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. DNA is in blue, microtubules in red and the centrosome in green. Altogether, our data show that human spermatozoa efficiently trigger the formation of bipolar spindles with centrosomes at their poles when incubated in XEE.
They suggest that patient sperm samples with different diagnosis have different spindle assembly efficiencies not associated with centrosome defects. The assays were performed independently four times with different XEE. The ratio of bipolar spindles in each experiment was then quantified as described above and normalised to the results obtained from parallel experiments with control Xenopus sperm nuclei incubated in the same XEE.
Human sperm samples have different capacities to trigger functional bipolar spindles.
Special Issue "Manufacturing a Female Gamete: An Oocyte Story"
Altogether, these results indicate that scoring spindle assembly efficiency of human sperm samples in XEE provides a novel test for assessing sperm functionality into the oocyte. Advances in this area are hampered by the lack of experimental systems that could provide functional information on the human gametes during and after fertilization. The present study describes a quantitative ex vivo heterologous system based on the use of XEE and human spermatozoa to evaluate the sperm capacity for driving microtubule nucleation and spindle assembly, providing a functional overview of the sperm behaviour during fertilization and early development.
Although previous studies had used XEE to study human spermatozoa 17 , here we have expanded the array of functional analyses and taken them to a quantitative level to define different chromatin and microtubule-associated events that must occur for a successful fertilization and early development.
We have demonstrated that this heterologous system recapitulates the human sperm DNA decondensation-condensation and replication events as well as the assembly of functional spindle structures. Furthermore, we found that it is sensitive enough to detect small differences mainly in spindle assembly in different samples. Our data suggest that these differences could be used as functional markers for sperm samples. However, to obtain statistically significant differences due to the natural variability of the sperm samples and the XEE, each sperm sample has to be analysed at least 4 times, and this could be particularly problematic for the analysis of samples with severe oligozoospermia.
Some of the sperm factors reported to be related with poor embryo development are severe DNA damage 18 , epigenetic abnormalities 19 , severe aneuploidies 20 and centrosome dysfunction 21 , among others. One of the earliest and critical events that occur in the zygote is the formation of a functional bipolar spindle to segregate correctly the genetic material into two daughter cells According to Simerly et al.
We validated our functional system by scoring 26 patient samples with a variety of diagnosis for bipolar spindle assembly in XEE, and found that samples with a higher percentage of motile spermatozoa, higher concentration and a normal morphology trigger bipolar spindle assembly more efficiently. They also provide some molecular evidences to the open question of whether the sperm selection criteria currently in use are effective clinically. Indeed, our results provide a validation of the current sperm selection criteria showing that normozoospermic samples with optimal parameters have higher probabilities to support fertilization and early development.
We are currently expanding the analysis of samples including sperm with severe abnormalities, such as DNA damage, to further validate our system and to explore its clinically application. To our knowledge this is the first assay that provides a correlation between semen diagnosis parameters and molecular data related to cell division in the oocyte.
With this validation, we posit that our system will be useful to study several sperm dependent processes as well as the causes of male idiopathic infertility. In ART, the timing of pronuclear formation and cell division is an important indicator of fertilization and embryo quality. This would indicate whether the timing of pronuclei formation is a limiting factor for embryo development. Several studies remarked the importance of the centrosome for pronuclei migration, syngamy and embryo cleavage 21 , 24 , Indeed, individual cases of severe asthenoteratozoospermia with abnormal head-to-tail attachment exhibit no microtubule nucleation from the centrosome once they are injected into a bovine oocyte Another study showed that the average rate of sperm aster formation was lower for spermatozoa from infertile patients injected in bovine oocytes than those produced by spermatozoa from fertile patients Our system could be very useful to analyse whether some idiopathic infertility cases are due to abnormal centrosome function as well as to obtain quantitative data for the basal body to centrosome transition and its microtubule nucleation activity in vitro for specific sperm samples.
Overall, we have developed and validated a quantitative heterologous system using human spermatozoa and XEE that mimics the events triggered by the human spermatozoa during fertilization. The system was validated using clinical data. It provides a quantitative approach to address molecular mechanisms that are very difficult to study in human oocytes due to ethical restrictions. Moreover, it reduces the bias originated by the heterogeneous human oocyte sample population, which in clinical studies cannot be ignored. It will be particularly interesting to use this system for cases of unexpected fertilization and early embryo development failures.
Biology and Pathology of the Oocyte
The system could provide some molecular evidences to address these infertility cases. All procedures performed were in accordance with the ethical standards of the institutional research committees and with the Helsinki declaration, as revised in Written informed consent to participate was obtained from all participants prior to their inclusions in the study.
The semen was classified according to the World Health Organization guidelines The ejaculate was then cryopreserved in straws with Sperm Cryoprotect II Nidacon, Molndal, Sweden and stored in liquid nitrogen up to sample preparation. Fertilization was assessed 14 to h post-ICSI by visualization of two pronuclei and two polar bodies. Xenopus spermatozoa were treated as previously described Xenopus laevis frogs were purchased from Nasco and used at an age between 1 and 3 years. Briefly, Xenopus laevis frogs were stimulated with and 1, I.
To analyse spindle assembly in cycled XEE, 3, sperm nuclei human or Xenopus together with 0.
Role in Fertility, Medicine and Nuclear Reprograming
Samples were visualised with an inverted DMI Leica wide-field fluorescent microscope using a 63x objective. The same microscope and image processing was used for all the experiments. The sample was then washed twice with PBS 0. Rhodamine-labelled tubulin was added to the XEE to visualise the nucleation of microtubules.
Centrioles of the bipolar spindles were detected by adding Nocodazole 0. The percentage of detected centrioles at both poles, one pole and no centrioles was quantified for Xenopus , human normozoospermic and asthenozoospermic sperm samples in four independent experiments. To assess the capacity of the human centrioles to recruit PCM and to nucleate microtubules, we used a previously published protocol with minor modifications The coverslips were quenched with 0. Microtubule and centrosomes were visualised by immunostaining with a rabbit monoclonal anti-beta-tubulin antibody ab Abcam, and a mouse polyclonal anti-centrin antibody 20H5 Millipore, , To apply the statistical analyses, first of all we evaluated the normality of the samples.
Visual inspection of the distribution using Q-Q plot quantile-quantile plot and Shapiro-Wilk test were performed.
For non-normal distributed variables, logarithm transformation was done before subsequent analyses i. DNA decondensation was still not normal, non-parametric tests Mann-Whitney test were applied. Dispersion data in all the analyses is given as Standard Deviation SD.
Biology and Pathology of the Oocyte: Role in Fertility, Medicine and Nuclear Reprogramming | Alumni
New York, USA. For each sperm sample triggering bipolar spindles in XEE, and sperm nuclei were counted for Xenopus and human samples, respectively in individual XEE a total of 4 independent XEE incubations with a total number of and Xenopus and human sperm nuclei analysed, respectively.
The microtubule structures associated with the spermatozoa DNA were classified as: bipolar spindles, abnormal structures or no structures. For spindle length measurements, the length of 20 spindles per sample was analysed using a straight line from ImageJ software. To analyse spindle assembly by sperm patients samples, the percentage of bipolar spindles was normalised on the percentage of Xenopus sperm bipolar spindles in the same XEE.
The linear relationship between bipolar spindles and the semen parameters was assessed using Pearson correlation, as well as the relationship between bipolar spindles and fertilization rate. Steptoe, P. Birth after the reimplantation of a human embryo. Lancet 2 , Palermo, G. Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection.
Biology and Pathology of the Oocyte: Role in Fertility, Medicine and Nuclear Reprograming.
Fertil Steril 59 , — Joergensen, M. Altered cleavage patterns in human tripronuclear embryos and their association to fertilization method: a time-lapse study. Liu, L. Clinical outcome of cycles with oocyte degeneration after intracytoplasmic sperm injection. Ebner, T. Planar embryos have poor prognosis in terms of blastocyst formation and implantation. Jin, H. Embryo developmental potential of microsurgically corrected human three-pronuclear zygotes. Clift, D. Restarting life: fertilization and the transition from meiosis to mitosis.
Johnson, G. The sperm nucleus: chromatin, RNA, and the nuclear matrix. Avidor-Reiss, T. Atypical centrioles during sexual reproduction. Bettencourt-Dias, M. Centrosome biogenesis and function: centrosomics brings new understanding. Cavazza, T. From meiosis to mitosis - the sperm centrosome defines the kinetics of spindle assembly after fertilization in Xenopus.
Karsenti, E. The mitotic spindle: a self-made machine. Hannak, E. Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts. Lohka, M. Induction of metaphase chromosome condensation in human sperm by Xenopus egg extracts. Exp Cell Res , — Ohsumi, K. Human sperm nuclei can transform into condensed chromosomes in Xenopus egg extracts.
Simerly, C. Nat Med 1 , 47—52 Biparental inheritance of gamma-tubulin during human fertilization: molecular reconstitution of functional zygotic centrosomes in inseminated human oocytes and in cell-free extracts nucleated by human sperm.