Systematic identification of Y-chromosome gene functions in mouse spermatogenesis

Abstract

The mammalian Y chromosome is essential for male fertility, but which Y genes regulate spermatogenesis is unresolved. We addressed this by generating 13 Y-deletant mouse models. In Eif2s3y, Uty, and Zfy2 deletants, spermatogenesis was impaired. We found that Uty regulates spermatogonial proliferation, revealed a role for Zfy2 in promoting meiotic sex chromosome pairing, and uncovered unexpected effects of Y genes on the somatic testis transcriptome. In the remaining single Y-gene deletants, spermatogenesis appeared unperturbed, but testis transcription was still altered. Multigene deletions, including a human-infertility AZFa model, exhibited phenotypes absent in single Y deletants. Thus, Y genes may regulate spermatogenesis even if they show no phenotypes when deleted individually. This study advances our knowledge of Y evolution and infertility and provides a resource to dissect Y-gene functions in other tissues.

Fig. 1. Generation and screening of thirteen Y-deletant mouse models for abnormal gamete production.

(A) Structure and gene content of the mouse Y chromosome. (B) Experimental strategy to generate and study Y-gene deletant mouse models. (C) Mean numbers of male and female pups born per litter from control and Y-deletant matings. n = total number of pups born. Error bars = 95% confidence interval. (D) Mean testis weight in controls and Y-deletants. n = number of males. Error bars = standard deviation. (E) Median seminiferous tubule area in control and Y-deletants. n = number of biological replicate males. At least 40 tubules were counted per replicate. Error bars = standard deviation. (F) Mean sperm count in controls and Y-deletants. Sperm was collected from the cauda epididymis. n = number of males. Error bars = standard deviation. (G) PAS-stained testis sections of control, Eif2s3y-KO, Uty-KO, AZFa-KO Zfy2-KO, and Zfy1&2-DKO. Tubules are circled by dotted lines. Asterisks indicate tubules with severe germ cell depletion. Black inset: control metaphase, red insets: dark cytoplasmic signal is visible, indicative of apoptosis. Scale bars = 20 μm. Statistical analysis by two-sided Chi-Square test for C, Mann Whitney test for D and F and two tailed t-test for E. Significant p values (<0.05) are shown.

Fig. 2. Screening for reduced gamete quality in thirteen Y-deletant mouse models.

(A) Median sperm head area for control and Y-deletants. n = number of males. Error bars = standard deviation. On average, 260 sperm were analysed per male. (B) Mean percentage of motile sperm in the cauda epididymis of controls and Y-deletants. n = number of males. Error bars = standard error of the mean. (C) Mean sperm curvilinear velocity (VCL) in controls and Y-deletant. n = number of males. Error bars = standard error of the mean. (D) Mean fertilization rate of sperm from control and Y-deletant males in IVF assays. n = number of males. Error bars = standard deviation. Statistical analysis by Mann Whitney test, with significant p values (<0.05) shown. (E) DAPI-stained sperm heads from Zfy2-KO, Zfy1&2-DKO, and AZFa-KO showing a range of abnormalities, from least (top) to most severe (bottom), compared to a control sperm head with stereotypical shape. Scale bars = 2.5 μm. The average sperm profile of the three deletants is compared to that of the control. n = number of sperm heads used to build the average outline. (F) Sperm from control, Zfy2-KO, and AZFa-KO tracked over one second using computer assisted sperm analysis (CASA). Red trajectory represents a rapid progressive sperm and blue trajectories slow, non-progressive sperm. Scale bars = 50 μm.

Fig. 3. Defects in the establishment and differentiation of the spermatogonial pool in Uty-deleted mice.

(A) Number of undifferentiated spermatogonia per tubule in control and Uty-KO P1, P10, and adults, quantified in testis immunostaining for LIN28A and cKIT. n = number of tubules counted across three biological replicates. p values calculated by Mann Whitney test. Scale bars = 2.5 μm. (B) P10 testis sections immunostained for LIN28A. Dashed lines encircle tubules with no germ cells. Scale bars = 100 μm. (C) Adult PAS-stained testis sections, with Uty-KO exhibiting different missing generations compared to control. Epithelial stages are shown in roman numerals. Spermatocytes, round and elongated spermatids are expected at all these stages. Scale bars = 20 μm. (D) Percentage of tubules exhibiting stage-specific missing generations. n = number of tubules counted across four biological replicates. (E) Quantification of percentage of tubules with differentiating In-B spermatogonia using P1, P10, and adult testis sections immunostained with LIN28A and cKIT. Scale bars = 2.5 μm. Tubules lacking progenitor cells (with no LIN28A+ cells) were excluded. n = number of tubules counted across three biological replicates. All error bars = standard deviation. Statistical analysis by Mann Whitney test for A and D and two tailed t-test for E. Significant p values (<0.05) are shown.

Fig. 4. Defects in meiotic sex chromosome pairing in the absence of Zfy2.

(A) cPARP immunostaining in testis sections to quantify metaphase apoptosis in control and Zfy deletants. Arrows indicate apoptotic metaphase cells. Arrowheads point to misaligned chromosomes. Insets show example metaphase cells. Scale bars = 10 μm. n = number of metaphases counted across three-four biological replicates. Error bars = standard deviation. (B) Percentage of metaphase cells with aligned and misaligned chromosomes in control and Zfy deletants. n = number of metaphases counted across three biological replicates. Error bars = standard error of the mean. (C) Representative aligned and misaligned metaphase plates in testis sections painted with X (green) and Y (magenta) whole chromosome paints. Arrowheads point to misaligned chromosomes. Scale bars = 2 μm. (D) Pachytene spermatocytes immunostained for SYCP3 (green), CREST (magenta), and γH2AX (grey) to quantify X-Y asynapsis in control and Zfy deletants. Insets show sex chromosomes, with synapsed and asynapsed X-Y in control and Zfy2-KO, respectively. Scale bars = 20 μm. n = number of pachytene cells counted across three biological replicates. Error bars = standard error of the mean. Statistical analysis by unpaired two-tailed t test, with significant p values (<0.05) shown.

Fig. 5. Transcriptional deregulation in Y-deletant testes.

(A) Quantification of differentially expressed (DE) genes in bulk RNAseq of Y-KO testes compared to controls. A threshold of Log2 fold change ≥0.5 was used. Asterisks indicate Y-genes not annotated in the mouse genome assembly which thus won’t appear as downregulated. (B-C), Uniform Manifold Approximation and Projection (UMAP) visualization of testis single nuclei RNA sequencing (snRNA-seq) datasets. (B) shows integrated P10 UMAP from control, Eif2s3y, and Uty KO samples (see Fig. S8E for individual UMAPs) and (C) displays integrated adult samples from control, Zfy1, Zfy2, Zfy1&2, and AZFa KOs (see Fig. S8F for individual UMAPs). Undifferentiated and differentiating (Undif and Dif) spermatogonia (SG); leptotene (Lep); zygotene (Zyg); pachytene (Pach); diplotene (Dip); metaphase (M) and secondary (2ry) spermatocytes (SC); round and elongating spermatids (rSD and eSD); foetal Leydig; adult Leydig; Sertoli; peritubular (PT); endothelial (EC) and macrophage (MP) cells are shown. Asterisks show unknown clusters with no clear signatures. (D) Quantification of DE genes for each cell type in P10 Eif2s3y and Uty KOs and adult Zfy1, Zfy2, Zfy1&2, and AZFa KOs. Cell types with less than 10 nuclei in the mutants are marked as na. (E) Expression of the differentiation markers Dmrt1, Uchl1 and the progenitor marker Gfra1 in Eif2s3y-KO and control Undif SG1. (F) Expression of the differentiation markers Dmrt1 and Dazl in Uty-KO, AZFa-KO, and control Dif SG. (G) Expression of members of the steroid synthesis pathway in Eif2s3y-KO, Uty-KO, and control Leydig cells. (H) Expression of the recombinases Dmc1 and Mnd1 in Zfy1, Zfy2, Zfy1&2 KOs and control zygotene cells. Statistical analysis by Kolmogorov Smirnov test, with significant p values (<0.05) shown. For box plots, center line is the median; box limits, 25th and 75th percentile; whiskers, minimum to maximum; points, outliers.