Computer-assisted motility analysis of spermatozoa obtained after spermatogonial stem cell transplantation in the mouse

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1 Computer-assisted motility analysis of spermatozoa obtained after spermatogonial stem cell transplantation in the mouse Ellen Goossens, Ph.D., Gert De Block, and Herman Tournaye, Ph.D., M.D. Research Centre for Reproduction and Genetics, University Hospital of the Dutch-speaking Brussels Free University (UZ Brussel), Brussels, Belgium Objective: To study the motility characteristics of epididymal spermatozoa after spermatogonial stem cell transplantation. Design: Testicular cells from fertile donor mice were transplanted to the testis of genetically sterile recipient mice. Three to nine months later, the epididymal spermatozoa were isolated and used for a computer-assisted sperm motility analysis. Spermatozoa from fertile adult mice were used as control. Setting: Murine transplantation model in an academic research environment. Animal(s): Donors, 6-day-old male C57Bl WBRej F1 mice; acceptors, 4- to 6-week-old W/W v mice of the same genetic background. Intervention(s): Two to 10 ml from a 30-million/mL testicular cell suspension was injected through the efferent duct in the rete testis. Main Outcome Measure(s): Vitality, concentration, motility, individual sperm movement, and hyperactivity of the spermatozoa. Result(s): Vitality was comparable between the two groups; the concentration, motility, and hyperactivity of posttransplantation spermatozoa were significantly reduced. The movement pattern of the individual spermatozoon was normal at the time of isolation but decreased more rapidly during in vitro culture, compared with the case in controls. This difference already had reached a significant level 3 hours after culture, which is comparable with the duration of an IVF procedure. Conclusion(s): The reduced fertilization rate after IVF can thus be explained by a lower number of motile spermatozoa and the faster decrease of the individual sperm movement parameters. (Fertil Steril Ò 2008;90: Ó2008 by American Society for Reproductive Medicine.) Key Words: Motility, vitality, spermatogenesis, spermatozoa, transplantation The introduction of spermatogonial stem cell transplantation by Brinster and Avarbock (1) and Brinster and Zimmerman (2) in 1994 was a great step forward in the prevention of infertility. This method was very promising in the case of childhood cancer, because in theory, spermatogonial stem cell banking is the only way to preserve the future fertility of boys undergoing a sterilizing chemotherapy (3). Spermatogonial stem cell transplantation has proven to be effective in restoring fertility in mice (4). However, in studies published elsewhere, we observed that the fertilization rate after assisted conception was reduced significantly in the transplanted group (5, 6). This difference was assumed to be the result of reduced motility in the spermatozoa obtained from transplanted animals. Received June 28, 2007; revised July 30, 2007; accepted August 13, Supported by grants from the Fund for Scientific Research Flanders (Brussels, Belgium) and the Dutch-speaking Brussels Free University (Brussels, Belgium; OZR 318). Reprint requests: Ellen Goossens, Ph.D., Research Centre for Reproduction and Genetics, University Hospital of the Dutch-speaking Brussels Free University (UZ Brussel), Laarbeeklaan 101, 1090 Brussels, Belgium (FAX: ; ellen.goossens@uzbrussel.be). Adequate sperm motility is a requirement for successful fertilization both in vivo and in vitro. Computer-aided sperm motility analysis systems are able to determine sperm concentrations and especially the proportions of motile (and progressively motile) spermatozoa (7, 8). Computer-aided sperm motility analysis is also a valuable tool for accurately assessing sperm kinematics (9). Although sperm motility is generated by flagellar beating, computer-aided sperm motility analysis technology quantifies flagellar motion indirectly, through sperm head motion, which is easier to measure than flagellar movement. Data obtained from computer-aided sperm analysis are time-average parameters; that is, they are computed over a known time interval. The energy of motion is measured by three velocities or sperm swimming speeds. Curvilinear velocity (VCL, mm/s) is the time-average velocity of the sperm head along its actual curvilinear trajectory. Average-path velocity (VAP, mm/s) is the time-average velocity of the sperm head along its average trajectory and is computed via a mathematical smoothing of the actual curvilinear path. The time-average velocity of the sperm head along a straight line from its first to its last position is the straight-line velocity (VSL, mm/s). To some extent, the energy of motion also is measured by the beat cross /08/$34.00 Fertility and Sterility â Vol. 90, Suppl 2, October doi: /j.fertnstert Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 frequency (BCF, Hz), that is, the time-average rate at which the actual sperm trajectory crosses the average path. The pattern of sperm head motion is measured by the dimensionless ratios linearity (linearity ¼ VSL/VCL) and straightness (STR), that is, the effective linearity of the path. In addition, the pattern of sperm head motion also is described by the amplitude of lateral head displacement (ALH, mm), that is, the lateral distance of the sperm head from its average path (10). In this study, we examined the motility, the individual movement pattern, and the vitality of spermatozoa after spermatogonial stem cell transplantation and compared these data with those of fertile controls. MATERIALS AND METHODS Breeding Procedure and Ethics Two WBRej W/þ female mice were housed together with one C57Bl W -v /þ male mouse under a 12:12-hour light dark cycle. Food and water were provided ad libitum. All experimental procedures were approved by the Animal Care and Use Committee at the Brussels Free University. Transplantation Donor cells were obtained from 6-day-old C57Bl WBRej F1 mouse pups (þ/þ). The testes were decapsulated and the testicular tissue digested as described elsewhere (11). We used 4- to 6-week-old W/W v -mice of the same genetic background (Jackson Labs, Bar Harbor, ME) as recipient animals. Transplantation was performed through the efferent duct, as described elsewhere (11). Two to 10 ml from a 30 million/ml suspension was injected in each recipient testis. Immediately after transplantation, animals were given a 100-mL dose of antibiotic (1,900 ml of phosphate-buffered saline þ 100 ml of Baytril 2.5%, SC [Bayer, Brussels, Belgium]). Isolation of Sperm Cells Transplanted mice were killed when the first offspring were born, or 9 months after transplantation. Sperm cells were isolated from transplanted males, which were maintained between 4 and 9 months after transplantation. The epididymides were removed and transferred into one well of a four-well plate containing 500 ml of Dulbecco s modified Eagle s medium supplemented with 3% bovine serum albumin (Sigma, Bornem, Belgium) covered with oil. Incisions were made to allow spermatozoa to move into the medium. After 5 minutes of incubation at 37 C, the epididymides were discarded. Analysis of Sperm Cell Vitality The staining solution for the one-step eosin-nigrosin staining technique contained 0.67% eosin Y and 10% nigrosin (12), dissolved in 0.9% sodium chloride in distilled water. The solution was brought to a boil and then allowed to cool to room temperature, after which it was filtered and stored in a dark, sealed glass bottle. One droplet of the sperm suspension (12 ml) was mixed with one droplet of the eosin-nigrosin staining solution (12 ml). The suspension was incubated for 30 seconds at room temperature. Then, a 12-mL droplet was transferred to a microscope slide, where it was smeared by sliding a coverslip over it. The smears were air dried and examined directly. At least 200 sperm were assessed at a magnification of 400. Sperm that were white (unstained) were classified as alive, and those that showed any pink or red coloration were classified as dead, with the sole exception made for sperm with a slight pink or red appearance that was restricted to the neck region (socalled leaky necks), which were assessed as alive. Analysis of Sperm Cell Kinematics Cells were analyzed by using the Integrated Visual Optical System sperm analyzer (version 10.7; Hamilton-Thorne Biosciences, Beverly, MA) at different time points. A drop of the sperm suspension (2 ml) was transferred to the incubation chamber, which was set at a temperature of 37 C. At least 200 sperm cells were analyzed using the following parameters: negative phase-optics; recording at 60 frames per second; minimum contrast, 60; minimum cell size, 6 pixels; STR threshold, R50%; cutoff of VAP and VSL, 25 and 30 mm/s, respectively; minimum progressive VAP, 75 mm/s; slow cells motile, no (this limit avoids counting sperm moved by others or by Brownian motion and low-velocity non-progressive cells); and minimum static contrast, 15 pixels (13). The following motility parameters were analyzed: total motility, progressive motility, VAP, VSL, VCL, ALH, BCF, STR, and LIN. In addition, the concentration and hyperactivity of the sperm cells were analyzed. The results observed with spermatozoa obtained after spermatogonial stem cell transplantation were compared with the results observed with control spermatozoa that were obtained from the epididymides of 3-month-old C57Bl WBRej F1 mice. Histology To analyze the histological appearance of the tubules after transplantation, the testes were fixed overnight in Bouin s fixative at 4 C and were embedded in paraffin. Five-micrometer-thick sections were cut and stained with eosin and hematoxylin. The percentage of tubular cross-sections with complete spermatogenesis was recorded and expressed as median (interquartile range, 1 3). One hundred crosssections were counted per testis. The slides were analyzed under an inverted microscope at a magnification of 200. Statistical Analysis Comparisons between experimental and control groups were performed by using the SPSSþ Statistics Package for Windows (version 13; SPSS Inc., Chicago, IL). Repeated measures analysis of variance was used to measure the effect of treatment and time on vitality and on parameters of 1412 Goossens et al. Motility of post-transplantation sperm Vol. 90, Suppl 2, October 2008

3 motility and individual sperm movement. If no sphericity was obtained by using the Mauchly s test for sphericity, Huynh- Feldt values were recorded. The Mann-Whitney U test was used to compare the concentration and data for motility characteristics at one specific time point. To evaluate the effect of concentration on sperm motility, the correlation between concentration and motility was measured by using the Spearman correlation coefficient. RESULTS Transplantation and Histology A testicular cell suspension was transplanted successfully in 23 testes of 14 W/W v -mice. At the time of evaluation, spermatogenesis was observed in 14 testes, and sperm cells were observed in 12 epididymides (of 23, or 52%) of eight mice. Transplanted mice that had fathered offspring showed spermatogenesis in 73.5% (interquartile range, 60.3% 88.0%) of the testicular cross-sections (Table 1). Analysis of Sperm Cell Vitality No differences were found in vitality between post-transplantation and control spermatozoa at any evaluated time point. Furthermore, there was no effect of the transplantation on vitality of spermatozoa (Fig. 1). Analysis of Sperm Cell Kinematics The concentration of sperm cells after transplantation was lower than that in the control group (51.6 vs million/ ml), although significance was not reached. The total and progressive motility both were significantly lower at the time of isolation (52.1% vs. 27.7%, P¼.011 for total motility and 35.5% vs. 17.6%, P¼.031 for progressive motility) and decreased more rapidly during the observed time, compared with the case of spermatozoa from fertile mice (Fig. 1). The concentration of the sperm sample was found to have an effect on sperm motility (P¼.02; Fig. 2). The individual parameters of sperm movement (VAP, VSL, VCL, ALH, BCF, STR, and LIN) in the transplanted group were not different from those in the control group at the time of isolation, but during culture, the post-transplantation spermatozoa lost their motility more rapidly, compared with control spermatozoa. The difference between experimental and control spermatozoa reached significance after 2 hours of culture for ALH and after 3 hours of culture for VAP, VSL, VCL, and LIN. The other parameters (BCF and STR) did not reach the significant level within the observed time period. The effect of transplantation was evident for motility, progressive motility, VAP, VSL, ALH, BCF, STR, and hyperactivity. Time had a significant effect on every parameter measured (P<.001). A combined effect was found for motility, progressive motility, VAP, VSL, STR, and LIN (Fig. 3). DISCUSSION Our studies elsewhere showed that after intracytoplasmic sperm injection with spermatozoa obtained after spermatogonial stem cell transplantation, the fertilization rate was comparable to those for spermatozoa from an adult fertile male donor mouse. On the contrary, the fertilization rate after IVF was significantly reduced (5, 6). This may suggest a motility problem in the spermatozoa that were obtained after TABLE 1 Spermatogonial stem cell transplantation in the mouse. Mouse no. Age (mo) Tubules with No. of spermatogonia injected spermatogenesis (%) Sperm concentration L R L R (millions/ml) Offspring , Yes ,891 12, No ,313 10, Yes ,810 14, No ,525 2, No ,525 5, Yes ,810 5, No ,333 11, No , Yes ,000 7, No , No , No ,163 6, Yes , Yes Fertility and Sterility â 1413

4 FIGURE 1 Vitality, motility, and progressive motility of spermatozoa obtained from fertile control male mice (Ctrl) and mice that had undergone spermatogonial stem cell transplantation (WW). Spermatozoa were cultured for 24 hours. Significance data for treatment effect by repeated measures analysis of variance were as follows: for vitality, effect of treatment was P¼.336, effect of time was P<.001, and combined effect was P¼.899; for motility, effect of treatment was P¼.012, effect of time was P<.001, and combined effect was P¼.003; and for progressive motility, effect of treatment was P¼.012, effect of time was P<.001, and combined effect was P¼.003. FIGURE 2 The effect of the concentration of the sperm sample on its motility. The Spearman correlation coefficient for not-normally divided data was WW ¼ mice that had undergone spermatogonial stem cell transplantation. spermatogonial stem cell transplantation. Therefore, we analyzed the motility characteristics of the spermatozoa in more detail. Computer-assisted sperm motility analysis is a very easy, quick, and reliable technique. The transplanted mice were evaluated when the first litter was born or, in other words, when spermatogenesis reached a degree sufficient for reproduction. In analogy to this idea, we used control mice from 3 months of age, which also resembles the age at which the first or second litters are born. In the present study, we found that the amount of motile spermatozoa was significantly reduced in the experimental group. Although the individual sperm motility parameters were comparable in both groups at the time of isolation, they decreased more rapidly in the transplanted group. This difference already was significant 3 hours after isolation, which coincidences with the timing of the IVF procedure. Velocity and the ALH of sperm were found to correlate with the fertilization rate in human IVF (14, 15). This finding may explain the reduced fertilization rate after spermatogonial stem cell transplantation in our mouse model as well. Flagellar motion is essential for the reproductive function of the spermatozoon. The spermatozoa in the seminiferous tubules are still immotile and unable to fertilize an oocyte. It is only in the epididymides that the spermatozoa acquire the ability to move forward. The final pattern of flagellar movement depends on a wide range of factors, that is, intracellular ph; the sperm membrane; the flagellar elements; and external factors such as substrate availability, ionic signals, and physical elements in the sperm microenvironment. Which factors are responsible for the reduced motility of spermatozoa in the transplanted recipient remains unclear. An explanation may be found in the formation of spermreactive antibodies as a result of the surgical procedure. Although the testis is an immunologically privileged site and no antigen recognition occurs in the seminiferous tubules, foreign antigens still can be detected in the epididymis, which contains T-helper cells (16). Because it is in the epididymis that spermatozoa achieve motility, the formation of spermreactive antibodies in the epididymis may explain the observed disturbances in motility (17). It was found, in autoimmune patients, that sperm-reactive antibodies impair the 1414 Goossens et al. Motility of post-transplantation sperm Vol. 90, Suppl 2, October 2008

5 FIGURE 3 Individual sperm movement characteristics of spermatozoa obtained from fertile control male mice (ctrl) and mice that had undergone spermatogonial stem cell transplantation (WW). Spermatozoa were cultured for 24 hours. Significance data for treatment effect by repeated measures analysis of variance were as follows: for VAP, effect of treatment was P¼.017, effect of time was P<.001, and combined effect was P¼.032; for VSL, effect of treatment was P¼.017, effect of time was P<.001, and combined effect was P¼.026; for VCL, effect of treatment was P¼.030, effect of time was P<.001, and combined effect was P¼.118; for ALH, effect of treatment was P¼.006, effect of time was P<.001, and combined effect was P¼.070; for BCF, effect of treatment was P¼.091, effect of time was P<.001, and combined effect was P¼.293; for STR, effect of treatment was P¼.049, effect of time was P<.001, and combined effect was P¼.023; for LIN, effect of treatment was P¼.062, effect of time was P<.001, and combined effect was P¼.013; and for hyperactivity, effect of treatment was P¼.029, effect of time was P<.001, and combined effect was P¼.255. Fertility and Sterility â 1415

6 sperm oocyte interaction (18, 19), resulting in a reduced fertilization rate after IVF (20). It also was suggested that sperm-reactive antibodies impaired embryonic development (21). However, these adverse effects were not observed after intracytoplasmic sperm injection (22). It is obvious that for any clinical application, an autologous transplantation will be performed instead of a heterologous one, which implies that any adverse immune responses will be avoided. However, the results of this study may not be due to the transplantation itself but rather to the mouse model used. The deterioration of the sperm movement may be due to the host W/W v mouse itself. The inner microenvironment of the seminiferous tubules and the epididymis may be different from that in wild-type mice, because of the original mutation of the c-kit receptor but also because of secondary effects. Seminiferous tubules of the acceptor never have supported spermatogenesis, and their epididymides have never accepted and stored spermatozoa. Because an effect of concentration on the motility of the sperm sample was observed, we may assume that when a higher sperm concentration can be obtained, a higher motility also may be found. This finding may have important consequences for fertility treatment after cancer. If the spermatogonial stem cells can be transplanted as soon as the boy has been cured, the time to establish spermatogenesis will be longer, and thus, the concentration of the sperm will be higher. According to the data in this study, better motility also may be achieved. Taking into account these thoughts, we believe that spermatogonial stem cell transplantation can be more successful in human beings. Acknowledgments: The authors are grateful to Professor Leonard Kaufman, Ph.D., and Mr. Micha el Saenen, BS, for their statistical advice. REFERENCES 1. 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