Mouse spermatozoa with higher fertilization rates have thinner nuclei

Animals

All animal experiments were approved by the Animal Care and Use Committee at the Research Institute for Microbial Diseases at Osaka University [permit number: 2589, 3514]. C57BL/6N (B6N) and BDF1 mice (12 weeks old) were obtained from SLC (JapanSLC, Inc. Shizuoka, Japan).

Abnormality test

Based on the morphological criteria for sperm abnormality that were described previously (Touré et al., 2004; Wyrobek & Bruce, 1975; Bruce, Furrer & Wyrobek, 1974; Watanabe & Endo, 1991), we defined abnormal sperm as size outliers that showed a PC1 score > 0.55 and largely lacked the hook-shaped heads common to most sperm.

In vitro fertilization and spermatozoon collection

Females (N = 16) were superovulated by injecting 5 IU of pregnant mare serum gonadotropin (PMSG, ASKA Pharmaceutical Co., Ltd. Tokyo, Japan), and then, 5 IU of human chorionic gonadotropin (HCG, ASKA Pharmaceutical Co., Ltd. Tokyo, Japan) 48 h after the PMSG injection. The ovulated oocytes were collected from the oviducts 14 h after the HCG injection. Cumulus-enclosed oocytes were placed in 100 µl drops of TYH medium (Toyoda, Yokoyama & Hoshi, 1971) covered with paraffin oil (Nacalai Tesque, Kyoto, Japan). The spermatozoa collected by mechanically-dissecting cauda epididymides (N = 5) were placed in 100 µl drop of TYH medium. After a 2-h incubation, the sperm suspension in TYH was added to the TYH drop containing eggs at a concentration of 5 × 10⁵ sperm/ml. After 2–8 h co-insemination of the spermatozoa and oocytes, the zona-bound spermatozoa were carefully removed using a holding needle. Because of the technical difficulty associated with collecting sperm in the zona pellucida of an oocyte by manipulation (Inoue et al., 2011), we obtained zona-penetrated spermatozoa using acidic Tyrode’s solution after in vitro fertilization. Acidic Tyrode’s solution treatment did not affect the sperm head aspect ratio. Zona pellucida was removed from the oocyte in 20 µl acidic Tyrode’s solution drops on a glass slide. The spermatozoa attached to the egg surface were removed by pipetting, and subsequently, the eggs were removed from the drops.

Epididymal spermatozoa were collected by dissecting the caput, corpus and cauda epididymides (N = 5 for each experiment) and placed into 400 µl phosphate buffered saline (PBS). Spermatozoa were collected from the oviduct and uterus by flushing out these structures with PBS 4 h after coitus.

Spermatozoon imaging and analysis

The spermatozoa were coated onto a glass slide (Matsunami Glass, Osaka, Japan) and stained with 65 µM Hoechst 33342 (Life Technologies, Carlsbad, CA, USA). The slides were viewed using an Olympus IX-70 fluorescence microscope with a 10× eyepiece and 100× objective lens (Numerical aperture is 1.4). The sperm head contours were derived from image binarization using the discriminant analysis method (Otsu, 1975) in openCV (available also in ImageJ, http://imagej.nih.gov/ij/), which was customized using the C programing language.

Elliptic Fourier descriptors and principal component analysis

Each sperm head was transferred to EFDs with two-dimensional coordinates given by: (1)${\displaystyle \left\{\begin{array}{c}X\left(t\right)=\sum _{n=1}^N\left(a_{n}cos\frac{2n\pi t}{T}+b_{n}sin\frac{2n\pi t}{T}\right)\hfill \\ Y\left(t\right)=\sum _{n=1}^N\left(c_{n}sin\frac{2n\pi t}{T}+d_{n}cos\frac{2n\pi t}{T}\right)\hfill \end{array}\right.}$ where n, N, t, and T denoted the harmonic number, the maximum harmonic number, the displacement along the contour, and the total displacement, respectively. At N = 1, (X(t), Y(t)) represented an ellipse. We set a_n, b_n, c_n, and d_n as parameters of the PCs. The number of coefficients was provided as 4N-3 because normalization was carried out for the size and angle of the first harmonic ellipse with a₁ = 1, b₁ = 0, and c₁ = 0. The variable d₁ denoted the aspect ratio. We approximated the contours of the heads of spermatozoa up to 20 ellipses (N = 20), and we performed the PCA on 77 parameters and the data reconstruction on the PC scores using SHAPE (http://lbm.ab.a.u-tokyo.ac.jp/ iwata/shape/index.html) (Iwata & Ukai, 2002).

Statistics

Kolmogorov–Smirnov test, Shapiro–Wilk test for checking normality, F-tests for checking homoscedasticity, t-tests and Steel-Dwass tests were performed using custom R programs. A P-value > 0.05 was considered not significant (n.s.), whereas P-values < 0.05 (*), <0.01 (**), and <0.001 (***) were considered significant.

EFD and PCA revealed sperm head aspect ratio as unique fertility indicator

In order to compare zona penetrated spermatozoa and ejaculated sperm, we focused on contour of the nucleus, which does not change by spontaneous acrosome reaction (See Figs. S2A–S2C). To quantify the variations in sperm head morphology, we first collected spermatozoa (n = 179) from dissected B6N male cauda epididymides (Fig. 1A) and tracked the sperm head contour (Fig. 1B) by taking the pictures of each spermatozoon nucleus (Fig. S3). The tracked contour was sequentially input into a quantitative descriptor EFD method (see “Image analysis” and “Elliptic Fourier descriptors and principal component analysis” in ‘Materials and Methods’; Fig. S4). To extract normal sperm characteristics, we subsequently performed PCA after subtracting out the abnormal spermatozoa (see “Abnormality test” in ‘Materials and Methods’; Fig. S5). Applying this protocol, we quantified the variation in head morphology of normal spermatozoa, as optimally separated into multiple principal components (PCs; Fig. S6), among B6N (n = 170, Fig. 2A) and BDF1 spermatozoa (n = 163, Fig. 2B). PC1 of the B6N spermatozoa highlighted variations in width (Fig. 2A and S6), whereas PC2 highlighted variations in the hook shape of the tip (Fig. S6).

To identify the PCs that correlate with fertilization ability, we focused on PC1, PC2, and PC3, whose contribution rates were above 10% for B6N normal sperm (49.8%, 19.3%, and 12.7%, respectively; Fig. S6). First, we identified the PCs that distinguished the BDF1 from the B6N spermatozoa and the epididymis-isolated from the zona-penetrated B6N spermatozoa (see “In vitro fertilization” in ‘Materials and Methods’). Regarding mouse strain, the BDF1 and B6N spermatozoa collected from the cauda epididymis showed differences in PC1, PC2, and PC3 (Fig. 2C and S7). However, regarding spermatozoa from different collection sites of spermatozoa, the epididymis-isolated and zona-penetrated B6N spermatozoa differed in PC1, but not in PC2 or PC3 (Fig. 2C and S7). Thus, PC1 uniquely distinguished spermatozoa between mouse strains and between collection sites of spermatozoa.

Next, we tried to identify the morphological parameters contributing to PC1. The width of the sperm head, defined as the longest part of the head perpendicular to its length, seemed to be highlighted in PC1 (Fig. 2A), but the error in width measurement was large. On the other hand, the major and minor axes of the ellipse representing the lowest mode of the sperm head EFD were almost precisely overlapped with the antero-posterior (length) and dorso-ventral (width) axes of the sperm head, respectively (Fig. 3A and Fig. S8A). Thus, we used these major and minor axes to calculate the aspect ratios (minor axis divided by the major axis, equivalent to d₁ in Eq. (1); Fig. 3A). We found that the sperm head aspect ratios of BDF1 spermatozoa (n = 330) were reduced compared with those of B6N spermatozoa (Fig. 3B; n = 298; one-tailed t-test, P = 2.1 × 10⁻¹⁶), whereas the sperm head areas were not significantly different between the two groups (Figs. S9A and S9B; n = 687 for BDF1; n = 465 for B6N; two-tailed t-test, P = 0.53). Moreover, the factor loading of the aspect ratio on PC1, which was proportional to the correlation coefficient with PC1, was more than 2-fold higher than that of any of the other parameters of the EFDs (x-coordinate in Fig. 3C). Thus, by combining PCA and EFDs, we established a quantitative evaluation pipeline of multi-dimensional morphological variation, and using this pipeline, we identified a low aspect ratio, i.e., a thin sperm head, as a unique and optimal morphological indicator of the fertilization ability of spermatozoa.

The sperm head aspect ratio decreased during the progression of fertilization

We next compared the sperm head aspect ratio from cauda epididymis-isolated B6N spermatozoa (n = 118, green in Fig. 4) with zona-penetrated B6N spermatozoa isolated from the perivitelline space (PVS) (n = 133, red in Fig. 4 and Fig. S10). The sperm head aspect ratio of zona penetrated spermatozoa was significantly smaller than that of cauda spermatozoa (Fig. 4; unpaired, one-tailed t-test, P = 2.2 × 10⁻¹⁶), further confirming the validity of the sperm head aspect ratio as a fertilization indicator. Moreover, the B6N zona penetrated spermatozoa had an aspect ratio that was similar to that of BDF1 cauda epididymis-isolated spermatozoa (Fig. 4, Kolmogorov–Smirnov test, P = 0.57), which have a higher fertilization rate than B6N epididymis-isolated spermatozoa. Importantly, the mean aspect ratio of spermatozoa decreased throughout the progression of fertilization, and it was consistently negatively correlated with the fertilization ability of the spermatozoa. To investigate whether the sperm head aspect ratio decreased during sperm maturation in male mice prior to ejaculation, we collected spermatozoa from each region of the epididymis, including the caput, corpus, and cauda (Fig. 1A). The mean aspect ratio of spermatozoa from the caput (n = 69, purple box in Fig. 4) was 3.0% larger than that from the corpus (n = 85, sky blue in Fig. 4; Steel-Dwass test, P = 0.01) and 2.6% larger than that from the cauda (n = 118, green in Fig. 4; Steel-Dwass test, P = 0.008); however, the mean aspect ratios of spermatozoa from the corpus and cauda were not different from each other (Steel-Dwass test, P = 0.98). In addition, the minor axis length of the ellipse was decreased by 3.1% from the caput to the cauda (Fig. S8B; two-tailed t-test, P = 0.015), whereas the major axis length was not significantly different between those groups (two-tailed t-test, P = 0.58). Similar to the aspect ratio means, the coefficients of variation (i.e., SD normalized by mean) of the aspect ratios of B6N spermatozoa continuously decreased from the caput (8.4%), to the corpus (6.0%), to the cauda (5.3%).

Finally, we also collected B6N spermatozoa from the uteri (n = 155) and the oviducts (n = 65; Fig. 1A) of females post-coitus, and we did not observe a difference in mean aspect ratio between these two locations (unpaired two-tailed t-test, P = 0.22). Moreover, to address whether the acrosome reaction (Dan, 1952) causes the decreased aspect ratio of the zona-penetrated spermatozoa, we analyzed sperm morphology before and after the acrosome reaction by using transgenic Acr-EGFP (EGFP fused to the acrosome migrating signal; Fig. S2) mice and found that the acrosome reaction did not affect the aspect ratio in sperm nucleous. Taken together, these data demonstrate that the aspect ratio of the sperm head did not change during entry of spermatozoa into the oviduct, but decreased during spermatozoon zona penetration.

Possible mechanisms of the decreasing sperm head aspect ratio throughout the fertilization process

During spermatozoon maturation in the epididymides of B6N male mice, we found major morphological changes including decreases in head aspect ratios and minor axis lengths of the normal spermatozoa (Fig. 4 and Fig. S8B). These decreases are likely due to structural changes that take place inside the sperm nucleus, which largely occupies the sperm head. During spermatogenesis in the testis, the DNA packaging histones are replaced by protamines, which more densely package every 50–60 kb of DNA into a toroid (donut) shape (Braun, 2001). In the caput, the toroids are subsequently cross-linked by disulfide (SS) bonds, resulting in further DNA compaction (Bedford & Calvin, 1974), which is consistent with our observed decrease in the sperm head minor axis length and aspect ratio in the caput-isolated spermatozoa. Thus, we hypothesize that the decreased aspect ratio in maturing spermatozoa from the epididymis is caused by the formation of SS bonds, which can be tested using dithiothreitol to reduce the SS bonds (Bedford & Calvin, 1974) and determine whether they are indeed responsible for these morphological changes.

In the female reproductive tract, the ratio of abnormal to normal spermatozoa was previously reported to decrease considerably at the utero-tubal junction (UTJ; junction of uterine and oviduct) (Krzanowska, 1974; Nestor & Handel, 1984). Here, we showed that the normal spermatozoa isolated from the uteri and oviducts after coitus had similar sperm head aspect ratios (Fig. 4), suggesting that UTJ plays a role in eliminating the abnormal spermatozoa but not in altering normal sperm morphology. The decreases in sperm head aspect ratios that we observed during zona penetration (Fig. 4) could be due to the selection of a fractional sperm population (Utsuno et al., 2013; Beletti, Da Fontoura Costa & Viana, 2005) or to the deformation of individual sperm. Sperm deformation has been attributed to the acrosome reaction (Dan, 1952), which, in our case, we showed to be unlikely (Fig. S2), or to the mechanical force loaded at the time of the zona penetration. Thus, determining whether sperm subpopulation selection or mechanical deformation occurs during zona penetration, using methods such as time-lapse imaging and/or measuring the yield stress on the sperm head during zona penetration, will be important avenues for future examination.

Strategies for identifying the most fertile spermatozoa and implication for therapeutic applications

The correlation of sperm head morphology and fertilization ability (Figs. 2C and 4) suggests that spermatozoon morphology can be used as a strategy to screen mouse spermatozoa suitable for fertilization. Here, we showed that populations of spermatozoa with higher fertilization abilities (BDF1 cauda and B6N zona-penetrated in Fig. 4) had smaller aspect ratios, i.e., thinner heads, than other populations. The generality of this correlation should be further validated by applying our morphometry to zona-penetrated spermatozoa of other strains whose fertilization rates are well known, including DBA/2, BALB/c, 129S3/SvIm, and FVB/N. Moreover, even in spermatozoon populations with a large mean aspect ratio, there could be subpopulations with smaller aspect ratios and increased fertilization abilities. For instance, in our study, we observed a fraction of B6N cauda spermatozoa that morphologically overlapped with BDF1 cauda spermatozoa and B6N zona-penetrated spermatozoa, which have higher fertilization abilities (PC1 = 0.3–0.4 in Fig. 2C). Thus, evaluating whether these morphologically unique subpopulations have correspondingly altered fertilization abilities will be another important avenue for future examination that will ultimately enable us to screen heterogeneous spermatozoon populations to identify spermatozoa that are suitable for fertilization by their morphological characteristics, such as their sperm head aspect ratio.

Applicability to human sperm

The correlation between aspect ratio and the success of fertilization in mice is consistent with a study showing that elongated sperm is more favorable for fertilization than rounded sperm (Ramón et al., 2014). Human spermatozoa have been represented mainly by ellipses using Elliptic Fourier descriptors (Utsuno et al., 2013). Therefore, the present pipeline, which integrates geometric morphometrics with comparative morphology (Figs. 2 and 3), could be applied under microscope in clinic laboratories, as long as edge detection of multiple spermatozoa is performed with sufficiently low error rate (Fig. 1B) so as to quantitatively examine the correlation between the aspect ratio normal sperm and fertilization success among human patients. This would greatly facilitate and enhance current reproductive technologies in an objective, high-throughput manner.