Nonrandom contribution of left and right testes to germline transmission from mouse spermatogonial stem cells

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1 Biology of Reproduction, 2017, 97(6), doi: /biolre/iox141 Research Article Advance Access Publication Date: 9 November 2017 Research Article Nonrandom contribution of left and right testes to germline transmission from mouse spermatogonial stem cells Mito Kanatsu-Shinohara 1,2,, Honda Naoki 3 and Takashi Shinohara 1 1 Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 2 Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kyoto, Japan and 3 Imaging Platform for Spatio-temporal Information, Graduate School of Medicine, Kyoto University, Kyoto, Japan Correspondence: Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto , Japan. Tel: ; Fax: ; mshinoha@virus.kyoto-u.ac.jp Grant Support: Financial support for this research was provided by The Uehara Memorial Foundation, The Takeda Foundation, The Naito Foundation, Grants-in-aid for Scientific Research on Innovative Areas Epigenome Dynamics and Regulation in Germ Cells ( ) and Stem Cell Aging and Disease (17H05639) from The Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Japan Science and Technology Agency (PRESTO), and the Platform for Dynamic Approaches to Living Systems from Japan Agency for Medical Research and Development (AMED). Edited by Dr. Kyle Orwig, PhD, University of Pittsburgh. Received 26 June 2017; Revised 15 August 2017; Accepted 7 November 2017 Abstract Vast amounts of sperm are produced from spermatogonial stem cells (SSCs), which continuously undergo self-renewal. We examined the possible effect of laterality in male germline transmission efficiency of SSCs using a spermatogonial transplantation technique. We transplanted the same number of wild-type and Egfp transgenic SSCs in the same or different testes of individual recipient mice and compared the fertility of each type of recipient by natural mating. Transgenic mice were born within 3 months after transplantation regardless of the transplantation pattern. However, transgenic offspring were born at a significantly increased frequency when wild-type and transgenic SSCs were transplanted separately. In addition, this type of recipient sired significantly more litters that consisted exclusively of transgenic mice, which suggested that left and right testes have different time windows for fertilization. Thus, laterality plays an important role in germline transmission patterns from SSCs. Summary Sentence Nonrandomness of germline transmission pattern of spermatogonial stem cells was examined by spermatogonial transplantation. Key words: spermatogenesis, spermatogonial stem cells, transplantation, testis. Introduction It has been estimated that spermatozoa are ejaculated for fertilization in mice [1]. Both right and left testes contribute to spermatogenesis and generate mature spermatozoa. However, it has remained unknown whether these testes contribute equally to offspring production. Previous studies have shown conflicting observations of fertility after orchidectomy of unilateral testis [2 4], and the effect of laterality in germline transmission remains unknown. On the other hand, ovulation had been considered to occur from alternating ovaries in mono-ovular species since the original observations by Rühl in 1925 [5]. Although more recent studies do not support this conclusion [6], the effect of laterality has been analyzed extensively in females. While ovulation is relatively easily identified by morphological analyses of ovaries, it has been difficult to study the 902 C The Author(s) Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please journals.permissions@oup.com

2 Nonrandom male germline transmission, 2017, Vol. 97, No differences in the contribution of each testis to germline transmission due to lack of appropriate experimental systems. Spermatogonial stem cells (SSCs) provide the foundation of spermatogenesis [7, 8]. In mouse testes, SSCs comprise only % of total germ cells [8, 9]. However, these cells continuously undergo self-renewal and produce sperm for the life span of the male animal. Once SSCs are committed to differentiate, spermatogonia progenitor cells undergo a limited number of mitotic divisions and enter into meiosis, and then transform into spermatids and sperm via spermiogenesis. Sperm produced in the testis are then ejaculated for fertilization after transportation through epididymis and vas deferens. Therefore, any difference in laterality can occur not only in the testis but also in epididymis and vas deferens. Unfortunately, SSCs and committed progenitors are morphologically indistinguishable, but SSCs have a unique ability to reconstitute empty seminiferous tubules of infertile mouse testes after microinjection into the seminiferous tubules [10]. Transplanted SSCs regenerate colonies of germ cells and eventually produce donor cell-derived offspring [11]. We recently developed a robust system to produce offspring using germline stem (GS) cell cultures, in which SSCs can expand indefinitely in vitro in the presence of self-renewal factors, including glial cell line-derived neurotrophic factor [12]. Although the frequency of SSCs in these cultures is 1 2% [13], SSCs in this culture continue to increase their number, which makes it possible to collect a large number of SSCs for biochemical and molecular biological studies. The most remarkable property of GS cells is fertility restoration. We were able to restore fertility to both congenitally infertile WBB6F1-W/W v (W) mice and busulfan-treated infertile mice [14]. Within 4 months after transplantation, 60 and 100% of W and busulfan-treated mice sired donor-derived offspring, respectively. This experimental system created a unique platform to study the effect of candidate factors involved in fertility restoration by SSCs. In this study, we analyzed the effect of laterality on the germline transmission pattern from SSCs. Wild-type (WT) GS cells and those with a homozygous Egfp transgene encoding enhanced green fluorescent protein (EGFP) were separately transplanted into each testicle of W mice. We also produced another type of recipient mice by transplanting both EGFP GS cells and WT GS cells into the same testis. Because W mice do not have endogenous spermatogenesis, all offspring born from W mice must be derived from transplanted donor SSCs. The recipient mice were caged with WT females for at least 7 months, and the genotype of the offspring was examined to determine the origin of SSCs. Materials and methods Cell culture GS cells that express the Egfp transgene were established from a transgenic mouse strain C57BL/6Tg14(act-EGFP)OsbY01 in a DBA/2 background, as previously described [14]. Wild-type GS cells were established from 5 to 10-day-old pup testes using the same procedure. These cells have the same genetic background except for the absence of the transgene. Cells were cultured in Iscove modified Dulbecco Medium (Invitrogen, Carlsbad, CA), which was supplemented with 10 ng/ml human FGF2, 15 ng/ml recombinant rat GDNF (both from Peprotech, London, UK), and 1% fetal bovine serum, as previously described [15]. Cultures were maintained on mitomycin C-treated mouse embryonic fibroblasts. Transplantation Spermatogonial transplantation was carried out as previously described using 4 6-week-old W mice (Japan SLC, Shizuoka, Japan). GS cells were dissociated into single cells by 0.25% trypsin/1 mm EDTA solution, and approximately cells were microinjected into the efferent duct of each testis, as previously described [16]. Each injection filled 75 85% of all seminiferous tubules. The recipient mice were treated with anti-cd4 antibody (GK1.5) intraperitoneally on days 0, 2, and 4 after transplantation to avoid rejection of the donor cells [17]. Recipients were housed with WT B6 females 4 weeks after transplantation to produce offspring. The Institutional Animal Care and Use Committee of Kyoto University approved all of our animal experimentation protocols. Analysis of recipient mice After mating for 7 months, recipients were sacrificed between 8 and 11 months after transplantation. Donor cell colonies in recipient testes were examined under UV light. For evaluation of donor cell colonization levels, testis samples were embedded in paraffin, and histological sections were stained with hematoxylin and eosin. The number of tubules with spermatogenesis, defined by the presence of multiple layers of germ cells in the entire circumference of the tubules, was recorded for one section from each testis. Lectin staining Testis samples were fixed in 2% paraformaldehyde for 3 h. Then they were embedded in Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan) for cryosectioning. Lectin staining of cryosections was carried out by treating the samples with 0.1% Triton-X in phosphate-buffered saline (PBS). After immersing the slides in blocking buffer (0.1%Tween 20, 3% bovine serum albumin and 10% goat serum in PBS) for >1 h, samples were incubated with rhodamineconjugated peanut agglutinin (PNA; Vector Laboratories RL-1072, Burlingame, CA). Samples were counterstained with Hoechst (Sigma, St. Louis, MO). Statistical analyses Significant differences between means for single comparisons were determined by a Mann Whitney U-test. Multiple comparison analyses were carried out using ANOVA followed by Tukey Honest Significant Difference test. The method for statistical evaluation of laterality is provided in Supplemental Information. Results Fertility restoration by transplantation of enhanced green fluorescent protein and wild-type GS cells into W mice In our previous study, we used GS cells that have homozygous Egfp transgene to obtain offspring [14]. We used the same GS cells and compared the fertility potential with WT GS cells that do not show fluorescence. Because these two types of GS cells do not show apparent differences in their morphology and proliferation speed during in vitro culture, they were indistinguishable unless they were exposed to UV light. For recipients, we used congenitally infertile W mice. W mice have a very small number of SSCs and undifferentiated spermatogonia, but completely lack differentiated spermatogonia and subsequent stages of germ cells due to mutations in the Kit gene. Therefore, any offspring produced by these mice must be derived from donor cells. We collected the same number of GS cells by trypsin digestion and single-cell suspensions were made. We produced two types of recipients (Figure 1A). In one group of recipients (designated as mixed

3 904 M. Kanatsu-Shinohara et al., 2017, Vol. 97, No. 6 Figure 1. Transplantation of GS cells into W mice. (A) An experimental scheme showing the transplantation of EGFP or WT GS cells into the seminiferous tubules of congenitally infertile W mice. Recipient males were continuously mated with WT females for offspring production. (B) Offspring produced by a recipient male under UV light. (C) Macroscopic appearance of recipient testis. Green tubules represent donor cell colonization. (D) Testis weight (n = 4 for WT and EGFP, n = 5 for Mix). (E) Histological appearance of the recipient testis. (F) Lectin staining of recipient testis. (G) Quantification of tubules with spermatogenesis. Tubules with PNA + spermatogenic cells were counted. At least 181 tubules in four testes were counted (n = 4 for WT and EGFP, n = 5 for Mix). Bar = 1mm(C), 100 μm (E, F). or M recipients), EGFP and WT GS cells were mixed in a 1:1 ratio, and the cells were transplanted into both sides of six recipient mice. In another group ( separate or S recipients), the same number of recipients underwent transplantation with two different types of cells in separate testes. Three recipients were transplanted with EGFP cells in the right testis, while WT cells were transplanted into the left testis. Four recipients underwent transplantation in the opposite manner. Two months after transplantation, these recipient mice were caged with 3 4 WT females to produce offspring from donor cells. A total of two transplantation experiments were performed. M recipients sired offspring as early as 83 days after transplantation (Table 1). Five of six M recipients became fertile. Similarly, S recipients sired offspring as early as 82 days after transplantation, and six of seven S recipients restored fertility upon transplantation. In both cases, the remaining recipients did not sire progeny for at least 7 months. Although the first transgenic mice were born at comparable speed, the average date of first offspring production from all recipients was somewhat delayed in S recipients (86.8 days vs days). The number of litters produced by S recipients ranged from 1 to 29 (Supplemental Figure S1A), whereas those with M recipients ranged from 16 to 18 (Supplemental Figure S1B). This was because three of the S recipients died within 5 months after transplantation. However, when the experimental animals that survived the whole experimental period were compared, the frequency of offspring production did not show apparent differences between the two types of recipients. In average, M recipients produced 16.8 litters, while S recipients produced 14.2 litters. These values were not significantly different. However, when the total offspring numbers were compared, M recipients sired an average of 9.2 offspring per litter, whereas 7.0 offspring were born from S recipients. This difference was statistically significant. We determined the genotype of the offspring by examining their fluorescence under UV light illumination. Although there is a controversy concerning the effect of laterality on sex ratio and ovulation

4 Nonrandom male germline transmission, 2017, Vol. 97, No Table 1. Offspring production after spermatogonial transplantation. Type Days Period Testis weight (mg) (type) % Spermatogenesis Recipient to first of analysis Litter EGFP + EGFP Total % GFP + / ID offspring (days) number offspring offspring offspring total Right Left Right Left Separate S (GFP) 58.2 (WT) 93.2 (GFP) 81.6 (WT) S ND ND ND ND S ND ND ND ND S (GFP) 63.3 (WT) 89.5 (GFP) 93.5 (WT) S (WT) 56.1 (GFP) 98.7 (WT) 90.2 (GFP) S (WT) 62.3 (GFP) 94.1 (WT) (GFP) Total Mix M ND 100 M M M M Total ND, not determined because these animals did not survive the whole experimental period. [18], we did not find an apparent bias: both left and right testes produced comparable numbers of males and females from S recipients. EGFP and WT SSCs also did not show apparent differences in sex ratio of the offspring. However, we noted a biased transgenic offspring production. Overall, 43.2% of the offspring were transgenic mice from S recipients, while 20.1% of the offspring were transgenic mice from M recipients (Figure 2). On the other hand, the average number of transgenic offspring per litter was 1.8 and 3.0 for M and S recipients, respectively. In both types of recipients, significantly more WT mice were born than transgenic mice (P = ), suggesting that WT SSCs have a competitive advantage over EGFP SSCs. The number of WT offspring per litter was 7.3 and 4.0 for M and S recipients, respectively. This difference between the wild-type litters was also statistically significant (P < ). These results suggested that the kinetics of SSCs in M and S recipients are different. Statistical validation of biased production of offspring by mathematical modeling In addition to the changes in the frequency of transgenic mouse production, we noted that a significant number of litters consisted of only one genotype. M recipient mice sired a total of 84 litters, of which one (1.2%) litter consisted of only transgenic mice (Figure 2). In contrast, S recipients sired 85 litters, of which eight (9.4%) litters consisted of only transgenic mice. Because S recipients contain only one genotype in each testis and sired 12 (14.1%) litters with the wild-type genotype, a total of 20 litters (23.5%) from S recipients were born with only one genotype. Since this finding suggested that left and right testes contribute to offspring production separately, we performed a following statistical analysis to determine whether transgenic or WT offspring were born unevenly from S and M recipients. In this analysis, we quantitatively evaluated the degree of bias of the offspring production in each litter based on a rarity of the offspring production. It should be noted that biased-offspring production patterns in litters with many offspring (e.g., six EGFP/zero WT and one EGFP/seven WT) are considered rare event (low probability), whereas the biases observed in litters with a small number of offspring (e.g., three EGFP/zero WT and zero EGFP/two WT) must be common (high probability), and also that unbiased-offspring production patterns (e.g., one EGFP/two WT and three EGFP/three WT) are considered common (high probability) events. These probabilities were calculated in a null condition where each offspring was evenly selected from a pool of SSC clones at random (Figure 3A) (see Supplemental Information). We then measured the degree of bias of the offspring production by negative log probability, i.e., Shannon information content, which gives a quantitative measurement of the degree of unpredictability. We found that the offspring production from S recipients was more biased than those from M recipients (P = 0.02; Mann Whitney U-test) (Figure 3B and C). Furthermore, we statistically assessed whether the biased offspring production from M recipients did not occur by chance. Under a null hypothesis that each offspring was randomly selected from a pool of SSC clones (Figure 3A), we sampled the offspring birth patterns using a Monte Carlo method and averaged the degree of bias of the offspring production over all litters. By iterating this sampling 1,000,000 times, we obtained a distribution of the average degree of bias, which serves as a null distribution (Figure 3DandE). By comparing this null distribution with actual averages calculated from the real data, we concluded that the offspring production from S recipients was significantly biased (P = 0) (Figure 3D). In contrast, no statistical difference was observed in M recipients (P = 0.21) (Figure 3E). Because there appeared to be litters with relatively high number of offspring with one genotype that were found more frequently in the early phase than in the later phase, we calculated the degree of bias, and also carried out a Monte Carlo method to evaluate the statistical differences in both early and later phases of the experimental period (Supplemental Figure S2A H). However, we found that offspring production from S recipients was still significant in both phases (Supplemental Figure S2C and G). Offspring production patterns of M recipients did not show statistical difference (Supplemental Figure S2D and H). These results suggested that S recipients sired transgenic offspring in a nonrandom pattern from each testis. Analysis of recipient testes after mating experiment We sacrificed all surviving animals after the experimental period and recovered their testes (Figure 1C). The average weights of the testis

5 906 M. Kanatsu-Shinohara et al., 2017, Vol. 97, No. 6 Figure 2. A summary diagram showing the offspring production pattern. All offspring born from S and M recipients are indicated. Each column indicates the offspring born from recipient males. The x-axis indicates the size of the litter. Numbers indicated on the left side of the y-axis represent the date of birth. containing WT, EGFP, and mixed GS cells were 61.9, 53.5, and 58.0 mg, respectively (Figure 1D). Although the value was slightly smaller for testes with EGFP GS cells, there were no significant differences among the three types of testes. All of these testes were much larger than untransplanted control W mouse testes ( 11 mg). Histological sections of the recipient testes showed normal appearing spermatogenesis (Figure 1E). Immunostaining of the recipient testes with an acrosome (PNA) marker also showed normal distribution during spermatogenesis, and no apparent abnormalities or differences were noted among the three types of samples. We also quantified the number of tubules of mature germ cells by staining with PNA staining (Figure 1F). However, comparable numbers of tubules contained multiple layers of germ cells (Figure 1G). These results indicate that

6 Nonrandom male germline transmission, 2017, Vol. 97, No Figure 3. A null model for the analysis and flow to evaluate the degree of bias of offspring production in each litter. Each progeny was assumed to be randomly selected from a pool of EGFP and WT SSC clones. (A) The probability of offspring production patterns from each litter was calculated based on the null model. Each progeny was assumed to be randomly selected from a pool of EGFP and WT SSC clones. The degree of bias of offspring production was evaluated by Shannon information content. Note that the degree of bias changes according to the total number of offspring. For example, the degree of bias in litter 3, which consisted of only two GFP clones, is However, it was 3.69 when all six offspring were WT in litter N. (B, C) Histograms of the information content in S(B) and M (C) recipients. Arrows indicate the average values. Significant difference was found between S and M recipients (P = 0.02; Mann Whitney U-test). (D, E) Statistical tests for the chance of the biased-offspring production. By Monte Carlo sampling in the null model (A), artificial dataset of offspring production patterns were generated following the observed numbers of offspring from recipients, and average of the information contents was calculated according to the same procedure. Probability distributions of the average information content in S (D) and M (E) recipients were obtained by repeating the Monte Carlo sampling 1,000,000 times. Red lines indicate the actual information content average calculated by experimental data. Statistical significance was determined using P values, which were calculated by empirical probabilities greater than the actual information content average (total areas of blue columns above the red bar). While the chance of the biased-offspring production was not statistically significant in mixed conditions (P = 0.21), separate conditions showed statistical significance (P = 0). colonization of the three types of GS cells in testes occurred at the same levels. Discussion The laterality of ovulation has long been studied in females [6, 19 21], and many studies are available on the fertility of orchidectomized human patients, particularly in relation to the treatment of tumors or varicoceles [22]. However, the effect of laterality on the germline transmission pattern has not been analyzed in the male, including experimental animals. One study using mice showed declining fertility after unilateral vasectomy, but not after orchiectomy [3]. Another more recent study showed that left/right differences in vascular architecture are associated with a decrease

7 908 M. Kanatsu-Shinohara et al., 2017, Vol. 97, No. 6 in hemoglobin saturation and increased levels of HIF1A in the left testis, but no effect on fertility has been reported [23]. Because unilateral spermatogonial transplantation could restore fertility [24], it is apparently not necessary to have spermatogenesis in both testes for fertility restoration, which is expected from reports on fertility of hemicastrated mice and humans. However, it is possible that each testis may influence or interact with the other and this possibility has not been evaluated. Despite its importance, an appropriate method to study the effect of laterality for SSCs has not been developed. In this study, we used spermatogonial transplantation and GS cell culture to address this problem. Spermatogonial transplantation is particularly useful because it is the only method to produce offspring from SSCs [10]. However, because SSC frequency is very low ( % in adult testis cells) [8, 9], direct transplantation of fresh testis cells is not very efficient for fertility restoration due to limited number of transplanted SSCs. Although the frequency of SSCs in GS cell culture is 1 2% [13], the concentration of SSCs in this culture is significantly higher than in fresh testis cells ( %), which greatly improves fertility restoration of recipients. In our recent study, we estimated that at least GS cells can restore fertility, which corresponds to SSCs per testis [14]. Thus, the combination of spermatogonial transplantation and GS cell culture techniques allows for assessment of the effect of laterality in a functional manner for the first time. An important finding in this study was the altered germline transmission pattern as a result of the transplantation procedure. The overall efficiency of transgenic offspring production was significantly higher in S recipients compared with M recipients (43.2% vs. 20.1%). Moreover, S recipients produced a total of 20 litters (23.5%) that contained only one genotype. In particular, 8 of 20 litters consisted of only transgenic offspring. Because an average of seven pups were included in a litter from S recipients, the chance of getting such a result would be extremely low if SSCs from both testes contribute randomly to fertilization. Thus, EGFP and WT SSCs not only changed the frequency of transgenic mouse production but also the pattern of their appearance according to the site of transplantation. Indeed, when we carried out mathematical simulation and compared the results with real data, we confirmed that the biased transgenic offspring production from S recipients occurred in a statistically significant manner. Because normal spermatogenesis continued in both testes at the same levels, this result was unexpected. One of the potential factors that are involved in this biased offspring production is competition between SSCs. Competition of spermatogonia was originally suggested in aggregation chimera studies [25], which likely has important implications in humans [26]. By making allophenic chimera mice, it was reported that the genotype of offspring born from male chimeric animals can change during their life time. For example, when C57BL/6 (B6) and C3H embryos were mixed to produce chimeras, male chimeras usually produced more offspring with C3H-derived sperm than B6 sperm and 15% of offspring were derived from B6 germ cells. However, the proportion of B6 germ cells among their progeny continued to decline, although fertility was sustained. It was suggested that such selection might occur at a stage in gametogenesis when successive proliferations are still occurring, i.e., in the diploid spermatogonial period. It was speculated that germ cells of the two strains might compete for available materials with different efficiencies, which confers a selective advantage for the C3H genotype. Although we did not find apparent functional differences between WT and EGFP GS cells during in vitro culture, this example suggests that a slight difference in growth/differentiation rate between WT and EGFP cells may cause competition in M recipient testes. Although spermatogonia competition may occur continuously in the testis, it still does not explain how the recipient could sire progeny with only one genotype from S recipients. It is possible that spermatogonia competition may gradually change the ratio of the transgenic offspring as shown in the chimera studies. However, our results showed that litters with one genotype were suddenly born without gradual change in the ratio of transgenic offspring. Because this occurred throughout the entire experimental period, spermatogonia competition alone does not appear to explain our results. Although we currently do not know the mechanism underlying this phenomenon, it is probably not regulated by the endocrine system because it would affect both sides in a similar manner. In this sense, we speculate that sperm transport through the male reproductive tract is influenced by nerve or vessels, which may distribute unevenly on the right and left side and differentially influenced fertilization patterns. Considering that ovulation is influenced by superior ovarian nerves [27], it is tempting to speculate that a similar mechanism may operate in sperm transport in males. A biased offspring production pattern was recently reported in our studies on lineage tracing of SSCs and may provide a clue to solve this problem [28]. In these experiments, we specifically marked SSCs by introducing a retro- or lentivirus before spermatogonial transplantation and found that offspring with the same viral integration patterns were frequently born in the same litters. The results indicated that a relatively small number of SSCs contribute to offspring production at a time. Given the results of the present experiments and the fact that relatively small numbers of SSCs contribute to a litter, it seems reasonable to speculate that sperm from right and left testes do not simply compete against each other but have different time windows for fertilization. EGFP SSCs may be outcompeted by more dominant WT SSCs in M recipients, but EGFP SSCs in S recipients do not have such competitors in the same testis and have an increased opportunity to participate in fertilization. If many SSCs from both testes compete equally to fertilization, the chance of producing a whole litter with EGFP SSCs would be very low. However, when only a few SSCs in one of the testes contribute to a given litter, the chance of EGFP SSCs to contributing to a whole litter becomes greater. Given these results, it is possible that a novel mutation in the germline may not be transmitted by simple competition of all spermatozoa; germline transmission may occur only when such a small number of SSCs obtain opportunity to fit in the time window for germline transmission. Perhaps, such machinery may prevent rapid expansion of newly generated mutations to the next generation. In addition, because sperm generated from one testis is sufficient for fertilization, it would be efficient to have another testis for subsequent fertilization if accumulation of sperm takes significant amount of time. This would increase the total fertility of mice. Because continuous collection of sperm is difficult from recipient testes and epididymides, these hypotheses are not easy to evaluate, but several approaches may clarify the potential machinery that underlies our observation. For example, it will be possible to identify specific stages of the seminiferous tubules (such as stage VII) and enumerate round spermatids per Sertoli cells to compare the sperm production efficiency between WT and EGFP SSCs. It would be ideal if it becomes possible to monitor the changes in the relative number of stages of seminiferous tubules in each testis as function of time. Absolute copy numbers of sperm genomes transiting the epididymis could be

8 Nonrandom male germline transmission, 2017, Vol. 97, No quantified by Digial Drop PCR. In addition, synchronization of the seminiferous tubules with WIN18,446, which inhibits the biosynthesis of retinoic acid from retinol, may also change the offspring production patterns [29]. Such approaches may help to better unveil novel cellular and molecular machineries that influence testicular laterality. Germline transmission from SSCs involves many steps. Currently, very little is known about the factors that are involved in this process. Because our experimental system is based on cell transplantation, we cannot exclude the possibility that analysis of transplanted SSCs may not fully mimic SSCs under physiological conditions. Nevertheless, analysis based on spermatogonial transplantation is currently the only method to study such process. We think that the early phase ( 3 4 months) may be reflecting the regeneration after transplantation, and the results from later stages probably represent stable spermatogenesis. This is because morphological abnormalities associated with spermatogonial transplantation, such as missing layers of germ cells in the seminiferous tubules, gradually disappear after 3 months [30]. To our knowledge, the current study provides a first functional analysis of effect of laterality on germline transmission patterns in the male and has an important implication in understanding the pattern of male side inheritance of genetic information. Our findings suggest that fertilization resulting from individual testis may have a potential time window, which suggests that regulation of male germline transmission is more sophisticated than is generally considered. Identification of key molecules that underlie this phenomenon is the next obvious goal. Further studies will increase our knowledge of the relationship between male fertility and SSCs, and such studies will also have important implications in genetic variation, disease onset, and human infertility treatment. Supplementary data Supplementary data are available at BIOLRE online. Supplemental Figure S1. Offspring production patterns of recipient mice. All offspring born from individual S (A) or M (B) recipients are shown. Each column indicates the offspring born from each recipient male. The x-axis indicates the size of the litter. Numbers indicated on the left side of the y-axis represent the litter number. Numbers in parenthesis indicate the date of birth after transplantation. Supplemental Figure S2. Comparison of early and late phase of biased offspring production. (A, E) Histograms of the information content in early (A) and late (E) phases of S recipients. (B, F) Histograms of the information content in early (B) and late (F) phases of M recipients. (C, G) Probability distributions of the average information content in early (C) and late (G) phases of S recipients by repeating the Monte Carlo sampling 1,000,000 times. (D, H) Probability distributions of the average information content in early (D) and late (H) phases of M recipients by repeating the Monte Carlo sampling 1,000,000 times. Arrows indicate the average values. Significant difference was found in both early and late phases of S recipients (P = 0; Mann Whitney U-test ), but no difference was found in both phases of M recipients. Acknowledgments We thank Ms Y. Ogata for technical assistance and Dr K. Nakae for giving advice on statistical analysis. References 1. Harper MJK. Sperm and egg transport. In: Austin CR, Short RV (eds.), Germ Cells and Fertilization. Cambridge: Cambridge University Press; 1982: Amelar RD, Dubin L, Hotchkiss RS. Restoration of fertility following unilateral orchiectomy and radiation therapy for testicular tumors. 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