Deficiency of TPPP2, a factor linked to oligoasthenozoospermia, causes subfertility in male mice

Abstract

Oligoasthenozoospermia is a major cause of male infertility; however, its etiology and pathogenesis are unclear and may be associated with specific gene abnormalities. This study focused on Tppp2 (tubulin polymerization promoting protein family member 2), whose encoded protein localizes in elongating spermatids at stages IV-VIII of the seminiferous epithelial cycle in testis and in mature sperm in the epididymis. In human and mouse sperm, in vitro inhibition of TPPP2 caused significantly decreased motility and ATP content. Studies on Tppp2 knockout (KO) mice demonstrated that deletion of TPPP2 resulted in male subfertility with a significantly decreased sperm count and motility. In Tppp2^-/- mice, increased irregular mitochondria lacking lamellar cristae, abnormal expression of electron transfer chain molecules, lower ATP levels, decreased mitochondrial membrane potential and increased apoptotic index were observed in sperm, which could be the potential causes for its oligoasthenozoospermia phenotype. Moreover, we identified a potential TPPP2-interactive protein, eEf1b (eukaryotic translation elongation factor 1 beta), which plays an important role in protein translation extension. Thus, TPPP2 is probably a potential pathogenic factor in oligoasthenozoospermia. Deficiency of TPPP2 might affect the translation of specific proteins, altering the structure and function of sperm mitochondria, and resulting in decreased sperm count, motility and fertility.

Keywords: TPPP2; energy metabolism; male subfertility; oligoasthenozoospermia; sperm function.

Figure 1

Distribution of TPPP2 in mouse tissues. (A) The presence of Tppp2 mRNA was evaluated in various tissues from adult mice using reverse transcription polymerase chain reaction (RT‐PCR). Tppp2 mRNA was detected in the testis and epidydimis. β‐Actin was amplified as an internal control. (B) The testicular Tppp2 mRNA expression profile was tested at the indicated time points after birth using RT‐PCR. β‐Actin was amplified as an internal control. Tppp2 mRNA expression was markedly increased at the third week. (C) Immunohistochemical localization of the TPPP2 protein in adult mouse testis. Each image shows a stage of the seminiferous epithelial cycle denoted by Roman numerals at the bottom of each image. (D) Immunohistochemical localization of the TPPP2 protein in adult mouse epididymis. (E) Mature sperm were subjected to immunofluorescent microscopy using an anti‐TPPP2 antibody (green) and Hoechst staining (blue), as described in the Materials and Methods. TPPP2 was expressed in the middle piece of the mature sperm. Scale bars, 20 µm

Figure 2

Changes in human sperm treated with anti‐TPPP2 antibodies. Sperm concentration = 30 × 10⁶ sperm/mL. (A‐B) 2, 8 and 20 µg/mL of TPPP2 antibody were respectively co‐incubated with sperm for 30 min and the corresponding concentrations of IgG were co‐incubated with sperm as controls. Motility was evaluated by counting 200 spermatozoa in randomly selected fields. Data are presented as the mean ± SD (n = 10). ***P < 0.001. (C) ATP contents were further measured in sperm between the IgG and 20 µg/mL TPPP2 groups (n = 10). ***P < 0.001. (D) Assessment of capacitation and acrosome reaction. Capacitated sperm underwent A23187‐induced acrosome reaction. Data are presented as the mean ± SD (n = 3). F: uncapacitated sperm; B: capacitated sperm; AR: acrosome reacted sperm

Figure 3

Changes in mouse sperm treated with anti‐TPPP2 antibodies. (A) 2, 8 and 20 µg/mL of TPPP2 antibody respectively, were co‐incubated with sperm for 30 min and the corresponding concentrations of IgG were co‐incubated with sperm as controls. Sperm samples were analysed using a Computer Assisted Sperm Analyzer (CASA) (n = 3), *P < 0.05. (B) ATP contents were further measured in sperm between the IgG and 20 µg/mL TPPP2 groups (n = 3), *P < 0.05. (C) When cumulus‐intact wild‐type (WT) eggs were inseminated with sperm from the IgG and 20 µg/mL TPPP2 group, the two‐cell cleavage rate at 24 h in 20 µg/mL TPPP2 group was significantly lower than that in the IgG group (n = 3), *P < 0.05

Figure 4

Generation of Tppp2 gene knockout (KO) mice. (A) Schematic strategies for the generation of KO mice using CRISPR/Cas9 technology. 62 bp of Tppp2 were deleted from Exon 1 (n = 3). (B) Genotype of each KO mouse was confirmed using PCR (n = 3). (C) TPPP2 levels in the testes of KO and wild‐type (WT) mice were evaluated using western blotting. β‐actin was used as a loading control (n = 4). (D‐F) TPPP2 levels in the testes and sperm of KO and WT mice were evaluated using immunohistochemical and immunofluorescent analysis. Scale bars, 20 µm (n = 3)

Figure 5

Fertility assay and sperm parameters. (A‐B) Each male was bred with two females. WT, wild‐type. Knockout (KO), Tppp2 ^−/− male mice. Tests were performed for average pups/litter between the WT and KO groups. Data are presented as mean ± SD (for the WT group, n = 9; for the KO group, n = 10). *P < 0.05, **P < 0.01, ***P < 0.001. (C‐D) Percentage of motile and progressively motile sperm from WT and KO males (n = 6). (E) Sperm counts per cauda epididymis of WT and KO males (n = 6)

Figure 6

Spermatogenesis in wild‐type (WT) and knockout (KO) mice. (A) Morphology and size of WT and KO mice testes. (B) Testis/body weight ratio for WT and KO mice (n = 6). (C) Periodic Acid‐Schiff's‐stained sections of testes from WT and KO male mice. Each image exhibits a stage of the seminiferous epithelial cycle denoted by Roman numerals at the bottom of the image. All showed normal spermatogenesis. (D) The number of spermatocytes and round spermatids from the testes of WT and KO males were counted as shown (n = 5). Scale bars, 20 µm. (E) Terminal deoxynulceotidyl transferase nick‐end‐labeling (TUNEL) staining in testes (n = 5). Scale bars, 20 µm

Figure 7

Assessment of ATP synthesis in sperm of wild‐type (WT) and knockout (KO) mice. (A) Transmission electron microscopy (TEM) images of sperm ultrastructures are shown. Scale bars, 0.5 µm. (B) Percentage of abnormal mitochondria in WT and KO male mouse sperm (n = 3). *P < 0.05. (C) The sperm samples from WT and KO mice were treated with the lipophilic cationic dye JC‐1 and were checked for their corresponding mitochondrial membrane potential (MMP) through flow cytometry analysis. P2, red‐stain sperm; P3, green‐stain sperm. Neg and carbonyl cyanide 3‐chlorophenylhydrazone were respectively used as a negative control and positive control respectively. (D) Fluorescence microscope observation of sperm after JC‐1 staining to determine the difference in MMP between WT and KO group. The red fluorescence sperm were normal and the green sperm had a low MMP. Scale bars, 50 µm. (E) Detection of mRNA expression levels of key markers in the mitochondrial electron transfer chain between WT and KO male mice sperm (n = 5). *P < 0.05, **P < 0.01. (F) Measured levels of ATP between WT and KO male mice sperm (n = 5)

Figure 8

Assessment of apoptosis in sperm of wild‐type (WT) and knockout (KO) mice. (A) Detection of apoptosis in sperm samples from WT and KO mice using western blotting (n = 4). (B) Gray intensity analysis showing the expression level of pro‐ and anti‐apoptotic markers. *P < 0.05.

Figure 9

The discovery of TPPP2 interaction protein. (A) Localization of green fluorescent protein (GFP) and TPPP2‐GFP in HEK293T cells. Cells were stained with rhodamine phalloidin and observed using fluorescence microscopy 48 h after transfection. (B) The level of TPPP2 in HEK293T cells was verified with western blotting. (C) Immunoprecipitation of TPPP2‐GFP coimmunoprecipitated eEF1b, as assessed using mass spectrometry. (D) Immunoprecipitation of TPPP2 coimmunoprecipitated eEF1b in testes