The Odad3 Gene Is Necessary for Spermatozoa Development and Male Fertility in Mice

Abstract

Odad3 gene loss-of-function mutation leads to Primary Ciliary Dyskinesia (PCD), a disease caused by motile cilia dysfunction. Previously, we demonstrated that knockout of the Odad3 gene in mice replicates several features of PCD, such as hydrocephalus, defects in left-right body symmetry, and male infertility, with a complete absence of sperm in the reproductive tract. The majority of Odad3 knockout animals die before sexual maturation due to severe hydrocephalus and failure to thrive, which precludes fertility studies. Here, we performed the expression analysis of the Odad3 gene during gonad development and in adult testes. We showed that Odad3 starts its expression during the first wave of spermatogenesis, specifically at the meiotic stage, and that its expression is restricted to the germ cells in the adult testes, suggesting that Odad3 plays a role in spermatozoa formation. Subsequently, we conditionally deleted the Odad3 gene in adult males and demonstrated that even partial ablation of the Odad3 gene leads to asthenoteratozoospermia with multiple morphological abnormalities of sperm flagella (MMAF) in mice. The analysis of the seminiferous tubules in Odad3-deficient mice revealed defects in spermatogenesis with accumulation of seminiferous tubules at the spermiogenesis and spermiation phases. Furthermore, analysis of fertility in heterozygous Odad3^+/- knockout mice revealed a reduction in sperm count and motility as well as abnormal sperm morphology. Additionally, Odad3^+/- males exhibited a shorter fertile lifespan. Overall, these results suggest the important role of Odad3 and Odad3 gene dosage in male fertility. These findings may have an impact on the genetic and fertility counseling practice of PCD patients carrying Odad3 loss-of-function mutations.

Keywords: MMAF; Odad3 gene; Odad3 gene dosage; Primary Ciliary Dyskinesia; cilia; male infertility; mouse model of PCD.

Figure 1

Expression analysis of Odad3 during development and in adult testes. (A) Quantitative PCR analysis of the Odad3 transcript during development. (B) Histological analysis of Odad3 expression in adult murine testes. Cellular Odad3 expression was visualized by the analysis of β-galactosidase activity using X-gal staining in Odad3-LacZ and wild-type (WT) testes. Left panels—X-gal staining; middle panels—DAPI nuclear staining; right panels—DAPI/X-gal overlay. Upper row image scale bar: 25 μm; lower row scale bar: 10 μm. Arrows indicate round spermatids; arrowhead indicates elongated spermatids Insert in the upper right image is a seminiferous tubule of WT testes—DAPI/X-gal overlay (insert’s magnification 40×). No X-gal activity was detected in the WT testicular epithelium. (C) IF analysis of testicular section with y-H2AX antibodies: (a) y-H2AX staining (red); (b) cellular nuclei were counterstained with DAPI (blue); (c) y-H2AX/Nuclei/X-gal overlay, y-H2AX staining is red, DAPI nuclear staining is black for presentation purposes and X-gal is blue. The white arrowheads indicate X-gal and y-H2AX double-positive primary spermatocytes at the pachytene stage of meiosis I. The black arrowhead points to double-positive primary spermatocytes at the leptotene stage of meiosis I. The black arrow indicates elongated spermatid. Elongated spermatids are weakly positive for y-H2AX and X-gal staining. Scale bar: 12.5 μm. (D) IF staining of Sertoli cells with SOX9: (a’) Sox9 staining (red), nuclear DAPI staining (blue); (b’), X-gal staining (blue); (c’), SOX9/Nuclei/X-gal overlay: Sox9 immunostaining is red; DAPI-stained nuclei are in black and X-gal staining is blue. The double positive for Sox9 and X-gal Sertoli cells were not detected. The arrow indicates Sertoli Sox9 immunopositive and X-gal negative cell. Scale bar: 12.5 μm.

Figure 2

Analysis of spermatozoa from caudal epididymis of animals with the conditional Odad3 deletion. (A) Quantitative PCR analysis of the Odad3 transcript from homozygous Odad3^icKO and heterozygous Odad3^icKO/+ testes. All animals are also heterozygous for the ROSA26ERT2-Cre allele, for induction of the conditional deletion by tamoxifen. Error bars represent the mean ± SEM; N = 4 per genotype; unpaired two-tailed t-test * p = 0.028. (B) Analysis of sperm counts prepared from caudal epididymis of Odad3^icKO and Odad3^icKO/+ males. Error bars represent the mean ± SEM; unpaired two-tailed t-test analysis was performed; N = 7 animals per independent group/genotype were analyzed. (C) Motile sperm counts. Error bars represent the mean ± SEM; N = 7 per independent group; unpaired two-tailed t-test * p = 0.039. (D) H&E staining of spermatozoa spread. The representative images of different morphological groups of analyzed spermatozoa are presented. The magnification of the images is 40×. (E) Quantification of morphological analysis of the spermatozoa spreads isolated from cauda. At least 150 sperm per independent sample (animal) were counted. Error bars represent the mean ± SEM; N = 4 per genotype; unpaired two-tailed t-test: normal sperm—* p = 0.026; folded—* p = 0.012; headless—* p = 0.016; amorphous—* p = 0.044.

Figure 3

Histological analysis of seminiferous tubules in Odad3^icKO animals. (A) H&E staining of the testicular sections from Odad3^icKO and Odad3^icKO/+ demonstrated the clogged seminiferous tubules in Odad3^icKO males. Scale bar: 50 μm. Arrows indicate sloughed round spermatids. (B–D) Quantitative analysis of the morphology of the seminiferous tubules: tubule diameter (B), luminal diameter (C), and epithelial thickness (D). Error bars represent the mean ± SEM; N = 4 per genotype; unpaired two-tailed t-test **** p ≤ 0.0001.

Figure 4

Histological staging analysis of seminiferous tubule cross-sections. (A) Stage analysis of the DAPI-stained seminiferous tubule cross-sections from the wild-type animals. Examples of seminiferous tubules at different stages. Stages I–VIII contain two generations of spermatids: round spermatids (RS) and elongated spermatids (ES). In stages I–VI, the elongated spermatids do not line the lumen yet and appear in bundles. During stages VII–VIII, elongated spermatids lined the lumen. In seminiferous tubules staged as IX–XII, no RSs are present. Stage IX is characterized by the start of spermatid elongation and no visible ES; Stages X–XI are characterized by elongates spermatids that are not fully condensed and have a hooked tip. Stages XII—presence of cells undergoing meiotic divisions (M). Scale bar: 50 μm. (B) Quantitative analysis of seminiferous tubule staging. Stages IX–XII were counted together since we initially divided between tubules with two types of spermatids and stages where only one type of spermatids is detected. At least 100 seminiferous tubules from Odad3^icKO (N = 3) and Odad3^icKO/+ (N = 3) animals were counted. Error bars represent the mean ± SEM; unpaired two-tailed t-test, stages I–VI * p = 0.0187; stages VII–VIII ** p = 0.003; stages IX–XII * p = 0.014. (C) Representative image of DAPI-stained seminiferous tubule at VII–VIII stage in Odad3^icKO and Odad3^icKO/+ testes. Scale bar: 50 μm.

Figure 5

Ablation of the Odad3 does not affect the ciliogenesis of ED epithelium. (A) Schematic representation of the male reproductive system in mice, adopted from Hoque et al., 2021 [19]. The fragments of the reproductive tract dissected for mRNA preparation and q-PCR analysis are indicated by arrows. (B) q-PCR analysis of Odad3 expression in the different regions of the C57BL/6N male reproductive system: mean ± SEM average of the N = 3 independent analysis. ED—efferent ducts; CA—Caput; CD—Cauda; VS—vas deferens. (C) Odad3 q-PCR product separated by gel electrophoresis. One-third of the q-PCR reaction obtained from the testes and an entire q-PCR reaction from the other regions of the male reproductive system were loaded on the gel. (D) H&E-stained sections of the efferent ducts: Odad3^icKO (a,a’) and Odad3^icKO/+ (b,b’). The black boxes are magnified on the right side of the corresponding image. Scale bar: images a,b—500 μm; images a’,b’—10 μm. (E) IF analysis with anti-acetylated alpha tubulin antibodies of ED cross-sections: Odad3^icKO (c,c’) and Odad3^icKO/+ (d,d’). The anti-acetylated alpha tubulin staining is red and DAPI-stained nuclei are blue. Scale bar: c,d—50 μm; c’,d’—10 μm (F) Representative H&E-stained sections from different regions of the reproductive system of Odad3^icKO (N = 3) and Odad3^icKO/+ (N = 3) animals. Scale bar: 50 μm.

Figure 6

Analysis of fertility in heterozygous Odad3 knockout males. (A) Quantitative PCR analysis of Odad3 mRNA transcript from adult testes of Odad3^+/− and wild-type (WT) littermates. Mean ± SEM; unpaired two-tailed t-test ** p = 0.007. (B) Fertility testing of the Odad3^+/− animals. The reciprocal breeding of Odad3^+/− females (N = 3) and males (N = 3) with C57BL/6N (background strain) males or females was set up at 8–12 weeks of age. The age (days) at which the last litter laid was plotted. After the last litter was delivered, the couples were maintained together in the breeding cage for at least 3 months. Mean ± SEM; unpaired t-test with Welsh’s correction ** p = 0.003. (C) Sperm count analysis. Spermatozoa from cauda of 8-month-old Odad3^+/− (N = 7) and wild-type (N = 7) littermates were counted; mean ± SEM; unpaired two-tailed t-test *** p = 0.0002. (D) Analysis of the sperm motility. The percentage of the motile, progressively moving, and vibrating sperm prepared from cauda was analyzed; mean ± SEM; N = 7 per genotype/experimental group; unpaired two-tailed t-test * p = 0.027; ** p = 0.007. (E) Number of motile spermatozoa prepared from cauda; mean ± SEM; N = 7 per genotype/experimental group; unpaired two-tailed t-test; Total motile sperm count ** p = 0.005; Total progressive motile sperm count ** p = 0.004. (F) Morphological analysis of the spermatozoa spreads isolated from cauda. Error bars represent the mean ± SEM; N = 7 per genotype/experimental group; unpaired two-tailed t-test * p = 0.013. When analyzing Odad3^+/− sperm, we observed trends to increase in the percentage of headless and amorphous sperm. However, the trends are not statistically significant. The category of headless sperm did not reach statistical significance with a p value of 0.058.