TCP11 is an evolutionary-conserved protein expressed in testis

Tcp11 is evolutionary conserved amongst most metazoans and contains an uncharacterized protein domain, the TCP11 domain, that comprises most of the protein (Figure 1A). In mouse, Tcp11 encodes two verified isoforms: the longer isoform consists of 567 amino acids (UniProt: B2KF24), while the shorter isoform is 488 amino acids (UniProt: Q5FWA2) (Figure 1B). The shorter isoform lacks 79 amino acids near the N-terminus. There are also two predicted isoforms at 454 and 447 amino acids long (UniProt: A0A3B2W7H0 and A0A3B2WD68, respectively) (Figure 1B). Reverse transcription polymerase chain reaction (RT-PCR) analysis from various mouse tissues confirms Tcp11 as testis-specific, with expression in the testis commencing at post-natal day 15 when the leading edge of the first wave of spermatogenesis enters the pachytene stage (Figure 1C). In contrast to the mouse, RT-PCR detects human TCP11 in the brain and epididymis in addition to strong expression in the testis ( Supplementary Figure S1). Tcp11 has undergone gene duplication in metazoans, with three paralogs present in mouse. Tcp11x2 (Tcp11l3) is present on the X-chromosome while Tcp11l1 and Tcp11l2 are on chromosome 2 and 10, respectively ( Supplementary Figure S2A). Homology between the mouse TCP11 paralogs ranges from 32 to 55% identity ( Supplementary Figure S2B). All paralogs contain the TCP11 domain ( Supplementary Figure S2C). RT-PCR shows that Tcp11x2 is also testis-specific with Tcp11l1 and Tcp11l2 having broader expression ( Supplementary Figure S2D).

Tcp11 knockout males are subfertile

To examine the role of Tcp11, we obtained mice carrying a knockout-first, conditional ready allele of Tcp11 (Tcp11^tm1a(WTSI), referred to as Tcp11^fl) [24, 25] and maintained the line on a mixed genetic background (Figure 2A). To obtain the post-cre null allele (Tcp11^tm1b, referred to as Tcp11^-), we crossed Tcp11^fl/+ mice with transgenic mice expressing iCre under the Gdf9 promoter [26] to produce heterozygous offspring that were intercrossed to obtain full-body knockouts. The WTSI allele targets exons 5–8 for deletion. From the annotated mouse genome, there is a processed pseudogene between exon 4 and 5 (4930526A20Rik) within the Tcp11 locus that encodes a tRNA splicing endonuclease. PCR genotyping readily distinguishes between the wild-type and tm1b allele (Figure 2B). Tcp11^fl/+ or Tcp11^-/+ mice were intercrossed to generate homozygous males. To confirm that Tcp11^-/- is a true null allele, we generated an antibody to amino acids corresponding to 15–32 of mouse TCP11. Western blot analysis shows the longer and shorter TCP11 isoforms running at approximately 62 and 54 kD, respectively (Figure 2C and  Supplementary Figure S3A). Tcp11^-/- testis lysate did not reveal expression of TCP11, indicating that the Tcp11^-/- mice are true knockout (KO; null) mice. Also, TCP11 was not detected in RIPA protein extracts from the epididymis, indicating that TCP11 is not present in mature spermatozoa (Figure 2C and  Supplementary Figure S3A). We were able to obtain a previously published anti-TCP11 antibody raised against full-length mouse TCP11 [20, 21]; western blot analysis with this antibody detects the longer (62 kD) and shorter TCP11 (54 kD) as well as a doublet running above 50 kD and possibly a shorter fifth isoform confirming four of the predicted TCP11 isoforms ( Supplementary Figure S3C and D). A commercially available anti-TCP11 antibody was also tested in testis and epididymal lysates from wild type and Tcp11-nulls and showed several non-specific bands ( Supplementary Figure S3E). To test whether Tcp11 might function in male fertility, we paired individual homozygous males with wild-type females (mixed genetic background) for three months and recorded the number of pups delivered. Fertility tests showed that the Tcp11^-/- (n = 5) mice were subfertile siring fewer pups compared to control males (n = 5), demonstrating that TCP11 plays an important role in male fertility (Figure 2D).

Tcp11-null sperm have decreased motility

To determine the cause of the subfertility in Tcp11^-/- male mice, we first examined spermatogenesis. The gross morphology is comparable, and no significant differences in testis weight were observed between adult control (heterozygous) and KO males (Figure 3A and B). Also, PAS stained testis and epididymis sections did not show noticeable differences between the two genotypes, suggesting no obvious defect in spermatogenesis (Figure 3C–F and  Supplementary Figure S4B). Closer examination of spermatogenesis with immunofluorescence also did not reveal obvious differences between control and KO ( Supplementary Movies S1–S4). Quantification of step 15–16 spermatozoa in seminiferous tubules (epithelial cycle VI–VIII) slightly more spermatozoa produced by the KO compared to the control ( Supplementary Figure S4C). To examine sperm quality, sperm from the cauda epididymis was isolated into TYH medium and incubated at 37 °C with 5% CO₂ for 10 and 120 min. After incubation in TYH medium, we visualized sperm morphology and utilized Computer Assisted Sperm Analysis (CASA) to examine sperm quality. Phase contrast microscopy revealed aberrantly shaped sperm heads in sperm examined from the KO mice when compared with wild-type mice (Figure 4A and B). Quantification revealed a significant decrease of morphologically normal sperm in the KO (Figure 4C). CASA analysis not only revealed a decrease in the percent of motile sperm in the KO, but also all velocity parameters of sperm motility (average path velocity, straight line velocity, and curvilinear velocity) were decreased (Figure 4E and F and  Supplementary Movies 5 and 6). These data indicate that TCP11 plays a role in sperm motility.

Figure 1.

Tcp11 is evolutionary conserved and expressed in testis. (A) Multiple sequence alignment of human, mouse, and rat TCP11. Darker color represents greater conservation. (B) Two verified variants of mouse TCP11 are annotated, one 567 amino acids long and a shorter version 488 amino acids long. Two additional TCP11 variants are predicted that are shorter. Most of TCP11 is composed of the TCP11 domain (black), which is specific to TCP11 homologs and is uncharacterized. Grey highlights the region specific to variant 1. Red highlights the region used to generate an antibody. (C) RT-PCR from various mouse tissues detects expression of Tcp11 in testis beginning at post-natal day 15. Hprt (hypoxanthine-guanine phosphoribosyltransferase) was used as a control.

To pinpoint which step in fertilization Tcp11-null sperm encounter problems, we carried out IVF. Tcp11-null sperm isolated from the cauda epididymis fertilized few cumulus-intact and cumulus-free oocytes but efficiently fertilizes zona pellucida-free oocytes (Figure 5). Successful IVF with zona pellucida-free eggs demonstrates that TCP11 does not function in sperm-egg fusion and is not required for the acrosome reaction. To test whether IVF failure with cumulus-intact and cumulus-free oocytes is due to the inability of the KO sperm to penetrate the zona pellucida, we pre-treated oocytes with CARD medium. CARD medium contains reduced glutathione that has been shown to loosen and expand the zona pellucida [27]. After treating oocytes with CARD medium, we observed a partial rescue of fertilization, with an average fertilization rate of 37% (n = 3). The partial rescue suggests IVF failure with cumulus-intact and cumulus-free oocytes is due to a failure of sperm to penetrate the zona pellucida, which further implicates reduced motility as explaining the subfertility seen in the Tcp11 KO males. To examine whether TCP11 functions in sperm capacitation, tyrosine phosphorylation [28–31] was examined in sperm incubated in TYH medium after 10 and 120 min. Western blot analysis using an anti-tyrosine phosphorylation antibody detects several bands in sperm proteins from wild type and Tcp11-null sperm ( Supplementary Figure S5), suggesting that capacitation may have occurred in null sperm.

Figure 2.

Tcp11-null males are subfertile. (A) Schematic of mouse Tcp11, the targeted Tcp11^tm1a(WTSI) floxed allele (Tcp11^fl), and the post-cre null allele Tcp11^tm1b (Tcp11^-). (B) Genotyping PCR distinguishing wild-type, heterozygous Tcp11^+/-, and Tcp11 knockout (Tcp11^-/-) mice. (C) Western blot analysis with an antibody raised against a region near the N-terminus, detects both the longer and shorter variants of TCP11 in wild-type testis lysate but not in epididymal lysates. IZUMO1 (a component of the acrosome) confirms the presence of spermatozoa in both the testis and epididymal lysate. (D) Mating tests show Tcp11^-/- sire fewer pups over a three-month mating period than controls when paired with wild-type females (n = 5 males).

TCP11 is expressed in the late stages of spermiogenesis and not present in mature sperm

To determine the localization of TCP11, we used our anti-TCP11 antibody to perform immunofluorescence on testis sections from control or Tcp11 KO males. To visualize the acrosome or flagellum, we stained with anti-IZUMO1 or anti-acetylated-TUBULIN antibody, respectively. In wild-type testis-sections, the TCP11 antibody signal appears to localize to the cytoplasm in the late steps of spermatid development (step 15,  Supplementary Figure S6) and does not colocalize with anti-IZUMO1 or anti-acetylated-TUBULIN signal (Figure 6A and C and  Supplementary Movies 7–9). The staining pattern indicates that TCP11 is a cytoplasmic protein, which is consistent with SignalP and transmembrane predictions of TCP11 as a non-secreted, non-transmembrane containing protein. In KO sections, there was little signal or what appears to be background staining from the anti-TCP11 antibody (Figure 6B and D). Our staining results are consistent with the previously reported localization of TCP11 [20]. To determine whether TCP11 may localize in mature sperm, we fractionated sperm extracted from the cauda epididymis. Sperm proteins were fractionated into a Triton X-100-, SDS-, and insoluble fraction corresponding to the membrane bound/cytoplasmic soluble, axonemal proteins, and fibrous sheath, respectively [32, 33]. Western blot analysis of fractionated sperm proteins does not detect the presence of TCP11 with our anti-TCP11 antibody (Figure 7) or with the previously published antibody ( Supplementary Figure S7) [20]. Western blot analysis and immunofluorescence indicates that TCP11 function is restricted to the cytoplasm in late stage spermatids and appears to be absent in mature sperm.

Tcp11-null sperm have decreased PKA activity

Previous reports implicated TCP11 in cAMP signaling by forming a complex with PKA in sperm [23, 34]. While we could not detect TCP11 in sperm, we wanted to determine whether PKA signaling was affected in the absence of TCP11. Sperm from the cauda epididymis from three wild-type and Tcp11 KO males were isolated into TYH media and incubated for 10 min at 37 °C with 5% CO₂. Sperm proteins were then isolated after the incubation and subjected to Western blot analysis using an antibody that recognizes the phosphorylated target motif of PKA (R-R-X-S/T-X). Western blot analysis revealed decreased signal from the Tcp11 KO samples when compared to the wild-type controls (Figure 8). It should be noted, while there is variability between the individual males with one male displaying higher signal in the Western blots, the overall trend is reduced signal compared to wild-type males. This suggests that phosphorylation of PKA targets is reduced, which further suggests that PKA signaling is reduced in the Tcp11 KO. These results are consistent with previous reports implicating TCP11 in cAMP/PKA signaling [23, 34].

Animals

Tcp11 ^{tm1a(KOMP)Wtsi/+} (referred to as Tcp11^fl/+) mice were obtained from the KOMP consortium. The null allele was generated using JM8A3.N1 ES cells, a pD223-DTA plasmid backbone, and homology arms 5.8kB upstream and 3.7kB downstream of Tcp11 (MGI: 98544). Tcp11^fl/+ were crossed with Gdf9-iCre to generate Tcp11^-/+ mice. Tcp11 mutant mice were maintained on a mixed genetic background (C57B6/129SvEv or B6D2F1), while wild-type mice were purchased from CLEA Japan (Tokyo, Japan) or Japan SLC (Shizuoka, Japan) or were produced from intercrosses of C57BL6 and 129S6/SvEv mice generated in the Matzuk laboratory at Baylor College of Medicine. All mice were housed in specific pathogen free animal facilities with a light:dark cycle of 12:12. All animals in this study were approved by the Institutional Animal Care and Use Committees of Baylor College of Medicine (Houston, TX) and the Research Institute for Microbial Diseases of Osaka University (Osaka, Japan).

Reverse transcription polymerase chain reaction

Mouse cDNA was prepared from multiple adult tissues of B6D2F1 and C57CL6/129SvEv hybrid mice and testes of 5 to 60-day-old mice. Human cDNAs were obtained from the Human Tissue Acquisition and Pathology core service (Baylor college of Medicine, USA). The primers and amplification conditions for each gene are summarized in  Supplementary Table S1.

Sequence comparisons

Sequence comparisons between Tcp11 homologs were done with BLAST or with Clustal Omega.

Mating tests

Five heterozygous (Tcp11^+/-) and Tcp11^-/- sexually mature males were paired with 6-week-old wild-type females for three months. The number of pups delivered was counted the day after birth.

Western blot analysis

Testis and epididymides were dissected from wild-type and Tcp11^-/- adults and placed into PBS. Testis samples were lysed in 1 mL RIPA (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT, 1% Nonident P-40, 0.5% deoxycholate, 0.1% SDS, protease inhibitors) using Qiagen's TissueLyser II (30 Hz, 2 min, RT). Epididymides were first minced with dissecting scissors in 1 mL RIPA (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM DTT, 1% Nonident P-40, 0.5% deoxycholate, 0.1% SDS, protease inhibitors) and then lysed with Qiagen's TissueLyser II (30 Hz, 2 min, RT). Lysates were clarified with a 10 min spin at 18 000×g at 4 °C. The protein concentration was determined with the Bradford method. Thirty microgram per sample were loaded per well. Western blot analysis was performed using BioRad's TransBlot Turbo. PVDF membranes were blocked with TBS with 0.05% Tween and 5% milk and washed with TBS with 0.05% Tween and 0.5% milk. Both primary and secondary antibodies were diluted in washing buffer. Nacalai's Super Signal was used for chemiluminescence and detected with Image Quant. Antibodies used include the following: goat anti-BASAGIN 1:1000 (Santa Cruz), mouse anti-AKAP4 1:1000 (BD Biosciences), and mouse anti-Acetylated Tubulin 11 000 (Sigma).

Figure 6.

TCP11 localizes to the cytosol in late spermiogenesis. (A and B) Anti-TCP11 antibody (green) displays cytoplasmic localization in testis cross sections from wild-type mice. In Tcp11 KO testis cross sections, anti-TCP11 antibody staining (green) is punctate and present in all cell types. Anti-acetylated-TUBULIN (magenta) localizes to the flagellum of spermatids in both wild-type and KO cross sections. White = Hoechst 33342. Scale bars = 50 µm. (C and D) Anti-TCP11 antibody (green) displays cytoplasmic localization in testis cross sections from wild-type mice. In Tcp11 KO testis cross sections, anti-TCP11 antibody staining (green) is diffuse in the testis and brightly stains the interstitial cells. Anti-IZUMO1 (magenta) localizes to the developing acrosome in round and elongated spermatids in both wild-type and KO cross sections. White = Hoechst 33342. Scale bars = 50 µm.

Figure 7.

TCP11 appears to not be present in mature sperm. Sperm isolated from the cauda epididymis were used for protein extraction into a Triton X-100- (membrane bound and cytoplasmic soluble), SDS- (axonemal components), and SDS-insoluble (fibrous sheath and outer dense fibers) pool. Western blot analysis against BASIGIN, acetylate-Tubulin, and AKAP4 demonstrates successful extraction of these three pools, respectively. Anti-TCP11 antibody only detects bands in the wild-type testis lysate.

Isolating sperm proteins for tyrosine phosphorylation and PKA target phosphorylation analysis and sperm fractionation

For analyzing tyrosine phosphorylation, sperm were incubated in TYH medium for 10 min or 2 h. For PKA target phosphorylation, sperm were incubated in TYH medium for 10 min. Sperm were then collected in 1 mL PBS and centrifugated at 2000×g for 2 min at room temperature. The collected sperm were lysed with sample buffer (66 mM Tris-HCl, 2%, SDS, 10% glycerol and 0.005% Bromophenol Blue), boiled for 5 min, and centrifugated at 15 000×g for 15 min at 4 °C. For Western blot analysis, 5 µL per sample were loaded per well. Blotting was done using BioRad's TransBlot Turbo. PVDF membranes were blocked with TBS with 0.05% Tween and 5% BSA and washed with TBS with 0.05% Tween and 0.5% BSA. Millipore's 4G10 monoclonal antibody against phosphorylated tyrosine and Cell Signaling's 100G7E antibody against PKA substrate were used at 1:1000 for Western blot analysis.

For sperm fractionation, sperm were extracted from the cauda epididymis from wild-type males (B6D2F1) into TYH media. Sperm were centrifuged at 2000×g for 2 min at room temperature. The sperm pellet was then resuspended in 100 µL of Triton X-100 extraction buffer (20 mM Tris pH 7.5, 50 mM NaCl, 1% Triton X-100, and Protease Inhibitors) and incubated for 2 h at 4 °C with gentle mixing. The sample was centrifuged for 10 min, 4 °C, 15 000×g. The pellet was resuspended in 100 µL SDS extraction buffer (75 mM NaCl, 24 mM EDTA, 1% SDS) and incubated at room temperature for 1 h with gentle mixing. The sample was then centrifuged for 10 min, 4 °C, 15 000×g. The pellet was resuspended in 100 µL 1X SDS PAGE sample buffer. For SDS PAGE, 1 µL per sample was loaded per well.

Figure 8.

Tcp11 KO sperm have decreased phosphorylation of PKA substrates. Western blot analysis using anti-PKA substrate phosphorylation antibody shows decreased signal in sperm proteins isolated from three Tcp11 KOs. Sperm were incubated for 10 min in TYH medium prior to protein extraction.

TCP11 antibody production

Sigma's antibody production service was utilized to produce our TCP11 antibody. The peptide sequence AAESASRESRGGNTRESA corresponding to amino acids 15–32 of mouse TCP11 was used to immunize one rabbit. After 54 days, the blood serum was extracted. Anti-TCP11 antibodies were purified using an affinity resin (Sulfo-Link) generated with the immunizing peptide. The previously published anti-TCP11 antibody was a kind gift from [20, 21]. Both antibodies were used at 1:1000 dilution for Western blot analysis. The Novus anti-TCP11 antibody (NBP1-57698) was used at 1:500 dilution for Western blot analysis [23].

Histology

For PAS staining, testis and epididymides were fixed in Bouin's fixative, processed, and embedded in paraffin. Sections were cut at 5 µm thickness. Sections were then deparaffinized, rehydrated, stained with Periodic Acid-Schiff's Reagent, counterstained with hematoxylin, dehydrated, and mounted with VectaMount or Permount.

In vitro fertilization

Seven-week-old B6D2F1 females were injected with 0.1 cc Hyper-Ova. Forty-eight hours later, the females were injected with 0.2 cc hCG. The next day oviducts were collected, and cumulus-oocyte-complexes were isolated into TYH medium or CARD medium. CARD medium was prepared according to manufacturer's instructions. The glutathione concentration recommended for IVF using frozen-thawed sperm was used for the CARD medium. Some eggs in TYH medium were treated with hyaluronidase to generate cumulus-free oocytes, while another batch was treated with collagenase to generate zona pellucida-free oocytes. Sperm from wild-type and Tcp11^-/- males was isolated and incubated in TYH medium at 37 °C with 5% CO₂ for 2 h. After the sperm incubation, 2×10⁵ sperm/mL was used for fertilization. Four hours after incubation, zygotes in CARD medium were washed with KSOM medium [45] and incubated until the next day. Six hours after incubation, zygotes with two or more pronuclei were counted from the zona-free condition. The next day, 2-cell embryos which developed in the cumulus intact, cumulus free, and CARD medium conditions were counted.

Sperm analysis

Sperm motility was analyzed as previously described [46]. Sperm from wild-type and Tcp11^-/- mice was extracted into TYH media and incubated at 37 °C with 5% CO₂. CASA (Hamilton Thorne) with CEROS I software was used to measure sperm motility after 10 min and 120 min incubation.

Immunofluorescence

For testis cryosections, immunostaining was performed as previously described [47]. Primary antibodies used include: mouse antiAcetTubulin 1:1000 (Sigma), rat anti-IZUMO1 1:200 [48], and rabbit anti-TCP11 1:500. Secondary antibodies used include: Alexa Fluor 488 anti-rabbit, Alexa Fluor 5465 anti-mouse, and Alexa Fluor 546 anti-rat used at 1:1000.

Statistical analysis

Statistical analysis was done when three or more males were used for experiments. Fisher's exact test or Student's t-test were used to examine statistical significance. P-values between 0.05 and 0.001 were considered significant (^*), while P-values below 0.001 were considered highly significant (^**).