Sperm Head

The electrophysiological properties of individual spermatozoa were investigated using the whole cell recording technique (Hamill et al., 1981 (link); Kirichok et al., 2006 (link); Lishko et al., 2011a (link)). The recording pipettes (10–18 MΩ) were fabricated from borosilicate glass and normally filled with standard pipette solution. Gigaohm seals were obtained by bringing the pipette tip into gentle contact with the cytoplasmic droplet, which lies just behind the sperm head, and the patch of membrane spanning the pipette tip then ruptured by applying suction in conjunction with 1 ms voltage pulses (see Lishko et al., 2010 (link)). Our standard recording conditions were designed to preserve physiologically relevant Na⁺, K⁺ and Cl⁻ gradients and V_m was held (pClamp 10 Software, Axon Instruments) at a hyperpolarized value (−92 mV) between test pulses. Initial experiments were undertaken by recording the membrane currents (I_m) evoked by ramping (250 ms) V_m from −92 mV to 68 mV at 1 Hz. To analyse the results of such experiments, I_m was first normalized to input capacitance (i.e. expressed as pA pF⁻¹) to ensure that variations between the sizes of different spermatozoa did not contribute to the variability in the presented data. All cited values of V_m were corrected for the liquid junction potential between the pipette/bath solutions (E_L), and for the voltage drop across the access resistance (R_a, 62.8 ± 0.8 MΩ, n = 476 cells from 29 donors). The latter correction was applied retrospectively using the expression V_m = V_Pip − R_a·I_m, where V_Pip is the pipette potential. Since the bath was grounded via a 4% agar/3 M KCl, bridge, the bath solution changes imposed during the present study had negligible effects upon E_L. Plots showing the relationship between I_m and V_m were constructed and, unless otherwise stated, cited values of membrane conductance (G_m, pS pF⁻¹) are derived by regression analysis (i.e. ΔI_m/ΔV_m) of data recorded at positive potentials. Resting V_m was either inferred from the reversal potential (V_Rev, i.e. the value of V_m at which I_m is zero, voltage clamp experiments) or measured directly by monitoring (5 KHz, data low pass filtered at 3 KHz) the zero current potential (see Hamill et al., 1981 (link)). Experiments that quantified the responses to step changes in V_m were undertaken using an experimental design that employed the standard features of pClamp software (V/4 protocol) to subtract leak/capacitive currents from all recorded data. The statistical significance of differences between control/experimental values were determined tested using Student's paired (repeated measurements on the same cells) or unpaired (comparison between different groups of cells) t-test. The results of experiments that followed more complex protocols were analysed by one way analysis of variance (ANOVA)/Dunnet's post hoc test. Data are cited as mean ± s.e.m. and values of n refer to the number of spermatozoa in each group. All observations were confirmed using spermatozoa from at least three different donors.

Fresh semen was dropped on glass, eosin-negrosin was added, mixed homogeneously, and a smear preparation was made and dried over the flame quickly. The viability of the sperm was examined with a microscope 400×. The percentage of dead and living spermatozoa was calculated in three microscopic fields of view. The assessment of spermatozoa viability was as follows: head of live spermatozoa appeared transparent or clear, meanwhile died spermatozoa suffered damage on the plasma membrane, caused increased permeability, dyes will enter the cell and head of spermatozoa look reddish [8 ].

Sperm samples were directly recovered from the cauda of both epididymides. Spermatozoa were fixed in glutaraldehyde solution. A sub-sample of 2 µl was used to prepare the smears. Semen smears were air-dried for one day, then immersed in the glutaraldehyde fixative solution for 5 min and immediately mounted, sealing the edges with dibutyl phthalate xylene (DPX). This method avoids floating cells on the slide (Figure 1A), which greatly helps sperm morphometry analysis. Sperm samples were photographed using a high-resolution camera DXM1200 (Nikon, Tokyo, Japan) under a phase-contrast microscopy using an Eclipse E600 microscope (Nikon, Tokyo, Japan), and a 40X objective (Nikon, Tokyo, Japan). The resolution of the pictures was 3840×3072 pixels (TIFF format). A scale of 10 µm (181 pixels) was used for the measurements. The pixel size was 0.055 µm in the horizontal and vertical axes. Sperm lengths were assessed using ImageJ software (National Institutes of Health, USA). The main structures of red deer spermatozoon are shown in Figure 1B. The following sperm morphometry parameters were determined: head width, head length, proximal midpiece width, distal midpiece width, midpiece length, flagellum length, and terminal piece length (Table S1). From these measurements, we calculated other morphometric parameters such as total sperm and principal piece lengths. The head area was calculated using the formula for the area of an ellipse [31] (link), [32] :
Head perimeter was calculated using the Ramanujan's formula for calculating the perimeter of an ellipse [33] :
In both formulae, L and W are the semi-major and semi-minor axis of the sperm head, respectively. Twenty-five representative sperm were measured for each male as described by Malo et al. [16] (link).

Air-dried smears were fixed in 3% buffered glutaraldehyde for 30 min and then stained with 5% aqueous aniline blue in 4% acetic acid (pH 3.5) for seven min. The staining intensity of the sperm head was divided into three categories, namely unstained (gray/white), partially stained, and entire sperm head stained dark blue.

The smears were fixed in methanol:glacial acetic acid (3:1) at 40 °C for five min and treated with 100 µL of 0.25 mg/mL chromomycin A3 in a McIlvaine
buffer containing 10 mM MgCl₂ (pH 7.0) for 20 min. The slides were mounted using buffered glycerol and observed with a fluorescent microscope. Two types of staining patterns of the sperm head were identified, namely bright green fluorescence and dull green staining (abnormal and normal chromatin packaging, respectively).

Sperm was prepared using ejaculated sperm, caput epididymal sperm obtained by PESA, or testicular sperm obtained by TESA. For PESA, after applying local anesthesia, a fine needle was used to puncture the epididymal head with a 1-ml sperm-washing syringe. The sperm was aspirated and analyzed under an optical microscope. If no sperm was recovered, TESA was immediately performed by percutaneous puncture of testicular tissue with a needle, extraction of a convoluted seminiferous tubule with fiber tweezers, and microscopic examination of sufficient sperm. If necessary, the operation was repeated until there was enough sperm for ICSI. For patients with NOA, TESA was performed directly. Then, the in vitro matured oocytes were inseminated with sperm by ICSI.