doi: 10.1038/s42003-023-05685-2.
Multiple ageing effects on testicular/epididymal germ cells lead to decreased male fertility in mice
Affiliations
- PMID: 38177279
- PMCID: PMC10766604
- DOI: 10.1038/s42003-023-05685-2
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Multiple ageing effects on testicular/epididymal germ cells lead to decreased male fertility in mice
Commun Biol.
.
Abstract
In mammals, females undergo reproductive cessation with age, whereas male fertility gradually declines but persists almost throughout life. However, the detailed effects of ageing on germ cells during and after spermatogenesis, in the testis and epididymis, respectively, remain unclear. Here we comprehensively examined the in vivo male fertility and the overall organization of the testis and epididymis with age, focusing on spermatogenesis, and sperm function and fertility, in mice. We first found that in vivo male fertility decreased with age, which is independent of mating behaviors and testosterone levels. Second, overall sperm production in aged testes was decreased; about 20% of seminiferous tubules showed abnormalities such as germ cell depletion, sperm release failure, and perturbed germ cell associations, and the remaining 80% of tubules contained lower number of germ cells because of decreased proliferation of spermatogonia. Further, the spermatozoa in aged epididymides exhibited decreased total cell numbers, abnormal morphology/structure, decreased motility, and DNA damage, resulting in low fertilizing and developmental rates. We conclude that these multiple ageing effects on germ cells lead to decreased in vivo male fertility. Our present findings are useful to better understand the basic mechanism behind the ageing effect on male fertility in mammals including humans.
© 2024. The Author(s).
Conflict of interest statement
The authors declare no competing interests.
Figures
a Appearance of C57BL/6 J male mice at 2 and 24 months of age. Scale bars, 10 mm. b Diagram of mating test. Single 2-month-old males (n = 5) were caged with two of 2-month-old females, and the females were replaced with new females every 4 months (arrowheads), until the males reached the age of 24 months (red box). Vaginal plugs in females were checked when the males reached the age of 2–4, 12–14, and 22–24 months. c Mating test (pup numbers per litter) with males from 2 to 24 months of age. Error bars, mean ± SD. Blue dots, biological replicates of males (n = 5). P = 0.0003 and r = −0.45 (Pearson’s correlation). d Diagram of days between birth and the next pregnancy. Green box, pregnancy period. Red bar, days between litters (births). Blue bars, days between birth and the next pregnancy. e Days between births. Each red dot represents the days between births when females (n = 30) were mated with males (n = 5) at 2–4, 12–14, or 22–24 months of age. NS, not significant (P > 0.05; Dunnett’s test). f Serum testosterone concentration (ng/mL) in males at 2 and 24 months of age. Blue dots, biological replicates of males at 2 (n = 11) and 24 (n = 12) months of age. NS, not significant (P > 0.05; one-tailed t test). g Testicular testosterone concentration (ng/g of testis) in males at 2 and 24 months of age. Blue dots, biological replicates of males at 2 (n = 5) and 24 (n = 5) months of age. NS, not significant (P > 0.05; one-tailed t test).
a Gross morphology of testes in males at 2 and 24 months of age. Scale bars, 5 mm. b Testis weight (mg) in males at 2, 6, 12, 18, and 24 months of age. Error bars, mean ± SD. Red dots, testis values in males at 2 (n = 5), 6 (n = 5), 12 (n = 5), 18 (n = 5), and 24 (n = 7) months of age. P = 0.16 and r = 0.13 (Pearson’s correlation). c Body weights (g) in males at 2, 6, 12, 18, and 24 months of age. Error bars, mean ± SD. Blue dots, biological replicates of males at 2 (n = 5), 6 (n = 5), 12 (n = 5), 18 (n = 5), and 24 (n = 7) months of age. P = 0.0004 and r = 0.60 (Pearson’s correlation). d Testis weight per body weights (%) in males at 2, 6, 12, 18, and 24 months of age. Error bars, mean ± SD. Red dots, testis values in males at 2 (n = 5), 6 (n = 5), 12 (n = 5), 18 (n = 5), and 24 (n = 7) months of age. P < 0.00001 and r = −0.61 (Pearson’s correlation). e Testis tubule cross-sections in males at 2 (Left; a stage VII tubule) and 24 (Right; a tubule showing germ cell depletion) months of age, stained with hematoxylin and periodic acid-Schiff (He-PAS). Upper panels enlarge black boxed regions in Lower panels. Green boxed regions (#1 and #4) indicate areas shown in higher magnification in Supplementary Fig. S2a. Scale bars, 20 μm. f Number of germ cell-depleted tubule cross-sections per total tubule cross-sections. Blue dots, biological replicates of males at 2 (n = 5) and 24 (n = 5) months of age. P = 0.0008 (one-tailed t test). g Testis tubule cross-sections in stage IX of males at 2 (Left) and 24 (Right) months of age, stained with He-PAS. Upper panels enlarge black boxed regions in Lower panels. Green boxed regions (#2 and #5) indicate areas shown in higher magnification in Supplementary Fig. S2a. Scale bars, 20 μm. h Number of stage IX-X tubule cross-sections with aligned spermatozoa per total tubule cross-sections. Blue dots, biological replicates of males at 2 (n = 5) and 24 (n = 5) months of age. P = 0.003 (one-tailed t test). i Number of stage IX-X tubule cross-sections with aligned spermatozoa per total stage IX-X tubule cross-sections. Blue dots, biological replicates of males at 2 (n = 5) and 24 (n = 5) months of age. P = 0.01 (one-tailed t test). j Testis tubule cross-sections in males at 2 (Left; a stage XII tubule) and 24 (Middle and Right; a tubule containing stages X, XI, and XII) months of age, stained with He-PAS. Upper (2 and 24 months) and Lower left (24 months) panels enlarge black boxed regions in Lower (2 months) and Lower right (24 months) panels. Green boxed regions (#3 and #6) indicate areas shown in higher magnification in Supplementary Fig. S2a. Scale bars, 20 μm. k Number of tubule cross-sections containing more than two stages per total tubule cross-sections. Blue dots, biological replicates of males at 2 (n = 5) and 24 (n = 5) months of age. P = 0.002 (one-tailed t test).
a Testis tubule cross-sections in stage VII of males at 2 (Left) and 24 (Right) months of age, stained with hematoxylin and periodic acid-Schiff (He-PAS). Upper panels enlarge black boxed regions in Lower panels. Scale bar, 20 μm. b Number of germ cells (type A spermatogonia) in stage VII tubule cross-sections per tubule section area (mm2). Blue dots, biological replicates of males at 2 (n = 3) and 24 (n = 5) months of age. P = 0.03 (one-tailed t test). c Number of germ cells (preleptotene and pachytene spermatocytes, round spermatids, and spermatozoa) in stage VII tubule cross-sections per tubule section area (mm2). Blue dots, biological replicates of males at 2 (n = 3) and 24 (n = 5) months of age. P = 0.02 and 0.01 (one-tailed t test). NS, not significant (P > 0.05; one-tailed t test). d Gross morphology of (caput and cauda) epididymides in males at 2 and 24 months of age. Scale bars, 5 mm. e Epididymis weight (mg) in males at 2, 6, 12, 18, and 24 months of age. Error bars, mean ± SD. Red dots, epididymis values in males at 2 (n = 5), 6 (n = 5), 12 (n = 5), 18 (n = 5), and 24 (n = 7) months of age. P = 0.0001 and r = 0.49 (Pearson’s correlation). f Epididymis weight per body weights (%) in males at 2, 6, 12, 18, and 24 months of age. Error bars, mean ± SD. Red dots, testis values in males at 2 (n = 5), 6 (n = 5), 12 (n = 5), 18 (n = 5), and 24 (n = 7) months of age. P = 0.43 and r = 0.02 (Pearson’s correlation). g Cauda epididymis tubule longitudinal-sections in males at 2 (Left) and 24 (Right) months of age, stained with He-PAS. Upper panels enlarge black boxed regions in Lower panels. Scale bars, 100 μm. h Total sperm numbers from the cauda epididymis (×106). Blue dots, biological replicates of males at 2 (n = 8) and 24 (n = 7) months of age. P = 0.02 (one-tailed t test).
a Immunostaining for STRA8, BrdU, and SOX9, with hematoxylin counterstain, on testis cross-sections of males at 2 (Left; a stage VII-VIII tubule) or 24 (Middle, a tubule in stages VII-VIII; Right, a tubule showing germ cell depletion) months of age. Arrows, Sertoli cell nuclei. Arrowheads, type A spermatogonia (white) and preleptotene spermatocytes (black). Scale bars, 20 μm. b Number of BrdU-positive type A spermatogonia per tubule section area (mm2). Blue dots, biological replicates of males at 2 (n = 3) and 24 (n = 5) months of age. P = 0.03 and 0.01 (Tukey-Kramer test). NS, not significant (P > 0.05; Tukey-Kramer test). P = 0.008 and 0.002 (one-tailed t test). NS, not significant (P > 0.05; one-tailed t test). c Testis tubule cross-sections of males at 2 (Left; a stage I-II tubule) or 24 (Middle, a stage I-II tubule; Right, a tubule showing germ cell depletion) months of age, stained with hematoxylin and periodic acid-Schiff (He-PAS). Arrowheads, degenerating spermatogonia. Scale bar, 20 μm. d Number of degenerating spermatogonia per tubule section area (mm2). Blue dots, biological replicates of males at 2 (n = 3) and 24 (n = 5) months of age. NS, not significant (P > 0.05; Tukey-Kramer test). e Senescence-associated β-galactosidase (SA-β-gal) staining, with Nuclear Fast Red counterstain, on testis cross-sections of males at 2 (Left) or 23 (Right) months of age. Insets (red) enlarge red boxed regions. Scale bars, 100 μm. f Cauda epididymis tubule longitudinal-sections in males at 2 (Left) and 24 (Middle and Right) months of age, stained with hematoxylin and periodic acid-Schiff (He-PAS). Upper panels enlarge black boxed regions in Lower panels. Red bars, epithelial cells. Asterisks, vacuoles. Scale bars, 20 μm. g Senescence-associated β-galactosidase (SA-β-gal) staining, with Nuclear Fast Red counterstain, on cauda epididymis tubule longitudinal-sections of males at 2 (Left) or 24 (Right) months of age. Insets (red) enlarge red boxed regions. Scale bars, 100 μm.
a Scatter plots of RNA-Seq data comparing the normalized FPKM values for all genes in testes (Left) or cauda epididymides (Right) of males at 2 (x-axis, n = 5) vs. 24 (y-axis, n = 5) months of age. Red dots, up-regulated genes (fold change ≥2). Blue dots, down-regulated genes (fold change ≤−2). Gray dots, no differential gene expression (−2 <fold change <2). b Gene Ontology (GO) terms (biological process) of up-regulated genes (fold change ≥2) on RNA-Seq data in testes (Left) or cauda epididymides (Right) of males at 2 (n = 5) vs. 24 (n = 5) months of age. The top 10 enriched terms are displayed based on the -log10 (P-value). Y-axis, −log10 (P-value). Blue graphs, GO terms related to inflammation. c Heatmap of differentially expressed genes related to cellular senescence, undifferentiated spermatogonia, spermatogonial differentiation, meiosis, spermiogenesis, Sertoli cells, and Leydig cells, on RNA-Seq data in testes or cauda epididymides of males at 2 (n = 5) vs. 18 (n = 5) vs. 24 (n = 5) months of age.
a Morphology of cauda epididymal spermatozoa in males at 2 (Left) and 24 (Middle and Right) months of age. Upper panels enlarge black boxed regions in Lower panels. Arrowheads, bent head (dark gray) and bent (or broken) midpiece (light gray). Scale bars, 20 μm. b Number of abnormal spermatozoa per total spermatozoa (%), collected from caput and cauda epididymides, in males at 2 (n = 5 biological replicates) and 24 (n = 6 biological replicates) months of age. Percentages of abnormal spermatozoa were increased from 1.2% (16/1291 at 2 months) to 9.1% (122/1344 at 24 months) in caput epididymides (P < 0.00001; χ2 test), and from 9.1% (158/1628 at 2 months) to 34.9% (566/1620 at 24 months) in cauda epididymides. Boxes, normal (white), bent head (dark gray), bent or broken midpiece (light gray), and other abnormalities (black). c Ultrastructural analysis of cauda epididymal spermatozoa, in males at 2 (Left) and 24 (Middle and Right) months of age, by scanning electron microscopy (SEM). Upper panels enlarge blue boxed regions in Lower panels. Arrowheads, bent head (yellow) and bent midpiece at the annulus (white). Scale bars, 5 μm. d Flagellar bending patterns of spermatozoa at 10 min (Upper) and 120 min (Lower) after incubation in capacitation media, in males at 2 (Far left) and 24 (Center left, normal; Center right, bent head; Far right, bent or broken midpiece) months of age. Single frames throughout one beating cycle or 20 frames are superimposed. e Progressively motile spermatozoa per total spermatozoa (%), at 10 min and 120 min after incubation in capacitation media, in males at 2 and 24 months of age. Error bars, mean ± SD. Blue dots, biological replicates of males at 2 (n = 6) and 24 (n = 9) months of age. P = 0.02 and 0.0001 (one-tailed t test). f Velocity parameters of motile spermatozoa (μm/sec) at 10 min and 120 min after incubation in capacitation media, in males at 2 and 24 months of age. VCL curvilinear velocity, VAP average path velocity, VSL straight line velocity. Error bars, mean ± SD. Blue dots, biological replicates of males at 2 (n = 6) and 24 (n = 9) months of age. P = 0.03, 0.02, 0.01, 0.009, and 0.00005 (one-tailed t test). NS, not significant (P > 0.05; one-tailed t test).
a Number of fertilized eggs per total oocytes (%) at 8 h after in vitro fertilization (IVF), by using spermatozoa in males at 2, 6, 12, 18, and 24 months of age. Error bars, mean ± SD. Blue dots, biological replicates of males at 2 (n = 13), 6 (n = 3), 12 (n = 3), 18 (n = 5), and 24 (n = 8) months of age. P = 0.0002 and r = −0.58 (Pearson’s correlation). b Number of two pronuclear (2PN) eggs per fertilized eggs (%), at 8 h after IVF, by using spermatozoa in males at 2, 6, 12, 18, and 24 months of age. Error bars, mean ± SD. Blue dots, biological replicates of males at 2 (n = 13), 6 (n = 3), 12 (n = 3), 18 (n = 5), and 24 (n = 8) months of age. P = 0.02 and r = 0.35 (Pearson’s correlation). c Number of embryos at each stage per total 2PN eggs (%), at Day 1–4 after IVF, produced using spermatozoa from males at 2 and ≥24 months of age. Error bars, mean ± SD. Blue dots, biological replicates of males at 2 (n = 9) and ≥24 (n = 12) months of age. P = 0.04 and 0.006 (one-tailed t test). NS, not significant (P > 0.05; one-tailed t test). d Blastocysts, at Day 4 after IVF, produced using spermatozoa from males at 2 (Far left) and ≥24 (Center left, Center right, and Far right) months of age. Insets, number of embryos at blastocyst stage per total 2PN eggs (%). Red dots, blastocysts. Scale bars, 100 μm. e Blastocyst diameter without zona pellucida (μm), at Day 4 after IVF, using spermatozoa from males at 2 (n = 5) and ≥24 (n = 5) months of age. Error bars, mean ± SD. Each red dot represents a blastocyst (n = 91, at 2 months; n = 79, at ≥24 months). P = 0.001 (one-tailed t test). f Blastocysts at Day 4 after IVF, using spermatozoa from males at 2 (Left) and ≥24 (Right) months of age, immunostained for OCT4 and CDX2 (merged), with DAPI counterstain. BF, bright field, were merged with DAPI. Z-stack images (10–12 μm thickness) were converted into single projection images. Scale bars, 30 μm. g Blastocyst cell number, at Day 4 after IVF, using spermatozoa from males at 2 (n = 3) and ≥ 24 (n = 5) months of age. Each red dot represents a blastocyst (n = 20, at 2 months; n = 35, at ≥24 months). P = 0.0006 and 0.0002, and <0.00001 (one-tailed t test). Total cells, DAPI-positive cells. Inner cell mass (ICM) cells, OCT4-positive and CDX2-negative cells. Trophectoderm (TE) cells, CDX2-positive cells. h Number of ICM cells per total cells (%) in blastocysts at Day 4 after IVF, by using spermatozoa in males at 2 (n = 3) and ≥24 (n = 5) months of age. Each red dot represents a blastocyst (n = 20, at 2 months; n = 35, at ≥24 months). NS, not significant (P > 0.05; one-tailed t test). i Number of blastocysts per total 2PN eggs (%), at Day 4 after IVF (c), using spermatozoa from males at ≥24 months of age. Error bars, mean ± SD. Blue dots, biological replicates of males at ≥24 (n = 12) months of age, were divided into two groups: high (90–73%) and low (71–22%) IVF rates at Day 0. NS, not significant (P > 0.05; one-tailed t test).
a Immunostaining for γH2AX, with hematoxylin counterstain, on testis cross-sections of males at 2 (Left, a stage VII-VIII tubule) or 24 (Middle, a tubule in stages VII-VIII; Right, a tubule showing germ cell depletion) months of age. Lower panels are the grayscale versions of Upper panels. Dots, Sertoli cell nuclei (purple), type A spermatogonia (blue), preleptotene spermatocytes (red), pachytene spermatocytes (yellow), and round spermatids (green). Brown areas, spermatozoa. Scale bars, 20 μm. b Immunostaining for γH2AX, with hematoxylin counterstain, on cauda epididymis tubule longitudinal-sections of males at 2 (Far left, a tubule area; Center left, an interstitial space) or 24 (Center right, a tubule area; Far right, an interstitial space) months of age. Red bars, epithelial cells. Arrowheads, γH2AX-positive cells. Scale bar, 20 μm. c Comet assay images using spermatozoa from males at 2 (n = 3) and ≥24 (n = 6) months of age. White bars, comet tail lengths, represent the fragmented sperm DNAs. Scale bars, 10 μm. d Comet tail length (μm), using spermatozoa from males at 2 (n = 3) and ≥24 (n = 6) months of age. Error bars, mean ± SD. Each red dot represents a spermatozoon (n = 85, at 2 months; n = 250, at ≥24 months). P < 0.00001 (one-tailed t test). e A proposed model for ageing effects on in vivo male fertility and on germ cells and somatic cells in testes and epididymides.
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