Nonrandom Germline Transmission of Mouse Spermatogonial Stem Cells Mito Kanatsu-Shinohara, Honda Naoki, Takashi Shinohara  Developmental Cell  Volume 38, Issue 3, Pages 248-261 (August 2016) DOI: 10.1016/j.devcel.2016.07.011 Copyright © 2016 Elsevier Inc. Terms and Conditions

Developmental Cell 2016 38, 248-261DOI: (10.1016/j.devcel.2016.07.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Evaluation of Sperm Production Efficiency by SSCs (A) Experimental procedure. Testis cells from 8- to 10-day-old green and WT mice were transplanted into 8- to 10-day-old W mice. Testes were then recovered 2, 4, and 8 months after transplantation for colony counts. Spermatozoa were also collected from epididymides to evaluate Egfp levels using real-time PCR. (B) Macroscopic appearance of recipient testis 8 months after transplantation. A representative histological appearance is shown in the inset. Scale bar, 1 mm. (C) EGFP+ colony counts in recipient testes (n = 4–6). (D) Relative frequency of EGFP+ sperm (n = 4–6). (E) Estimation of total colony numbers. Numbers (WT + EGFP) estimated by colony/sperm ratio ([number of colonies × 100]/[percentage of Egfp+ sperm × 2]) (indicated as “Sperm”) or transplantation ratio ([colony counts] × 7 [ratio between total and EGFP+ cells]) (indicated as “Donor cells”) are shown (n = 4–6). Note that only half of the sperm will carry the transgene since the donor mice were heterozygous for the transgene. Error bars indicate SEM. Developmental Cell 2016 38, 248-261DOI: (10.1016/j.devcel.2016.07.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Transgenesis of SSCs and Analyses of Transgenic Progeny (A) Experimental procedure. Testis cells from 8- to 10-day-old pups were infected overnight with LacZ-expressing retro- or Venus-expressing lentivirus vectors prior to transplantation into 8- to 10-day-old W mice. (B) Macroscopic and histological appearance of untransplanted W testes. (C and D) Macroscopic and histological appearance of the recipient testes that were transplanted with testis cells transduced with retro- (C) or lentivirus (D) vectors. Testes of progenies showing LacZ or Venus transgene expression are shown in insets, showing germline transmission. (E and F) Immunostaining of recipient testes that were transplanted with Venus-infected cells. Meiotic (SYCP3; E) and haploid (PNA; F) cell markers indicated normal spermatogenesis. (G) Histological appearance of caput epididymis (top) and motile spermatozoa collected from cauda epididymis (bottom). Stain: H&E (B, C, G); Hoechst 33342 (E, F). Scale bars: testes, 1 mm (B–D); sections, 50 μm (B, C), 200 μm (D), 20 μm (E, F), and 50 μm (G). See also Figure S1. Developmental Cell 2016 38, 248-261DOI: (10.1016/j.devcel.2016.07.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Clonal Analysis of the SSC Contribution to Spermatogenesis (A) Screening of transgenic offspring by southern blot analysis using LacZ (left: recipient A) or Venus probes (right: recipient K). The litter number is indicated above the gel (e.g., A9 indicates the ninth litter of recipient A), and birth dates after transplantation are shown in parentheses. The enzymes that were used to digest DNA are shown below the gel. EcoRI digests the LacZ probe, while BamHI does not cut LacZ or Venus probes. Therefore, each band after BamHI digestion indicates the integration of a single virus. For example, two viral integrations were found in two males in the 19th litter from the K recipient male, while three females from the same litter showed single integrations. (B) Temporal changes in transgenic offspring production patterns by southern blot analyses using LacZ (left: recipient A) or Venus probes (right: recipient L). Each band represents a single integration event since NcoI and BamHI do not cleave the probes. Therefore, one of the females born in litter A12 and two males born in litter L3 contained two viral integrations. The remaining offspring showed single integrations. (C) Confirmation of the clones in different litters by southern blot analyses using LacZ (left: recipient A) or Venus probes (right: recipient J). Transgenic DNA that was identified by the initial screen was digested with multiple restriction enzymes. While EcoRI digested the probe, the rest of the enzymes did not. The same clones were also observed in another litter from both recipient types. Since the two females from recipient A were born at the same time, this also represented a case of clonal coincidence. (D) A summary diagram showing transgene integration patterns in progeny from recipient J. Shown are the litter number and birth date after transplantation. Each square represents offspring born at the indicated time points and filled orange squares indicate transgenic offspring. This recipient mouse produced 242 offspring with 126 different integration patterns, siring 144 transgenic mice. Identical lowercase letters within the squares (a to h) indicate offspring with the same pattern of transgene integration. Offspring born on the same date were recorded as the same litter. (E) The time interval between the first and last appearance of a clone based on all the offspring born from LacZ- or Venus-transfected SSCs. Developmental Cell 2016 38, 248-261DOI: (10.1016/j.devcel.2016.07.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Mathematical Analysis of Offspring Production Patterns (A) Probability of birth from the same clones in the same litter. Using the estimated active SSC number (Table 1), the chance of producing offspring with the same transgene integration pattern in the same litter was statistically examined. The null hypothesis was that each transfected progeny was randomly selected from a pool of active SSC clones. Blue columns indicate the probabilities of the number of the clonal coincidence determined using Monte Carlo samplings. Red bars indicate the actual number of litters with offspring having the same transgene integration pattern (coincident clones). Statistical significance was determined using a Monte Carlo test in which p values were calculated by the sum of the probabilities of higher than or equal to the observed number of clonal coincidences (total areas of blue columns above the red bar). When we used SSC number by colony/sperm ratio, A, J, and K sired litters with coincident clones more frequently than was expected based on Monte Carlo samplings. J and K showed statistical significance when we used SSC number by transplantation ratio. p Values in the figure are based on the estimated active SSC number (Table 1). (B) Experimental data showing the minimum interval between births of offspring with identical transgene integration patterns based on all the offspring born from LacZ- or Venus-transfected SSCs. Shown is a histogram of the interval between births with identical virus integration patterns. The left y axis indicates the actual number of offspring born within bin sizes of 34 days. The 0-day interval (i.e., the number of the clonal coincidence) was counted with 1-day bin size. The right y axis indicates the probability density. The blue line indicates the probability density function (PDF) based on a random interval between births with the same integration patterns. The green line indicates the PDF based on a model that assumes SSCs divide on a continuous 8.6-day cycle, in accordance with the seminiferous tubule epithelial cycle. The red line indicates the PDF based on a model incorporating spermatogenic burst and rest (corresponding to Equation 14). The model parameters were estimated by regression analyses using the data on the number of offspring. Statistical differences were not observed between the red line and the experimental data using a Pearson's Chi-Squared test for goodness of fit: p > 0.89 for red line; p < 0.01 for blue and green lines. Differences in bin for observed and theoretical frequencies were compared. (C) The model for spermatogenic activity (ability of producing offspring) in a single hypothetical clone. SSCs go through cycles of dormancy in terms of spermatogenic activity. The probability of producing offspring fluctuates, but our theory predicts that there is always a rest period. Each SSC will repeat this cycle of burst and rest during its lifetime. See also Figure S2 and Table S1. Developmental Cell 2016 38, 248-261DOI: (10.1016/j.devcel.2016.07.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Lineage Analysis of SSCs Using H2B-GFP Labeling (A) Experimental procedure. Six- to seven-week-old TetOP-H2B-GFP transgenic mice were pulsed with Dox for 5 weeks or 4 months to induce GFP expression. GFP expression patterns were analyzed by immunohistochemistry and flow cytometry. (B) Immunohistochemical analysis of GFP expression in CDH1+ undifferentiated spermatogonia after Dox withdrawal. At day 0, a total of 60 CDH1+ cells in 16 tubules all showed GFP fluorescence. Stain: Hoechst 33342. Scale bars, 20 μm. (C) Quantification of GFP+ cells in CDH1+ spermatogonia after Dox withdrawal. (D) Staining patterns and quantification of cell numbers in five spermatogonial populations (I to V) found in the spermatogonia gate (defined by forward scatter/side scatter in C; n = 3). Results are normalized to the cell number in population I (undifferentiated spermatogonia). (E) Flow cytometric analyses of apoptotic cells. Cells were stained with ANXA5 and the indicated spermatogonial markers. (F) Quantification of apoptotic cells in the five gates using ANXA5 staining (as in D; n = 3). Asterisks indicate statistical significance by ANOVA (p < 0.05). Error bars indicate SEM. See also Figures S3–S6. Developmental Cell 2016 38, 248-261DOI: (10.1016/j.devcel.2016.07.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 A Model Showing Spermatogonia Progenitor Selection SSCs repeat cycles of transient bursts of spermatogenic activity that led to the birth of offspring. However, progenitors from most SSCs undergo clonal apoptosis mostly at differentiating spermatogonia stage and fail to produce sperm (indicated by the crosses). The same clone can resume progeny production at later time points. Developmental Cell 2016 38, 248-261DOI: (10.1016/j.devcel.2016.07.011) Copyright © 2016 Elsevier Inc. Terms and Conditions