Functional Analysis of Human Pathological Semen Samples in an Oocyte Cytoplasmic Ex Vivo System

Abstract

Human fertilization and embryo development involve a wide range of critical processes that determine the successful development of a new organism. Although Assisted Reproduction Technologies (ART) may help solve infertility problems associated to severe male factor, the live birth rate is still low. A high proportion of ART failures occurs before implantation. Understanding the causes for these failures has been difficult due to technical and ethical limitations. Diagnostic procedures on human spermatozoa in particular have been limited to morphology and swimming behaviours while other functional requirements during early development have not been addressed due to the lack of suitable assays. Here, we have established a quantitative system based on the use of Xenopus egg extracts and human spermatozoa. This system provides novel possibilities for the functional characterization of human spermatozoa. Using clinical data we show that indeed this approach offers a set of complementary data for the functional evaluation of spermatozoa from patients.

Figure 1

Human spermatozoa nuclei reorganise and replicate their DNA in XEE. (A) Schematic representation of the experimental design. 3,000 human (or Xenopus) sperm nuclei and calcium were incubated in the XEE to mimic fertilization. These incorporations induce the resumption of the cell cycle, first by 90 min of interphase followed by 60 min of mitosis. (B,C) Time course of sperm nucleus decondensation. Xenopus and human spermatozoa (normozoospermic and asthenozoospermic) incubated in XEE were retrieved at different time points along the 150 min of the experiment, to measure the nuclei area. The graph shows the area evolution in µm² of the three different sperm samples. ^aSignificantly different p-value of 0.010. ^bNo significantly different p-value of 0.89. Dispersion data are given as SD. The images on the right are representative pictures of the nuclei decondensation per time point. Scale bar, 10 µm. (D,E) Time course of sperm DNA replication. Xenopus and human (normozoospermic and asthenozoospermic) spermatozoa, calcium and biotin-labelled dUTPs were incubated in the XEE and retrieved every 30-min. The graph shows the percentage of incorporated dUTPs per sample. ^aSignificantly different p-value of 0.017. ^bSignificantly different p-value of 0.013. Dispersion data are given as Standard Deviation (SD). The images are representative pictures of the dUTPs incorporation. dUTPs are in green, DNA in blue and microtubules in red. Scale bar, 10 µm.

Figure 2

The human sperm basal body is converted into a fully functional centrosome in the oocyte cytoplasm. (A) Human sperm basal body actively nucleates microtubules in XEE. Immunofluorescence images of KI treated sperm samples incubated with XEE and pure tubulin. Microtubule asters are in red and centrioles in green. Scale bar, 5 µm. (B,C) Capacity of the Xenopus and human spermatozoa to assemble microtubule mitotic structures. The microtubules are in red and the DNA in blue. The structures associated with the DNA were classified as bipolar spindles, abnormal structures and no structures as shown in the images on the right. Scale bar, 10 µm. The graph on the left shows the analysis of 100 and 200 sperm nuclei for Xenopus and human samples respectively. No significant differences were found in any of the samples and microtubule structures. Dispersion data are given as Standard Deviation (SD).

Figure 3

Human spermatozoa are able to assemble bipolar spindles in XEE independently of the sperm diagnosis. (A) Capacity to assemble microtubule mitotic structures of human spermatozoa samples with different diagnosis. The graph shows the percentage of bipolar spindles, abnormal structures and no structures per sample. Only significant differences were detected when comparing abnormal structures with normozoospermic and asthenozoospermic samples (p = 0.010). Dispersion data are given as Standard Deviation (SD). (B,C) Human basal body duplicates in XEE. Bipolar spindles assembled in XEE were treated with Nocodazole and processed for immunofluorescence. The number of bipolar spindles with centrosomes at both poles were analysed in 2 normozoospermic and 2 asthenozoospermic samples and normalised by the number of bipolar spindles with centrosomes at both poles in Xenopus spermatozoa. Dispersion data are given as Standard Deviation (SD). DNA is in blue, microtubules in red and the centrosome in green. Scale bar, 10 µm.

Figure 4

Human sperm samples have different capacities to trigger functional bipolar spindles. (A–D) The percentage of bipolar spindles per XEE was normalised by the percentage of bipolar spindles triggered by Xenopus spermatozoa in the same XEE. The average of bipolar spindles of each sample in the four XEE was correlated with each sperm sample characteristics and clinical outcomes for their respective IVF/ICSI cycle.