Kit (Qiagen). Finally, WISH was performed as described using probes including rpl18, gata1, hbae1.1, hbbe1.1, lyz, mpx, p53, p21, and mdm2. The stained embryos were photographed using a microscope (ZEISS, Imager.A1).

The ability of spermatozoa to undergo acrosome reaction was assessed by previously described calcium ionophore (A23187)–induced acrosome reaction (CIAR) assay [22 (link)] with minor modifications. Sperm suspension was treated with or without 5 μM calcium ionophore (Cat. No. C7522, A23187, Sigma-Aldrich, USA) for 30 min at 37 °C and 5% CO₂. Post-incubation, washed cells were smeared on the coverslip permeabilized in 100% cold methanol. Cells were stained with FITC-conjugated Pisum sativum agglutinin (FITC PSA; Cat. No. L0770, Sigma-Aldrich, USA) at a concentration of 25 μg/mL at room temperature for 30 min. Washed sperm cells were then counterstained with 7 μg/mL of propidium iodide (PI; P4170, Sigma-Aldrich, USA) and mounted on a clean slide using Dako mounting medium. A minimum of 500 spermatozoa were evaluated under a fluorescence microscope (Imager-A1; Zeiss, Gottingen, Germany). Acrosome-reacted spermatozoa had no green acrosome cap (Supplementary figure 3).

For the collection of embryos, embryos of different periods were fixed in 4% PFA solution overnight at 4 °C. Embryos can be stored at −20 °C for a long time after fixation and gradient. The cDNA fragment for nop56 was cloned into pUC19 plasmid (TaKaRa Bio, Shiga, Japan) using nop56-probeF: 5′-TAATACGACTCACTATAGGGAGAGCTGCGAGATTTGGTGC-3′ and nop56-probeR: 5′-GGATCCACGTAACTGAGTGCGTTCTTTCCC-3′. Probes for other genes (scl, lmo2, gata1, c-myb, hbae1.1, lyz, lcp1) were used as previously described in [5 (link),6 (link)]. A whole mount in situ hybridization was performed as described in [22 (link)].The stained embryos were photographed using a microscope (ZEISS, Imager.A1, Oberkochen, Germany).