Extended Data Fig. 7: Changes in testes features in response to gut microbiota perturbation.

(a) Testes seminiferous tubule sections from control males or after 6wk nABX, stained with hematoxylin and eosin (H&E). Shown are corresponding sections of CON and nABX testis from multiple different tubule stages (labelled in roman numerals), with the expected distribution of germ cell types for each stage shown (left). nABX testis routinely exhibited abnormalities and loss of entire cell-type compartments, potentially reflective of an impacted spermatogonial stem cell pool. Round spermatids (RS), Elongated spermatids (ES), Pachytene (P), Zygotene (Z), Preleptotene (Pl), Leptotene (L), Diplotene (D), Meiotic division (M). Scale bars: 100μm. (b) Sperm count from indpendent control and 6wk nABX-treated males. p-value by unpaired two-tailed t-test (CON n = 29, nABX n = 33). Bar indicates mean with 95% C.I. (c) Quantification of abnormal testes stratified by effect on individual testis (CON n = 7 testis, from N = 4 males; nABX n = 10, N = 5 males). All but one nABX testis had a higher mean rate of abnormalities relative to control average. Bar indicate mean with 95% C.I. (d) Quantification of total tubule diameter in control and nABX-treated (6wk) testis. p-value by nested unpaired two-tailed t-test (CON n = 854 tubules, nested into N = 4 males; nABX n = 1061, N = 4 males). Bar represents median and whiskers indicate 5-95th percentile. (e) Representative images of epididymis sections from independent nABX-treated males. Star indicates abnormality. (f) Assessment of sperm morphology. Shown are representative examples of normal sperm and those with abnormal head-piece, mid-piece and tail defects. Indicated right is quantification of overall level of abnormal sperm in control and nABX-treated males. All values are within the normal range, consistent with normal fecundity but altered molecular payload. Each male sperm sample was prepared in 3 slide smears at different concentrations and ~100 sperm cells were counted per slide (N = 2 males; n = 300 sperm cells counted per male). Out of 600 sperm cells counted per group, 279 from the CON group and 339 from the nABX group showed one or more morphological defects.

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