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Nuclear volume differences between balanced and unbalanced spermatozoa in chromosomal translocation carriers
Reproductive BioMedicine Online (2015) 30, 290–295
w w w. s c i e n c e d i r e c t . c o m w w w. r b m o n l i n e . c o m
ARTICLE
Nuclear volume differences between balanced and unbalanced spermatozoa in chromosomal translocation carriers Alexandre Rouen a,*, Alinoë Lavillaureix a, Capucine Hyon a, Solveig Heide a, Sylvain Clède b, Richard Balet c, Esther Kott a, Nino Guy Cassuto d, Jean-Pierre Siffroi a a
Service de Génétique et Embryologie Médicales, Hôpital Armand-Trousseau, AP-HP, Unité INSERM U933, 26 Avenue du Dr Arnold Netter, Paris 75012, France; b Ecole Normale Supérieure, Département de Chimie, Sorbonne Universités, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules, UMR7203, 25 rue Lhomond, 75231 Paris Cedex 05, France; c Service de Médecine de la Reproduction, Clinique des Bluets, 4 rue Lasson, 75571 Paris Cedex 12, France; d Laboratoire Drouot, Clinique des Bluets, 4 rue Lasson, 75571 Paris Cedex 12, France * Corresponding author.
E-mail address: [email protected] (A. Rouen). Alexandre Rouen, MD, is a medical geneticist at Trousseau Hospital, in Paris, France. He specializes in infertility of genetic origin, and conducts research focusing on improving reproductive care in patients with genetic defects, notably chromosomal rearrangements.
While chromosomal translocations are usually associated with a normal phenotype, they can still cause male infertility as well as recurrent miscarriages and fetal malformations related to their transmission in an unbalanced state. The distinction between balanced and unbalanced spermatozoa on morphological criteria is still unfeasible. However, we previously showed that: i) spermatozoa with an unbalanced content have a higher rate of DNA fragmentation; and ii) that density gradient centrifugation partially separates balanced from unbalanced sperm cells. We hypothesized that a chromosomal imbalance could alter the fine spermatic nuclear architecture and consequently the condensation of DNA, thus modifying normal sperm density. Spermatic nuclear volumes in four translocation carriers were analyzed using confocal microscopy. Secondarily, FISH analysis was used to establish the segregation mode of each spermatozoon. We found the average spermatic nuclei size to be higher among unbalanced spermatozoa in all patients but one. All the unbalanced modes were associated with larger nuclei in two patients, while this was the case for the 3:1 mode only in the other two, suggesting an abnormal condensation. This could be the first step in elaborating a procedure to completely eliminate unbalanced spermatozoa from semen prior to in vitro fertilization.
Abstract
© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.rbmo.2014.10.019 1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Unbalanced spermatozoa in translocation carriers have larger nuclei
291
KEYWORDS: chromosomal translocations, confocal microscopy, infertility, spermatic DNA
Introduction Chromosomal translocations are found in the general population with a prevalence of 1/500, and are of two types: reciprocal and Robertsonian translocations (Meschede et al., 1998). They are usually balanced, and associated with a normal phenotype. However, they can lead to infertility in both male and female carriers. The abnormal chromosomal segregation during meiosis leads to the presence of chromosomally unbalanced gametes, the proportion of which varies among patients. Fertilization of an unbalanced gamete can have different outcomes, depending on the size and density in genes of the involved chromosomal segments: spontaneous abortion, fetal malformations, developmental delay and learning disabilities (Ravel et al., 2006). There is presently no way of identifying unbalanced spermatozoa in male translocation carriers since no morphological character has been identified for them (Cassuto et al., 2011). The proportion of unbalanced spermatozoa in a given translocation carrier is established through fluorescent in situ hybridization (FISH), however this procedure does not allow sperm retrieval and using for subsequent IVF. Therefore, the only two therapeutic options for those couples are either natural conception followed by prenatal diagnosis and pregnancy termination of an affected fetus, or preimplantation genetic diagnosis. However, we previously described a simple way to improve sperm quality in translocation carriers (Rouen et al., 2013b). Indeed, apart from abnormal chromosomal segregation, translocations have been associated with abnormal seminal parameters in male carriers, as well as to increased spermatic apoptosis and DNA fragmentation (Brugnon et al., 2006, 2010; Perrin et al., 2009). Furthermore, an increase in DNA fragmentation rate has been reported in unbalanced versus balanced spermatozoa in these men (Rouen et al., 2013a). This led to using a sperm procedure called discontinuous gradient centrifugation (DGC), which is currently used in IVF procedures in some centers for partially eliminating apoptotic spermatozoa and improving sperm quality, in order to decrease the proportion of unbalanced spermatozoa. This procedure leads to a partial but significant decrease in the proportion of unbalanced sperm (Rouen et al., 2013b). A multicentric program has since been launched by our center and already led to the birth of one healthy child, in a couple with a 10-year history of infertility and recurrent miscarriages (Rouen et al., 2014). The efficiency of DGC in partially eliminating unbalanced spermatozoa suggests that those cells have a different density compared with their balanced counterparts. This difference could be explained by an altered nuclear condensation, related to an abnormal chromosomal architecture. Indeed, spermatic nuclei are characterized by an extreme condensation, which is essential in protecting the genetic information along the male and female genital tracts. This condensation is made possible by the replacement of somatic histones by protamines and by a fine and remarkably constant nuclear architecture (Mudrak et al., 2005): chromosomes are bent at their centromeres, with their telomeric extremities facing outward and bound to each other, and their centromeres gathered together in a structure termed chromocentre
(Ioannou and Griffin, 2011). Furthermore, each chromosome has a relatively constant location in the nucleus (Zalenskaya and Zalensky, 2004). Therefore, the question arises whether the presence of translocated chromosomes and/or the lack or excess of chromosomal segments could alter this fine nuclear architecture, and therefore impair the condensation process. This could lead to an increased nuclear volume and a change in cellular density which could explain the partial elimination of those cells during DGC. The aim of the present study was to investigate the relationship between nuclear volume, measured in three dimensions through confocal microscopy, and chromosomal segregation, in chromosomal carriers.
Materials and methods This study was conducted in the Medical Genetics and Embryology department at Trousseau Hospital and in the Assisted Reproduction Department at Clinique des Bluets (Paris, France). Genetic analyses were performed according to the French reglementation, edicted by the Agence de la Biomédecine. Informed written consent was obtained from each participant. Four translocation carriers were included. Patient P1, 25 years old, carried a t(1;18)(p22;q21.1) reciprocal translocation. It was diagnosed from a chromosomally unbalanced pregnancy, which was voluntarily interrupted. Semen analysis showed severe oligozoospermia (1 million/ml). Patient P2, 36 years old, carried a t(5;12)(q13;q13) reciprocal translocation. It was diagnosed after the occurrence of three spontaneous abortions. Semen analysis showed normal sperm parameters. Patient P3, 37 years old, carried a t(2;10)(p10,q10) reciprocal translocation. He and his partner had had four spontaneous abortions. Semen analysis showed asthenoteratozoospermia. Patient P4, 29 years old, carried a rob(13;14)(q10,q10) Roberstonian translocation which was diagnosed after the same translocation was found in his brother, who consulted for primary infertility. Semen parameters were within the normal limits. Samples were obtained by masturbation from those four patients, after a 3 to 5-day abstinence period. After 30 minutes at 37°C for liquefaction, spermatozoa are washed with PBS (centrifugation at 500 × g for 10 minutes), before fixation with methanol and acetic acid and freezing at −20°C. The main steps of the protocol are summarized in Figure 1. Confocal microscopy analysis: spermatozoa were placed on a slide, and their DNA was stained with DAPI (4′,6-diamidino2-phenylindole) and an antifading agent (Prolong Gold Antifade Reagent, Life Technologies, CA, USA). Acquisition was performed on a Nikon Eclipse 80i confocal microscope equipped with the Nikon EZ C1 acquisition software. Each acquisition, containing around 20 spermatozoa, comprised around 30 images with a resolution of 512 × 512 and a distance of 300 nm along the z axis between each image. Each set of images was
292
A Rouen et al.
Figure 1 Main steps of the study’s protocol. After DAPI staining of the spermatozoa, their nuclear volumes are measured through confocal microscopy and computer analysis (ImageJ software). Secondly, a FISH assays allows for the assessment of the segregation mode of each spermatozoon.
Table 1 Spermatic nuclear volumes (µm3) in all four patients, according to the spermatozoa’s genetically balanced or unbalanced status. The mean nuclear volume is higher in unbalanced spermatozoa than in balanced spermatozoa, in all patients but P1.
P1 P2 P3 P4
Balanced
Number of analyzed spermatozoa
Unbalanced
Number of analyzed spermatozoa
Volume variation
53.45 47.93 74.37 49.30
95 50 52 70
54.64 51.84* 84.15* 59.90*
100 76 70 30
+2% +8% +13% +22%
*P < 0.05.
accurately localized on the slide, and the x and y coordinates were noted down. Tridimensional computer analysis: open source software ImageJ, as well as open source plug in 3D Object Counter (Fabrice Cordelières, Institut Curie, Orsay, Paris), were used to perform tridimensional analysis of the spermatic nuclei volumes. They were first evaluated in pixels, then conversion to µm3 was made possible owing to the known values of resolution and step distance in µm for each set of images. For each aquisition, boundary decisions were automated in ImageJ, using a set threshold level. 3D analysis was conducted in a blinded manner, as it was performed prior to chromosomal segregation analysis. Confocal microscopy was used to measure spermatic heads volumes as this allowed secondary analysis of chromosomal segregation. Other volume measurement methods, such as atomic force microscopy, while possibly more accurate, would have led to spermatic DNA disruptions, preventing further chromosomal analysis. Chromosomal segregation analysis: for each spermatozoon, the chromosomal segregation mode (alternate, adjacent 1, adjacent 2, and 3:1 for reciprocal translocations, and alternate, adjacent, and 3:0 for Roberstonian translocations) was assessed through FISH assay. For Robertsonian translocations, two subtelomeric probes were used, as well as a centromeric probe for a chromosome not involved in the rearrangement in order to distinguish an abnormal sperm cell after a 3:0 translocation from a diploid cell. For reciprocal translocations, two subtelomeric probes for each chromosome were used, as well as a centromeric probe for one of those two chromosomes. Non-commercial subtelomeric probes were prepared from BACs (bacterial artificial chromosomes) using an RCA (rolling circle amplification) procedure (Berr and Schubert, 2006). Spermatozoa were deposited on a glass slide and left to incubate in 2 SSC NP40 (saline
sodium citrate, nonyl-phenoxypolyethoxyethanol) for 30 minutes. They were then dehydrated in ethanol (three successive washes : 70%, 85%, and 95%). Nuclear heads were decondensed in 1M sodium hydroxide and dehydrated again. Denaturation of both nuclear DNA and probes was conducted in hybridization buffer under cover glass on a plate at 73°C for 2 min 30s. Hybridization was then performed at 37°C for 24 hours on a programmable hot plate (Slidebooster®, Beckman Coulter, FL, USA) in a way to increase signal intensity. The next day, the cells were washed in 0.4 SSC-NP40 in 73°C for 30 seconds and for two minutes at 37°C in 2 SSC NP40, before being counterstained with DAPI. Each spermatozoon previously acquired on confocal microscopy was relocated, and its chromosomal content was assessed. Statistical analysis: a t student test was used for statistical analysis of both sizes and standard deviations, and results with P < 0.05 were considered as significant.
Results We examined spermatic nuclear volumes according to the genetically balanced or unbalanced status of the spermatozoa. We found significantly larger nuclei among unbalanced spermatozoa than in balanced ones in all patients but P1 (P < 0.05, Table 1). The average differences were 3.91 µm3 (+8%) for P2, 9.78 µm3 (+13%) for P3, and 10.6 µm3 (+22%) for P4. The same analysis was conducted for each segregation mode (Table 2). For P2 and P4, the spermatozoa from all the unbalanced modes had a significantly larger nuclear volume than the balanced spermatozoa (P2 all comparisons P < 0.05; for P2 adjacent versus alternate P < 0.05 and 3:0 mode versus versus alternate P < 0.01). For P1 and P3, spermatozoa from
Unbalanced spermatozoa in translocation carriers have larger nuclei
293
Table 2 Spermatic nuclear volumes (µm3) in all four patients, according to the spermatozoa’s segregation modes: alternate, adjacent 1, adjacent 2, and 3:1 in reciprocal translocation, and alternate, adjacent, and 3:0 in the Robertsonian translocation. In P1 and P3, the mean spermatic nuclear volumes are significantly increased, compared with balanced spermatozoa, for spermatozoa from the 3:1 segregation mode. In P2 and P4, the mean spermatic nuclear volumes are significantly increased in all unbalanced modes, compared with balanced alternate spermatozoa.
P1 P2 P3
P4
Alternate
n
Adjacent 1
n
Δ
Adjacent 2
n
Δ
3:1
n
Δ
53.45 47.93 74.37
95 50 52
51.10 51.28* 58.52
60 40 5
−4% +7% +21%
57.78 54.03* 84.16
9 28 5
+8% +13% +13%
60.76* 51.34* 85.70*
31 8 60
+14% +7% +15%
Alternate
n
Adjacent
n
Δ
3:0
n
Δ
49.30
70
59.05*
26
+20%
79.30**
4
+61%
*P < 0.05, **P < 0.01.
the 3:0 mode only were found to have significantly larger nuclei than balanced spermatozoa (both P < 0.05). The segregation mode with the largest spermatic nuclei was the 3:1 mode (or 3:0 for P4), except for P2, for whom it was the adjacent 2 mode.
Discussion Chromosomal translocation carriers produce both balanced gametes, either carrying the translocation or not, and unbalanced ones due to an abnormal segregation of the translocation during meiosis. In males, the proportion of unbalanced spermatozoa for a given patient is now routinely established by FISH. However, gametes analyzed by this technique are no longer available for further fertilization using insemination or in vitro methods (IVF, ICSI). Several authors, including our team, have tried to identify unbalanced spermatozoa in translocation carriers by searching for morphological characteristics in living gametes that would allow their use in assisted reproductive techniques (ART) (Cassuto et al., 2011). Unfortunately, no defining character had been found, except for an increased DNA fragmentation among unbalanced spermatozoa (Rouen et al., 2013a). This finding led us to test discontinuous gradient centrifugation (DGC), a well-established method performed in most ART laboratories prior to IVF, as a tool for separating balanced from unbalanced spermatozoa based on their DNA integrity. Indeed, DGC selects preferentially motile and nonapoptotic sperm cells (Rouen et al., 2013b). We showed that it increases the percentage of spermatozoa with a balanced nuclear content in translocation carriers as well, thus raising the question whether a chromosomal imbalance could slightly modify sperm head volume and consequently nuclear density. In our experiments, confocal microscopy was performed prior to FISH in order to avoid measurement bias due to the hybridization of probes. Indeed, FISH procedure includes a decondensation step which modifies sperm head volumes. Spermatic nuclear volumes were assessed after staining of nuclei with DAPI. Since most of the cytoplasm is eliminated from the spermatozoa’s heads after spermatogenesis, the measured volume grossly equals that of the spermatic head.
Approximately 30–40 images were acquired along the × axis for each spermatozoon, making the volume estimate as reliable as possible. Contradictory data exist in the literature about human sperm head volume. Using atomic force microscopy, Lee et al. found that the average volume of normal sperm heads was 5.02 ± 0.37 µm3 (Lee et al., 1997). Similar values were found by Coppola et al. who measured a mean sperm head nuclear volume of 8.03 ± 0.75 µm3 using digital holographic microscopy (Coppola et al., 2014). Values drawn from these two studies are in agreement with that by Maree et al. (2010) which found nuclear sperm area values ranging from 9.26 ± 0.99 to 12.87 ± 1.19 µm2 according to the staining method used in light microscopy. Owing to the fact that mature spermatozoa have a flat shaped head (Holstein and Roosen-Runge, 1981), such area values are compatible with nuclear volumes around 10 µm3. In contrast, Hazzouri et al. (2000) studied human sperm genome organization by FISH and confocal microscopy after various decondensation methods and found very high nuclear volumes, up to 283 µm3, with a minimal value of 70 µm3 obtained after treatment with heparin which is a milder decondensation agent than NaOH. Intermediate volume values were reported by Curry et al. (1996) who found a mean human sperm head volume of 22.2 ± 1.2 µm3 by using stereological methods, in agreement with older works. (Brotherton, 1975; Smith et al., 1988). Our own measurements of sperm head volumes give a mean value of nearly 57 µm3 in controls, which is more than two times higher than these latter values. We hypothesized that an overestimation of distances along the z axis in confocal microscopy could be responsible for this discrepancy. Indeed, a mismatch between the microscopic object and the immersion oil has been described to lead to overestimate the size of the objects (Visser and Oud, 1994). This was confirmed by performing acquisitions with the same procedure of other objects of known dimensions and shape, such as lymphocytes. They appeared elongated along the z axis and not spherical (data not shown), and had a measured volume twice as high as the actual volume. We however considered that such an overestimation of volume values did not cause any bias, since the effect was equally present for balanced and unbalanced cells. The observed differences therefore raise the question of whether the presence of
294 abnormal translocated chromosomes disrupt the spatial organization of DNA in the spermatic nucleus. Individual chromosome territories are known to be organized in a non-random way within the sperm nucleus (Hazzouri et al., 2000; Zalenskaya and Zalensky, 2004). Consequently, any disturbance in sperm chromosomal arrangement, due to changes in particular chromosomal segment positions in translocations or to an excess and/or a lack of chromosomal content in unbalanced gametes, may hinder the fine and precise architecture of sperm nucleus and could prevent the normal condensation of DNA, leading to spermatozoa with an increased head volume. According to the nature and/or the length of the translocated chromosomal segments, as well as the mode of segregation leading to unbalanced gametes, impairments in normal DNA condensation could be variable and, therefore, could have also variable consequences on sperm head volume. We observed such variability among our patients since P2 and P4 exhibited increased nuclear volumes in spermatozoa issued from all segregation modes while, in P1 and P3, this was observed only in sperm cells from the 3:1 mode. Interestingly, in reciprocal translocations, this mode of segregation is the only one associated with an abnormal number of chromosomes, and therefore an abnormal number of centromeres, which have a major role in the sperm nuclear organization through the constitution of the chromocenter (Ioannou and Griffin, 2011). We were only able to study four patients, which is admittedly low. Further work should aim at studying a greater number of subjects, as well as carriers of other chromosomal rearrangements, such as chromosomal inversions. We however obtained statistically significant results because of the high number of analyzed spermatozoa. In other words, each patient was his own control: unbalanced spermatozoa were compared with balanced spermatozoa, for each subject. The present study shows that in male chromosomal rearrangement carriers, genetically unbalanced spermatozoa have significantly larger nuclei than their balanced counterparts. This is especially true for the most unbalanced of the modes, the 3:1 (or 3:0) one. This finding is appealing on both the pathophysiological and clinical points of view. As mentioned before, it is likely that it is the disturbance of the fine spermatic nuclear organization that hampers normal DNA condensation, making the cell more vulnerable to DNA fragmentation and apoptosis. There could be implications in the clinical setting. With this study, we provide a possible pathophysiological explanation to the partial elimination of unbalanced spermatozoa by discontinuous gradient centrifugation. This could open up possibilities in selecting balanced spermatozooa prior to IVF.
Acknowledgement The authors would like to thank the patient association Valentin for their collaboration.
References Berr, A., Schubert, I., 2006. Direct labelling of BAC-DNA by rollingcircle amplification. Plant J. 45, 857–862.
A Rouen et al. Brotherton, J., 1975. The counting and sizing of spermatozoa from ten animal species using a Coulter counter. Andrologia 7, 169– 185. Brugnon, F., Van Assche, E., Verheyen, G., Sion B., Boucher D., Pouly, J.L., Janny, L., Devroey, P., Liebaers, I., Van Steirteghem, A., 2006. Study of two markers of apoptosis and meiotic segregation in ejaculated sperm of chro- mosomal translocation carrier patients. Hum. Reprod. 21, 685–693. Brugnon, F., Janny, L., Communal, Y., Darcha, C., Szczepaniak, C., Pellestor, F., Vago, P., Pons-Rejraji, H., Artonne, C., Grizard, G., 2010. Apoptosis and meiotic segregation in ejaculated sperm from Robertsonian translocation carrier patients. Hum. Reprod. 25, 1631–1642. Cassuto, N.G., Le Foll, N., Chantot-Bastaraud, S., Balet, R., Bouret, D., Rouen, A., Bhouri, R., Hyon, C., Siffroi, J.P., 2011. Sperm fluorescence in situ hybridization study in nine men carrying a Robertsonian or a reciprocal translocation: relationship between segregation modes and high-magnification sperm morphology examination. Fertil. Steril. 96, 826–832. Coppola, G., Di Caprio, G., Wilding, M., Ferraro, P., Esposito, G., Di Matteo, L., Dale, R., Coppola, G., Dale, B., 2014. Digital holographic microscopy for the evaluation of human sperm structure. Zygote. 22, 446–454. Curry, M.R., Millar, J.D., Tamuli, S.M., Watson, P.F., 1996. Surface area and volume measurements for ram and human spermatozoa. Biol. Reprod. 55, 1325–1332. Hazzouri, M., Rousseaux, S., Mongelard, F., Usson, Y., Pelletier, R., Faure, A.K., Vourc’h, C., Sèle, B., 2000. Genome organization in the human sperm nucleus studied by FISH and confocal microscopy. Mol. Reprod. Dev. 55, 307–315. Holstein, A.F., Roosen-Runge, E.C., 1981. Atlas of Human Spermatogenesis. Grosse, Berlin. Ioannou, D., Griffin, D.K., 2011. Male fertility, chromosome abnormalities, and nuclear organization. Cytogenet. Genome Res. 133, 269–279. Lee, J.D., 4th, Allen, M.J., Balhorn, R., 1997. Atomic force microscope analysis of chromatin volumes in human sperm with headshape abnormalities. Biol. Reprod. 56, 42–49. Maree, L., du Plessis, S.S., Menkveld, R., van der Horst, G., 2010. Morphometric dimensions of the human sperm head depend on the staining method used. Hum. Reprod. 25, 1369–1382. Meschede, D., Lemcke, B., Exeler, J.R., De Geyter, C., Behre, H.M., Nieschlag, E., Horst, J., 1998. Chromosome abnormal- ities in 447 couples undergoing intracytoplasmic sperm injection- prevalence, types, sex distribution and reproductive relevance. Hum. Reprod. 13, 576–582. Mudrak, O., Tomilin, N., Zalensky, A., 2005. Chromosome architecture in the decondensing human sperm nucleus. J. Cell Sci. 118, 4541–4550. Perrin, A., Caer, E., Oliver-Bonet, M., Navarro, J., Benet, J., Amice, V., De Braekeleer, M., Morel, F., 2009. DNA fragmenta- tion and meiotic segregation in sperm of carriers of a chromosomal structural abnormality. Fertil. Steril. 92, 583–589. Ravel, C., Berthaut, I., Bresson, J.L., Stiffroi, J.P., 2006. Prevalence of chromosomal abnormalities in phenotypically normal and fertile adult males: large-scale survey of over 10 000 sperm donor karyotypes. Hum. Reprod. 21, 1484–1489. Rouen, A., Pyram, K., Pollet-Villard, X., Hyon, C., Dorna, M., Marques, S., Chantot-Bastaraud, S., Joyé, N., Cassuto, N.G., Siffroi, J.-P., 2013a. Simultaneous cell by cell study of both DNA fragmentation and chromosomal segregation in spermatozoa from chromosomal rearrangement carriers. J. Assist. Reprod. Genet. 30, 383–390. Rouen, A., Balet, R., Dorna, M., Hyon, C., Pollet-Villard, X., ChantotBastaraud, S., Joyé, N., Portnoï, M.-F., Cassuto, N.G., Siffroi, J.P., 2013b. Discontinuous gradient centrifugation (DGC) decreases the proportion of chromosomally unbalanced spermatozoa in chromosomal rearrangement carriers. Hum. Reprod. 28, 2003–2009.
Unbalanced spermatozoa in translocation carriers have larger nuclei Rouen, A., Hyon, C., Balet, R., Joyé, N., Cassuto, N.G., Siffroi, J.-P., 2014. First birth after sperm selection through discontinuous gradient centrifugation and artificial insemination from a chromosomal translocation carrier. Case Rep Genet. 2014, 906145. Smith, D.C., Anderson, C.W., Barratt, C.L., Williams, M.A., 1988. Ultrastructural morphometric data of human spermatozoa. Andrologia 20, 396–403. Visser, T.D., Oud, J.L., 1994. Volume measurements in threedimensional microscopy. Scanning 16, 198–200.
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Received 3 July 2014; refereed 29 October 2014; accepted 29 October 2014.
w w w. s c i e n c e d i r e c t . c o m w w w. r b m o n l i n e . c o m
ARTICLE
Nuclear volume differences between balanced and unbalanced spermatozoa in chromosomal translocation carriers Alexandre Rouen a,*, Alinoë Lavillaureix a, Capucine Hyon a, Solveig Heide a, Sylvain Clède b, Richard Balet c, Esther Kott a, Nino Guy Cassuto d, Jean-Pierre Siffroi a a
Service de Génétique et Embryologie Médicales, Hôpital Armand-Trousseau, AP-HP, Unité INSERM U933, 26 Avenue du Dr Arnold Netter, Paris 75012, France; b Ecole Normale Supérieure, Département de Chimie, Sorbonne Universités, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules, UMR7203, 25 rue Lhomond, 75231 Paris Cedex 05, France; c Service de Médecine de la Reproduction, Clinique des Bluets, 4 rue Lasson, 75571 Paris Cedex 12, France; d Laboratoire Drouot, Clinique des Bluets, 4 rue Lasson, 75571 Paris Cedex 12, France * Corresponding author.
E-mail address: [email protected] (A. Rouen). Alexandre Rouen, MD, is a medical geneticist at Trousseau Hospital, in Paris, France. He specializes in infertility of genetic origin, and conducts research focusing on improving reproductive care in patients with genetic defects, notably chromosomal rearrangements.
While chromosomal translocations are usually associated with a normal phenotype, they can still cause male infertility as well as recurrent miscarriages and fetal malformations related to their transmission in an unbalanced state. The distinction between balanced and unbalanced spermatozoa on morphological criteria is still unfeasible. However, we previously showed that: i) spermatozoa with an unbalanced content have a higher rate of DNA fragmentation; and ii) that density gradient centrifugation partially separates balanced from unbalanced sperm cells. We hypothesized that a chromosomal imbalance could alter the fine spermatic nuclear architecture and consequently the condensation of DNA, thus modifying normal sperm density. Spermatic nuclear volumes in four translocation carriers were analyzed using confocal microscopy. Secondarily, FISH analysis was used to establish the segregation mode of each spermatozoon. We found the average spermatic nuclei size to be higher among unbalanced spermatozoa in all patients but one. All the unbalanced modes were associated with larger nuclei in two patients, while this was the case for the 3:1 mode only in the other two, suggesting an abnormal condensation. This could be the first step in elaborating a procedure to completely eliminate unbalanced spermatozoa from semen prior to in vitro fertilization.
Abstract
© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.rbmo.2014.10.019 1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Unbalanced spermatozoa in translocation carriers have larger nuclei
291
KEYWORDS: chromosomal translocations, confocal microscopy, infertility, spermatic DNA
Introduction Chromosomal translocations are found in the general population with a prevalence of 1/500, and are of two types: reciprocal and Robertsonian translocations (Meschede et al., 1998). They are usually balanced, and associated with a normal phenotype. However, they can lead to infertility in both male and female carriers. The abnormal chromosomal segregation during meiosis leads to the presence of chromosomally unbalanced gametes, the proportion of which varies among patients. Fertilization of an unbalanced gamete can have different outcomes, depending on the size and density in genes of the involved chromosomal segments: spontaneous abortion, fetal malformations, developmental delay and learning disabilities (Ravel et al., 2006). There is presently no way of identifying unbalanced spermatozoa in male translocation carriers since no morphological character has been identified for them (Cassuto et al., 2011). The proportion of unbalanced spermatozoa in a given translocation carrier is established through fluorescent in situ hybridization (FISH), however this procedure does not allow sperm retrieval and using for subsequent IVF. Therefore, the only two therapeutic options for those couples are either natural conception followed by prenatal diagnosis and pregnancy termination of an affected fetus, or preimplantation genetic diagnosis. However, we previously described a simple way to improve sperm quality in translocation carriers (Rouen et al., 2013b). Indeed, apart from abnormal chromosomal segregation, translocations have been associated with abnormal seminal parameters in male carriers, as well as to increased spermatic apoptosis and DNA fragmentation (Brugnon et al., 2006, 2010; Perrin et al., 2009). Furthermore, an increase in DNA fragmentation rate has been reported in unbalanced versus balanced spermatozoa in these men (Rouen et al., 2013a). This led to using a sperm procedure called discontinuous gradient centrifugation (DGC), which is currently used in IVF procedures in some centers for partially eliminating apoptotic spermatozoa and improving sperm quality, in order to decrease the proportion of unbalanced spermatozoa. This procedure leads to a partial but significant decrease in the proportion of unbalanced sperm (Rouen et al., 2013b). A multicentric program has since been launched by our center and already led to the birth of one healthy child, in a couple with a 10-year history of infertility and recurrent miscarriages (Rouen et al., 2014). The efficiency of DGC in partially eliminating unbalanced spermatozoa suggests that those cells have a different density compared with their balanced counterparts. This difference could be explained by an altered nuclear condensation, related to an abnormal chromosomal architecture. Indeed, spermatic nuclei are characterized by an extreme condensation, which is essential in protecting the genetic information along the male and female genital tracts. This condensation is made possible by the replacement of somatic histones by protamines and by a fine and remarkably constant nuclear architecture (Mudrak et al., 2005): chromosomes are bent at their centromeres, with their telomeric extremities facing outward and bound to each other, and their centromeres gathered together in a structure termed chromocentre
(Ioannou and Griffin, 2011). Furthermore, each chromosome has a relatively constant location in the nucleus (Zalenskaya and Zalensky, 2004). Therefore, the question arises whether the presence of translocated chromosomes and/or the lack or excess of chromosomal segments could alter this fine nuclear architecture, and therefore impair the condensation process. This could lead to an increased nuclear volume and a change in cellular density which could explain the partial elimination of those cells during DGC. The aim of the present study was to investigate the relationship between nuclear volume, measured in three dimensions through confocal microscopy, and chromosomal segregation, in chromosomal carriers.
Materials and methods This study was conducted in the Medical Genetics and Embryology department at Trousseau Hospital and in the Assisted Reproduction Department at Clinique des Bluets (Paris, France). Genetic analyses were performed according to the French reglementation, edicted by the Agence de la Biomédecine. Informed written consent was obtained from each participant. Four translocation carriers were included. Patient P1, 25 years old, carried a t(1;18)(p22;q21.1) reciprocal translocation. It was diagnosed from a chromosomally unbalanced pregnancy, which was voluntarily interrupted. Semen analysis showed severe oligozoospermia (1 million/ml). Patient P2, 36 years old, carried a t(5;12)(q13;q13) reciprocal translocation. It was diagnosed after the occurrence of three spontaneous abortions. Semen analysis showed normal sperm parameters. Patient P3, 37 years old, carried a t(2;10)(p10,q10) reciprocal translocation. He and his partner had had four spontaneous abortions. Semen analysis showed asthenoteratozoospermia. Patient P4, 29 years old, carried a rob(13;14)(q10,q10) Roberstonian translocation which was diagnosed after the same translocation was found in his brother, who consulted for primary infertility. Semen parameters were within the normal limits. Samples were obtained by masturbation from those four patients, after a 3 to 5-day abstinence period. After 30 minutes at 37°C for liquefaction, spermatozoa are washed with PBS (centrifugation at 500 × g for 10 minutes), before fixation with methanol and acetic acid and freezing at −20°C. The main steps of the protocol are summarized in Figure 1. Confocal microscopy analysis: spermatozoa were placed on a slide, and their DNA was stained with DAPI (4′,6-diamidino2-phenylindole) and an antifading agent (Prolong Gold Antifade Reagent, Life Technologies, CA, USA). Acquisition was performed on a Nikon Eclipse 80i confocal microscope equipped with the Nikon EZ C1 acquisition software. Each acquisition, containing around 20 spermatozoa, comprised around 30 images with a resolution of 512 × 512 and a distance of 300 nm along the z axis between each image. Each set of images was
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Figure 1 Main steps of the study’s protocol. After DAPI staining of the spermatozoa, their nuclear volumes are measured through confocal microscopy and computer analysis (ImageJ software). Secondly, a FISH assays allows for the assessment of the segregation mode of each spermatozoon.
Table 1 Spermatic nuclear volumes (µm3) in all four patients, according to the spermatozoa’s genetically balanced or unbalanced status. The mean nuclear volume is higher in unbalanced spermatozoa than in balanced spermatozoa, in all patients but P1.
P1 P2 P3 P4
Balanced
Number of analyzed spermatozoa
Unbalanced
Number of analyzed spermatozoa
Volume variation
53.45 47.93 74.37 49.30
95 50 52 70
54.64 51.84* 84.15* 59.90*
100 76 70 30
+2% +8% +13% +22%
*P < 0.05.
accurately localized on the slide, and the x and y coordinates were noted down. Tridimensional computer analysis: open source software ImageJ, as well as open source plug in 3D Object Counter (Fabrice Cordelières, Institut Curie, Orsay, Paris), were used to perform tridimensional analysis of the spermatic nuclei volumes. They were first evaluated in pixels, then conversion to µm3 was made possible owing to the known values of resolution and step distance in µm for each set of images. For each aquisition, boundary decisions were automated in ImageJ, using a set threshold level. 3D analysis was conducted in a blinded manner, as it was performed prior to chromosomal segregation analysis. Confocal microscopy was used to measure spermatic heads volumes as this allowed secondary analysis of chromosomal segregation. Other volume measurement methods, such as atomic force microscopy, while possibly more accurate, would have led to spermatic DNA disruptions, preventing further chromosomal analysis. Chromosomal segregation analysis: for each spermatozoon, the chromosomal segregation mode (alternate, adjacent 1, adjacent 2, and 3:1 for reciprocal translocations, and alternate, adjacent, and 3:0 for Roberstonian translocations) was assessed through FISH assay. For Robertsonian translocations, two subtelomeric probes were used, as well as a centromeric probe for a chromosome not involved in the rearrangement in order to distinguish an abnormal sperm cell after a 3:0 translocation from a diploid cell. For reciprocal translocations, two subtelomeric probes for each chromosome were used, as well as a centromeric probe for one of those two chromosomes. Non-commercial subtelomeric probes were prepared from BACs (bacterial artificial chromosomes) using an RCA (rolling circle amplification) procedure (Berr and Schubert, 2006). Spermatozoa were deposited on a glass slide and left to incubate in 2 SSC NP40 (saline
sodium citrate, nonyl-phenoxypolyethoxyethanol) for 30 minutes. They were then dehydrated in ethanol (three successive washes : 70%, 85%, and 95%). Nuclear heads were decondensed in 1M sodium hydroxide and dehydrated again. Denaturation of both nuclear DNA and probes was conducted in hybridization buffer under cover glass on a plate at 73°C for 2 min 30s. Hybridization was then performed at 37°C for 24 hours on a programmable hot plate (Slidebooster®, Beckman Coulter, FL, USA) in a way to increase signal intensity. The next day, the cells were washed in 0.4 SSC-NP40 in 73°C for 30 seconds and for two minutes at 37°C in 2 SSC NP40, before being counterstained with DAPI. Each spermatozoon previously acquired on confocal microscopy was relocated, and its chromosomal content was assessed. Statistical analysis: a t student test was used for statistical analysis of both sizes and standard deviations, and results with P < 0.05 were considered as significant.
Results We examined spermatic nuclear volumes according to the genetically balanced or unbalanced status of the spermatozoa. We found significantly larger nuclei among unbalanced spermatozoa than in balanced ones in all patients but P1 (P < 0.05, Table 1). The average differences were 3.91 µm3 (+8%) for P2, 9.78 µm3 (+13%) for P3, and 10.6 µm3 (+22%) for P4. The same analysis was conducted for each segregation mode (Table 2). For P2 and P4, the spermatozoa from all the unbalanced modes had a significantly larger nuclear volume than the balanced spermatozoa (P2 all comparisons P < 0.05; for P2 adjacent versus alternate P < 0.05 and 3:0 mode versus versus alternate P < 0.01). For P1 and P3, spermatozoa from
Unbalanced spermatozoa in translocation carriers have larger nuclei
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Table 2 Spermatic nuclear volumes (µm3) in all four patients, according to the spermatozoa’s segregation modes: alternate, adjacent 1, adjacent 2, and 3:1 in reciprocal translocation, and alternate, adjacent, and 3:0 in the Robertsonian translocation. In P1 and P3, the mean spermatic nuclear volumes are significantly increased, compared with balanced spermatozoa, for spermatozoa from the 3:1 segregation mode. In P2 and P4, the mean spermatic nuclear volumes are significantly increased in all unbalanced modes, compared with balanced alternate spermatozoa.
P1 P2 P3
P4
Alternate
n
Adjacent 1
n
Δ
Adjacent 2
n
Δ
3:1
n
Δ
53.45 47.93 74.37
95 50 52
51.10 51.28* 58.52
60 40 5
−4% +7% +21%
57.78 54.03* 84.16
9 28 5
+8% +13% +13%
60.76* 51.34* 85.70*
31 8 60
+14% +7% +15%
Alternate
n
Adjacent
n
Δ
3:0
n
Δ
49.30
70
59.05*
26
+20%
79.30**
4
+61%
*P < 0.05, **P < 0.01.
the 3:0 mode only were found to have significantly larger nuclei than balanced spermatozoa (both P < 0.05). The segregation mode with the largest spermatic nuclei was the 3:1 mode (or 3:0 for P4), except for P2, for whom it was the adjacent 2 mode.
Discussion Chromosomal translocation carriers produce both balanced gametes, either carrying the translocation or not, and unbalanced ones due to an abnormal segregation of the translocation during meiosis. In males, the proportion of unbalanced spermatozoa for a given patient is now routinely established by FISH. However, gametes analyzed by this technique are no longer available for further fertilization using insemination or in vitro methods (IVF, ICSI). Several authors, including our team, have tried to identify unbalanced spermatozoa in translocation carriers by searching for morphological characteristics in living gametes that would allow their use in assisted reproductive techniques (ART) (Cassuto et al., 2011). Unfortunately, no defining character had been found, except for an increased DNA fragmentation among unbalanced spermatozoa (Rouen et al., 2013a). This finding led us to test discontinuous gradient centrifugation (DGC), a well-established method performed in most ART laboratories prior to IVF, as a tool for separating balanced from unbalanced spermatozoa based on their DNA integrity. Indeed, DGC selects preferentially motile and nonapoptotic sperm cells (Rouen et al., 2013b). We showed that it increases the percentage of spermatozoa with a balanced nuclear content in translocation carriers as well, thus raising the question whether a chromosomal imbalance could slightly modify sperm head volume and consequently nuclear density. In our experiments, confocal microscopy was performed prior to FISH in order to avoid measurement bias due to the hybridization of probes. Indeed, FISH procedure includes a decondensation step which modifies sperm head volumes. Spermatic nuclear volumes were assessed after staining of nuclei with DAPI. Since most of the cytoplasm is eliminated from the spermatozoa’s heads after spermatogenesis, the measured volume grossly equals that of the spermatic head.
Approximately 30–40 images were acquired along the × axis for each spermatozoon, making the volume estimate as reliable as possible. Contradictory data exist in the literature about human sperm head volume. Using atomic force microscopy, Lee et al. found that the average volume of normal sperm heads was 5.02 ± 0.37 µm3 (Lee et al., 1997). Similar values were found by Coppola et al. who measured a mean sperm head nuclear volume of 8.03 ± 0.75 µm3 using digital holographic microscopy (Coppola et al., 2014). Values drawn from these two studies are in agreement with that by Maree et al. (2010) which found nuclear sperm area values ranging from 9.26 ± 0.99 to 12.87 ± 1.19 µm2 according to the staining method used in light microscopy. Owing to the fact that mature spermatozoa have a flat shaped head (Holstein and Roosen-Runge, 1981), such area values are compatible with nuclear volumes around 10 µm3. In contrast, Hazzouri et al. (2000) studied human sperm genome organization by FISH and confocal microscopy after various decondensation methods and found very high nuclear volumes, up to 283 µm3, with a minimal value of 70 µm3 obtained after treatment with heparin which is a milder decondensation agent than NaOH. Intermediate volume values were reported by Curry et al. (1996) who found a mean human sperm head volume of 22.2 ± 1.2 µm3 by using stereological methods, in agreement with older works. (Brotherton, 1975; Smith et al., 1988). Our own measurements of sperm head volumes give a mean value of nearly 57 µm3 in controls, which is more than two times higher than these latter values. We hypothesized that an overestimation of distances along the z axis in confocal microscopy could be responsible for this discrepancy. Indeed, a mismatch between the microscopic object and the immersion oil has been described to lead to overestimate the size of the objects (Visser and Oud, 1994). This was confirmed by performing acquisitions with the same procedure of other objects of known dimensions and shape, such as lymphocytes. They appeared elongated along the z axis and not spherical (data not shown), and had a measured volume twice as high as the actual volume. We however considered that such an overestimation of volume values did not cause any bias, since the effect was equally present for balanced and unbalanced cells. The observed differences therefore raise the question of whether the presence of
294 abnormal translocated chromosomes disrupt the spatial organization of DNA in the spermatic nucleus. Individual chromosome territories are known to be organized in a non-random way within the sperm nucleus (Hazzouri et al., 2000; Zalenskaya and Zalensky, 2004). Consequently, any disturbance in sperm chromosomal arrangement, due to changes in particular chromosomal segment positions in translocations or to an excess and/or a lack of chromosomal content in unbalanced gametes, may hinder the fine and precise architecture of sperm nucleus and could prevent the normal condensation of DNA, leading to spermatozoa with an increased head volume. According to the nature and/or the length of the translocated chromosomal segments, as well as the mode of segregation leading to unbalanced gametes, impairments in normal DNA condensation could be variable and, therefore, could have also variable consequences on sperm head volume. We observed such variability among our patients since P2 and P4 exhibited increased nuclear volumes in spermatozoa issued from all segregation modes while, in P1 and P3, this was observed only in sperm cells from the 3:1 mode. Interestingly, in reciprocal translocations, this mode of segregation is the only one associated with an abnormal number of chromosomes, and therefore an abnormal number of centromeres, which have a major role in the sperm nuclear organization through the constitution of the chromocenter (Ioannou and Griffin, 2011). We were only able to study four patients, which is admittedly low. Further work should aim at studying a greater number of subjects, as well as carriers of other chromosomal rearrangements, such as chromosomal inversions. We however obtained statistically significant results because of the high number of analyzed spermatozoa. In other words, each patient was his own control: unbalanced spermatozoa were compared with balanced spermatozoa, for each subject. The present study shows that in male chromosomal rearrangement carriers, genetically unbalanced spermatozoa have significantly larger nuclei than their balanced counterparts. This is especially true for the most unbalanced of the modes, the 3:1 (or 3:0) one. This finding is appealing on both the pathophysiological and clinical points of view. As mentioned before, it is likely that it is the disturbance of the fine spermatic nuclear organization that hampers normal DNA condensation, making the cell more vulnerable to DNA fragmentation and apoptosis. There could be implications in the clinical setting. With this study, we provide a possible pathophysiological explanation to the partial elimination of unbalanced spermatozoa by discontinuous gradient centrifugation. This could open up possibilities in selecting balanced spermatozooa prior to IVF.
Acknowledgement The authors would like to thank the patient association Valentin for their collaboration.
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Received 3 July 2014; refereed 29 October 2014; accepted 29 October 2014.