Spermatogonial asynchrony in Tex14 mutant mice lacking intercellular bridges

in Reproduction

Correspondence should be addressed to H Chiarini-Garcia; Email: heliochiarini@gmail.com
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The existence of cytoplasmic passages between germ cells and their potential function in the control of the spermatogenic process has long been an intriguing question. Evidence of the important role of such structures, known as intercellular bridges (ICB), in spermatogenesis has been implicated by the failure of spermatogenesis in testis-expressed gene 14 (Tex14) mutant mice, which lack the ICBs, to progress past the pachytene spermatocyte stage. Using these Tex14 mutants, the present study evaluated, for the first time, the behavior and synchrony of the spermatogonial lineage in the absence of ICBs. Our data suggest that the absence of these cytoplasmic connections between cells affects the expansion of the undifferentiated type A (Aundiff) spermatogonia compartment and their transition to A1, resulting in a significant numerical reduction of differentiating A1 spermatogonia, but did not interfere with cell amplification during subsequent mitotic steps of differentiating spermatogonia from A1 through intermediate (In). However, beginning at the type B spermatogonia, the synchrony of differentiation was impaired as some cells showed delayed differentiation compared to their behavior in a normal seminiferous epithelium cycle. Thus although spermatogonial development is able to proceed, in the absence of ICBs in Tex14−/ mutants, the yield of cells, specific steps of differentiation, the synchrony of the cell kinetics, and the subsequent progression in meiosis are quantitatively lower than normal.

Supplementary Materials

    • Supplementary Figure 1 – Numbers of undifferentiated type A spermatogonia in the six spermatogonial stages of the seminiferous epithelium of individual (A) Tex14+/- (Het) and (B) Tex14-/- (KO) mice. The numbers of Aundiff spermatogonia for each mouse, averaged over the 6 stages is presented at the right of the figure. It was apparent in both panels that, in all animals, the number of Aundiff declined between stage 6 and stages 2 to 3 in heterozygotes and stages 3 to 4 in Tex14-/- mutants and that the curves were shifted up or down consistent with the average numbers of Aundiff in each animal. Since the variability between animals (particularly in the case of Tex14-/- mutants) was primarily based on the supply of Aundiff spermatogonia, the values at each stage were normalized to the average in that animal (C). This normalization reduced the relative standard deviations (coefficients of variation) and now significant differences in Aundiff numbers between stages were observed (ANOVA, P<0.001). The highest numbers of Aundiff spermatogonia observed in stages 6 and 1 were significantly higher than that at the minimum at stage 3 (P<0.001). * Significant difference (P<0.05) from heterozygote (t-test); † Significant (P<0.05) increase from minimum stage for that genotype, (ANOVA, Tukey post-hoc test).
    • Supplementary Figure 2 – The percentages of seminiferous tubules within eight intervals of BrdU labeling index (LI) of spermatogonia in individual tubule cross-sections are presented as a measure of synchrony. Synchronous procession through the cell cycle is indicated by tubules with high LI (S-phase) and low LI (G2/M/G1 phases); asynchrony is indicated by tubules with intermediate (~50%) LI. Since the spermatogonial stages could not be accurately identified in the material used for immunochemical assessment of LI, the tubules were divided in approximately equal subsets based on the numbers of spermatogonia per tubules, with the lower numbers () generally representing tubules with A1 to A4 spermatogonia and the higher number () representing tubules with In to B spermatogonia. (A) Note that in the Tex14+/− heterozygotes, there is a bimodal distribution with tubules with low LI (<12.5%) and tubules with high LI (62.5 to 87.5%), indicating the tubules contain cells synchronized in the G2/M/G1 and in the S-phase of the cell cycle, respectively. The distributions of the tubules with A and with In/B spermatogonia overlap indicating that both spermatogonial subclasses are equally synchronized in their cell cycles. (B) In contrast, the Tex14−/− mutants, show a unimodal distribution with many tubules with intermediate labeling indices between 37.5% and 62.5%, indicating that not all cells within a tubule are in the same phase of the cell cycle. Comparison of the distribution of the tubules likely containing A spermatogonia in the Tex14−/− mutants with that in heterozygotes, indicates that there appears to be some cell-cycle asynchrony within these tubules. The tubules likely containing the In/B spermatogonia show even more in the intermediate range, supporting the histological data that asynchrony increases at the B spermatogonial stage in Tex14−/− mutants.

 

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