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,,1
Departments of
* Pathology,
Molecular and Human Genetics, and
Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA
1Correspondence: Department of Pathology, One Baylor Plaza, Baylor College of Medicine, Houston, Texas 77030, USA. E-mail: mmatzuk{at}bcm.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10-fold higher consumption of intracellular ATP in Tekt4-null sperm as compared to wild-type, and null spermatozoa rapidly lose progressive motility when incubated for 
1.5 h. Our studies demonstrate that TEKT4 is necessary for the proper coordinated beating of the sperm flagellum and male reproductive physiology.—Roy, A., Lin, Y.-N., Agno, J. E., DeMayo, F. J., Matzuk, M. M. Absence of tektin 4 causes asthenozoospermia and subfertility in male mice.
Key Words: coiled-coil • axoneme • outer dense fiber • knockout mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Despite similarities, certain accessory structures distinguish mammalian sperm flagella from cilia as well as flagella of nonmammalian species. These unique peri-axonemal structures, the outer dense fibers (ODF), and the fibrous sheath (FS), extend along the length of the flagellum to form an outer cytoskeleton around the axoneme. In the midpiece, nine ODFs (numbered 1–9) surround the nine outer doublets, and each ODF is attached to the corresponding microtubule doublet to form a 9+9+2 superstructure (1)
. The ODFs extend distally into the principal piece where they are encircled by the FS, which is composed of two longitudinal columns (that replace ODFs 3 and 8) interconnected by transverse ribs (2)
.
The physiological role(s) of ODFs are unknown, but two putative functions have been ascribed to them: in the "Geometric Clutch" model posited by Lindemann (3)
, ODFs act as force multipliers that amplify the bending torque generated by interdoublet sliding, thereby decreasing the cost of work and improving energy utilization in flagella. Baltz et al. (4)
have proposed that ODFs function as structural reinforcements that stiffen and enhance the tensile strength of flagella to thereby overcome shear forces in the female reproductive tract. Although the primary role of the FS was long believed to impose constraints on the plane of flagellar bending, recent studies indicate that the FS serves a scaffolding function for localizing glycolytic enzymes and signaling molecules along the flagellum (2)
. Significantly, loss of the major FS protein AKAP4 (protein kinase A anchor protein 4) in mice leads to disruption of flagellar motion (5)
.
Tektins are coiled-coil filamentous proteins that were originally purified from sperm flagella of the sea urchin Strongylocentrotus purpuratus as sarkosyl-insoluble components stably associated with 3–4 tubulin protofilaments in the wall of the A and B outer doublet microtubules (6
, 7)
. Although tektin paralogs from the same species show little similarity between each other, individual tektin family members, characterized by a nonapeptide signature sequence [RPNV/I/MELCRD] at the carboxyl terminus, have highly conserved orthologs in multiple species. In sea urchin sperm flagella, tektins were found to exist as longitudinal polymers with axial periodicities matching the tubulin lattice (8)
, which led to the "molecular ruler" hypothesis that these proteins serve as "signposts" for the periodic attachment of axonemal appendages along the length of the flagellum. In support of this notion, spermatozoa from mice null for the flagellar protein tektin 2 (Tekt2) demonstrate absence of inner dynein arms in the axoneme, leading to diminished flagellar motility and infertility in these knockout mice (9)
.
In contrast to sea urchins, where biochemical purification of flagellar axonemes could identify only three tektins named tektin A, B, and C, our group and others have identified at least five members of this family in mammals (10
11
12
13
14)
. Moreover, recent proteomic studies on the peri-axonemal structures of rodent spermatozoa have detected the presence of all five tektins in the accessory structures (14)
, suggesting that the family might have expanded in mammals to perform additional functions as components of the ODF and/or FS. In the present study, we report the expression and functional characterization of tektin 4 in spermatozoa, flagellar motility, and male reproductive physiology in mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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as a probe for basic local alignment search tool (BLAST) analysis across multiple species, multiple tektins including Tekt4 (UniGene cluster Mm.282330) were identified. Using reference sequences from expressed sequence tags (ESTs) deposited in Mm.282330, we used reverse-transcription polymerase chain reaction (RT-PCR) to clone the full-length mouse Tekt4 cDNA from total testis RNA of an adult (8 wk old) wild-type (WT) 129S5B6F1 hybrid mouse. Primers for amplifying Tekt4 cDNA were as follows: 5'-GTTCCTTAGGCAACCAGGAA-3' (forward) and 5'-AGCTCTTTATTTGAATGAAGCAGTGA-3' (reverse). The cDNA was cloned into pGEM T-vector (Promega, Madison, WI, USA) and sequenced. The full-length sequence was matched against a genomic contig NC_000083, and database mining was used to identify orthologous genes in humans, rats and pufferfish.
A 303-bp Tekt4 exon 1 fragment was used to probe a 129S6/SvEv mouse genomic lambda Fix II phage library (Stratagene, La Jolla, CA, USA) to clone the gene. Briefly, the library was plated onto NZCY plates at a density of 500,000 plaques and transferred onto HyBond-N filters (Amersham Biosciences, Piscataway, NJ, USA) that were hybridized with [32P]dCTP random-primed probe in Church buffer at 65°C. Filters were washed and exposed overnight at –80°C. Two overlapping lambda FIX II recombinant clones (T4–2 and T4–3) extending from 10 kb upstream of Tekt4 to intron 5 of the gene were obtained after a secondary screen and were subcloned into the NotI site of pBluescript SK (+) vector (Stratagene) for ease of manipulation.
Multiple tissue RT-PCR and in situ hybridization
Total RNA was extracted from multiple tissues of an adult (8 wk old) WT 129S5B6F1 hybrid mouse by using TriZOL reagent (Invitrogen, Carlsbad, CA, USA). Two micrograms of total RNA were DNaseI-treated, and first-strand synthesis was performed with Superscript III (Invitrogen) as per manufacturer’s instructions. For the developmental PCR (Fig. 1
C), total RNA was extracted from testes of 129S5B6F1 hybrid mice at the ages indicated. A 390-bp cDNA fragment encompassing exons 2–4 of mouse Tekt4 was amplified by the following primers: 5'-CCTGAAGAGGACGATAGGACA-3' (forward) and 5'-TGCAGCTTCTGGAGTGAGTC-3' (reverse) from 2 µl of first-strand reaction. For RT-PCR of human TEKT4 from multiple tissues, a human cDNA library (Clontech, Mountain View, CA, USA) was used with the exception of human sperm that was obtained from an infertile male patient. The 5'-AGGCCTACAACATCGACGAG-3' (forward) and 5'-GCTAACGCGCTGTTTATTTG-3' (reverse) primers amplify a 736-bp fragment in human TEKT4. The mouse Hprt1 and human ACTB served as loading controls for the PCR reactions that were carried out for 30 cycles.
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In situ hybridization (ISH) of mouse testis sections was performed as described previously (12)
with the 390-bp Tekt4 cDNA fragment. Briefly, the cDNA fragment in pGEM T-vector (Promega) served as template for generating sense and antisense probes with [35S]dUTP using the T7/SP6 combination system (Promega). Sections were hybridized with the probes, washed, and exposed to photographic emulsion (NBT-3; Kodak, Rochester, NY, USA) for 4–7 days at 4°C. After the slides were developed and fixed, they were counterstained with hematoxylin. The sense probe revealed no hybridization (data not shown).
Northern blot analysis
Northern blot analysis was performed on total testis RNA extracted from mice of different genotypes as indicated in Results. Briefly, 15 µg of total RNA were electrophoresed and transferred onto nylon membranes as described previously (12)
. A cDNA fragment that encompasses exons 3–6 was labeled with [32P]-dATP using the Strip-EZ kit (Ambion, Austin, TX, USA) and used as a probe. The membrane was hybridized in UltraHyb buffer (Ambion), washed, and subjected to autoradiography. A mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh) cDNA probe was used as a loading control.
Generation of anti-TEKT4 antibodies and immunohistochemistry
The complete mouse Tekt4 open reading frame (ORF) was subcloned into pET-23b (+) (Novagen, San Diego, CA, USA) and sequenced to confirm absence of any mutations. Recombinant mouse TEKT4 protein expressed in BL21 (DE3) pLysS bacterial cells (Novagen) formed inclusion bodies that were purified, solubilized, and refolded according to manufacturer’s instructions. Two guinea pigs and two rabbits were immunized with the His-tagged TEKT4 to produce polyclonal antibodies (Cocalico Biologicals, Reamstown, PA, USA).
For immunohistochemistry, mouse testes were fixed in Bouin’s solution for 4 h, embedded in paraffin, sectioned at 5 µm thickness, and mounted onto polylysine-coated slides. Microwave antigen retrieval was employed as described previously (15)
. After being blocked, an aliquot of 100 µl primary guinea pig antibody (Ab; GP 158) diluted at 1:1000 was applied to each section and incubated at 4°C overnight. Incubation with secondary Ab and visualization of positive cells were performed using Vectastain Elite-kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. Preimmune serum from the same animal was used in control sections.
Immunoblot analysis
For immunoblot analysis, 30 µg of total protein were electrophoresed under denaturing conditions, transferred onto nitrocellulose membranes (Whatman Schleicher & Schuell, Florham Park, NJ, USA), and sequentially probed with the primary anti-TEKT4 antiserum (1:1000) and a peroxidase-conjugated antiguinea pig secondary Ab (1:10,000) (Jackson ImmunoResearch, West Grove, PA, USA), and developed with enhanced chemiluminescence (ECL) reagents (GE Healthcare, Piscataway, NJ, USA).
Immunofluorescence
Immunofluorescence on mouse caudal sperm was performed as described previously (5)
. Briefly, cauda epididymides were minced in prewarmed M2 medium (Sigma-Aldrich, St. Louis, MO, USA) and sperm were allowed to swim out by incubation at 37°C under 5% CO2 in air for 30 min. Sperm were collected by centrifugation at 650 g for 10 min, adjusted to a concentration of 106/ml, spotted onto polylysine coated slides, and allowed to air dry. Dried slides were treated with ice-cold PBS containing 0.5% (v/v) TritonX-100 for 1 min, fixed with methanol:acetic acid (3:1) at –20°C for 20 min, and allowed to air dry. The slides were blocked with TBST (Tris-buffered saline containing 0.01% Tween-20) containing 5% (v/v) normal goat serum for 30 min and incubated with anti-TEKT4 antisera at a dilution of 1:100 in TBST for 1 h. The slides were then washed with TBST three times for 15 min each and incubated with Alexa-Fluor 488 labeled antiguinea pig secondary Ab (Molecular Probes, Invitrogen) at a dilution of 1:200 in TBST for 1 h. The slides were then washed again as described above and mounted with Vectashield mounting medium containing 4',6'-diam idino-2-phenylidole (DAPI; Vector Laboratories). All wash and incubation steps were carried out at room temperature. Immunostaining was visualized by fluorescence microscopy, photographed as described before, and formatted by Photoshop 7.0 software (Adobe Systems, San Jose, CA, USA).
Generation of Tekt4 knockout mice
A 4807 bp HincII-SnaBI fragment upstream of exon 1 and a 2.2 kb MscI-SacII fragment downstream of exon 2 were subcloned into the pPgk1-HPRT1 vector (16)
to create the 5' and 3' arms of the targeting construct. After insertion of the MC1-tk (thymidine kinase) cassette, the construct was electroporated into HPRT-deficient AB2.2 mouse ES cells derived from a 129S7/SvEvBrd-Hprtb–m2 (129S7) strain, and ES cell clones were selected in M15 medium containing hypoxanthine, aminopterin, and thymidine (HAT) and 1-(2'-deoxy-2'-fluoro-B-D-arabinofuranosyl)-5'-iodouracil (FIAU) as described previously (16)
. Homologous recombination was detected by Southern blot analysis with BglII-digested and SacI-digested DNA and 5' or 3'external probes, respectively. Four correctly targeted ES cell clones that carried the Tekt4tm1Zuk mutation (herein called Tekt4–) were expanded, and these mutant clones were injected into recipient C57BL/6J blastocysts to obtain several high-percent chimeric males (estimated from coat color mosaicism) from two different ES cell clones. Chimeric males were bred to females of both the C57BL/6J (B6) and 129S5/SvEvBrd (129S5) strains to obtain F1 mice heterozygous for the Tekt4 targeted mutant allele (Tekt4+/–). Male and female F1 heterozygotes were intercrossed to produce Tekt4 homozygous mutant (Tekt4–/–) male and female F2 progeny.
Fertility analysis
Mutant mice and control littermates were mated to WT females beginning at 42 days of age. The number of litters and pups per litter born over a 6 month period was recorded to estimate the mean litter size and the average litters born per month.
Electron microscopy
For transmission electron microscopy (TEM) on sperm flagella, caudal sperm were collected as described above. The sperm pellet was immediately fixed in 2.5% glutaraldehyde and 2.0% formaldehyde in 0.1 M cacodylate buffer containing 2 mM CaCl2 for 1.5 h at room temperature. After being washed three times in 0.1 M cacodylate buffer containing 2 mM CaCl2 for 5 min each, the supernatant was discarded after a brief spin and the pellet was postfixed for 1 h in 1% OsO4 in 0.1 M cacodylate buffer. After another series of washes in cacodylate buffer, the pellet was dehydrated with graded series of ethanol followed by propylene oxide. The pellet was gradually infiltrated with an increasing resin to propylene oxide ratio followed by several changes of pure resin. The pellet was then embedded in Spurr’s resin and cured in a 70°C oven for 2–3 days before semithin (500 nm) or thin sections (80 nm) were made and stained with uranyl acetate and lead citrate. EM was performed using Hitachi H7500 Transmission Electron Microscope (Hitachi-HTA, Pleasanton, CA, USA) at 80 kV, and pictures were formatted with Photoshop 7.0 software (Adobe Systems).
Sperm motility analysis and video microscopy
Caudal spermatozoa from adult (6 wk old) mice were prepared in prewarmed M2 or M16 medium as described above. For analysis of percent motility, 20 µl of an aliquot of sperm diluted to 106/ml were spotted onto a glass slide and covered with a 22 x 22 mm coverslip. After 2 min were allowed for the sperm to settle down, a total number of 100 sperm (both motile and immotile) were scored manually using differential interference contrast (DIC) optics and a x20 objective. The procedure was repeated twice for each sample and averaged.
For video microscopy of activated motility, aliquots of sperm were prepared as above and placed under a 22 x 22 mm coverslip on a glass slide. The glass slide was placed inside a P-type heated insert (PeCon GmbH, Erbach, Germany) on the microscope stage with the temperature regulated at 37°C during the procedure, and video images of sperm motility were captured with time-lapse DIC optics at 15 Hz for 50–300 frames under an Axiovert-200 (Carl Zeiss MicroImaging, Thornwood, NY, USA) x40 objective using an AxioCam mRM video camera and AxioVision software (Carl Zeiss MicroImaging). At least two different fields were recorded for each sample and video files were exported as AVI files.
For calculation of straight-line velocity (VSL), manual reconstruction of sperm trajectories was performed using the sperm head as the reference point in each frame (17)
. The distance between the first and the last points in each trajectory was calculated using a direct measurement tool in the AxioVision software and corrected over elapsed time to estimate the VSL (net space gain over time) as described previously (17)
.
Sperm ATP levels
Sperm ATP levels were measured for Tekt4-null mice on both 129-inbred and B6;129S5 mixed genetic backgrounds. After minced cauda epididymides were incubated in M16 medium (Sigma-Aldrich) at 37°C in 5% CO2 and air for 15 min, sperm were resuspended to a concentration of 107 cells/ml and either measured immediately (t=0.25 h) or incubated further. At different time-points, 50 µl aliquots were added to 950 µl of boiling extraction buffer (4 mM EDTA/0.1 M Tris-HCl, pH 7.8) and boiled at 100°C for a further 3 min. The extract was centrifuged at 20,000 g for 5 min, and ATP was measured in quadruplicate 50 µl aliquots of the supernatant by using a luciferase bioluminescence assay according to the manufacturer’s protocol (ATP Bioluminescence Assay kit CLS II; Roche Applied Science, Indianapolis, IN, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. By using Tekt3 as a probe for BLAST analysis, we identified several novel expressed and predicted tektins in the mouse genome, one of which was a RIKEN full-length clone (GenBank accession #AK005842) that mapped to chromosome 17A3.3. The UniGene cluster Mm.282330, under which AK005842 is grouped, contains 46 ESTs, 37 of which were from male genital tissue, 5 from cerebellum/mesencephalon, while 4 were from uncharacterized sources. Using these reference sequences, we cloned the full-length mouse cDNA (1,532 bp; deposited into GenBank as accession #AY485267) from total testis RNA of an adult (8-wk-old) 129S5B6F1 hybrid mouse. AY485267 is 100% identical to the genomic sequence, whereas the original RIKEN sequence, AK005842, has a spurious CG dinucleotide at the 5' end and a missense alteration in the ORF (1432C>A) that causes a R434S substitution in the predicted WT protein sequence. The Mouse Gene Nomenclature Committee at the Jackson Laboratory assigned AY485267 as tektin 4 (Tekt4). Mouse Tekt4 encodes a 447-amino acid protein with the conserved nonapeptide tektin motif near the C-terminus (Fig. 2
A) and is 92.2% identical to the recently published rat TEKT4 protein (NP_001013987.1) (13)
and 77.1 and 46.7% identical to the human (NP_653306) and predicted pufferfish orthologs, respectively.
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To examine the tissue distribution of the orthologous tektin four genes, we used RT-PCR analyses on RNA from multiple tissues in mice (Fig. 1A
) and humans (Fig. 1B
). Tekt4 is exclusively expressed in the testis in mice; in humans, TEKT4 transcripts were highly abundant in total RNA extracted from ejaculated spermatozoa, while low levels of expression could be identified from the human pancreas. RT-PCR analysis performed on RNA from developing mouse testes shows that Tekt4 mRNA is first detectable around postnatal day 18 (Fig. 1C
), coinciding with the appearance of haploid round spermatids in the testis (18)
and increases thereafter, suggesting that Tekt4 is expressed in spermatids. Consistent with these data, ISH analyses localize Tekt4 mRNA (Fig. 1D-G
) specifically to steps 6–8 round spermatids, with an abrupt absence of signal from step 9 spermatids. The narrow expression window of Tekt4 mRNA during mouse spermiogenesis is summarized in Fig. 1H
.
TEKT4 is a flagellar protein expressed in elongated spermatids and mature spermatozoa
To investigate the expression pattern of TEKT4 protein, a polyclonal antibody was raised against the full-length protein, which detected a 52 kDa band on a Western blot with testis total protein (Fig. 2B
). Preimmune serum did not react with testis total protein (Fig. 2B
) or the recombinant protein (data not shown), indicating that the polyclonal anti-TEKT4 was the result of a specific immune response.
Immunohistochemistry on testis sections localized TEKT4 to the adluminal compartment of stages XI-XVI seminiferous epithelium (Fig. 2C
); higher magnification images revealed that it was specifically expressed in the developing flagella of step 16 elongated spermatids with low levels of diffuse expression in round spermatids (Fig. 2D
). No signal was present in the head region of spermatids or in the basal compartment of the seminiferous epithelium or in the interstitial spaces, suggesting that the protein is specific to the flagella of spermatids. Preimmune serum on testis sections did not demonstrate any signal (Fig. 2E
).
To analyze the localization of TEKT4 in mature spermatozoa after spermiation, we performed immunofluorescence on epididymal spermatozoa. The anti-TEKT4 antiserum specifically stained the flagella with the principal piece showing the strongest signal and a weaker signal present in the midpiece (Fig. 2G
).
Generation of Tekt4 knockout mice
To define the physiological role of TEKT4 in sperm flagellar motility and male reproduction, we generated a targeted mutation in Tekt4 using homologous recombination in embryonic stem (ES) cells. The Tekt4 gene consists of 6 exons spanning 5 kb on mouse chromosome 17, and the human TEKT4 gene resides in a syntenic region of human chromosome 2q11.1. We isolated 2 overlapping genomic fragments containing Tekt4 sequences from a mouse 129 genomic library and designed a targeting construct (Fig. 3
A) that would delete 2.8 kb of the gene eliminating 400 bp of upstream promoter sequences and the first two exons that include the initiation ATG codon and 202 of 447 codons. We predicted that this construct would create a null allele for Tekt4.
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The construct was electroporated into HPRT-deficient AB2.2 mouse ES cells derived from a 129S7/SvEvBrd-Hprtb–m2 (129S7) strain, and double selection was used to screen for targeted clones as described previously (16)
. Proper targeting was verified by Southern blot analysis using both 5' and 3' external probes (Fig. 3B
). Four correctly targeted ES cell clones were obtained from 177 clones screened (2.25% targeting efficiency). Targeted Tekt4 ES cell clones were injected into recipient C57BL/6J blastocysts, and several high-percent chimeric males (estimated from coat color mosaicism) were obtained from two different ES cell clones. Chimeric males were bred to females of both the C57BL/6J (B6) and 129S5/SvEvBrd (129S5) strains to obtain mice heterozygous for the Tekt4 targeted mutant allele (Tekt4tm1Zuk). Male and female F1 heterozygotes were intercrossed to produce Tekt4 homozygous mutant male and female F2 progeny that were born with the expected Mendelian frequency and 1:1 ratio for both sexes (Fig. 3B
). Consistent with the limited expression of this gene beyond the testis, homozygous mutant mice were viable and had no apparent gross abnormalities. Northern blot hybridization (Fig. 3C
) and Western blot analysis (Fig. 3D
) demonstrated the absence of mRNA and protein in the testes of homozygous mutants, confirming that the Tekt4tm1Zuk mutant allele is a null allele (hereafter referred to as Tekt4–). In addition, indirect immunofluorescence analysis on caudal sperm with the anti-TEKT4 antiserum confirmed that the flagella of sperm produced by the null males were devoid of TEKT4 (Fig. 3G
).
Tekt4–/– males on an inbred 129 background mice display reduced fertility
To assess the potential roles of TEKT4 in male fertility, Tekt4–/–males and heterozygous littermates (Tekt4+/–) carrying the targeted allele on a mixed genetic background (B6;129S5-Tekt4tm1Zuk) were bred to WT females for 6 mo. Mating of 9 Tekt4+/– males with WT females over a 6 month period resulted in 49 litters with an average litter size of 7.98 ± 0.3 pups per litter; mating of 9 Tekt4–/– males with WT females resulted in a mean litter size of 7.11 ± 0.3 pups per litter (Fig. 4
A). These findings suggest that on a mixed background, Tekt4–/– males possess normal fertility.
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We then tested the fertility of Tekt4–/– males carrying the null mutation on a 129 genetic background (129S5-Tekt4tm1Zuk) by mating them to WT females. In contrast to null males on a mixed background, 129S5- Tekt4–/– males are subfertile. Whereas five 129S5-Tekt4+/– bred to WT females over 6 month produced 30 litters with an average litter size of 5.2 ± 0.3 pups per litter, Tekt4-null littermate males bred to WT females over a 5-month period showed reduced fertility and produced only 9 litters with an average litter size of 2.23 ± 0.5 pups per litter (P<0.01, Fig. 4B
). A significant reduction was also seen in the litters sired per month during the mating period (Fig. 4C
); Tekt4+/– males sired 1.0 ± 0.05 litters per month over a 6 month period, but Tekt4–/– males sired 0.3 ± 0.15 litters per month over a 5 month period of mating (P<0.003). Over the breeding period, null males showed a progressive reduction in fertility (Fig. 4D
). Four out of five null males sired at least one litter during the first month of breeding, but by the fourth month or earlier, they had stopped breeding altogether. One Tekt4–/– male was infertile and did not produce any pups over a 5 month period. Tekt4–/– null females on both genetic backgrounds had normal fertility (data not shown).
Tekt4–/– males have asthenozoospermia with reduced progressive motility
To investigate the fertility deficit of the 129S5- Tekt4–/– male mice, we analyzed the reproductive physiology of the mutant strain in more detail. Mean testis weights of WT and null males were not significantly different at 6–8 wk of age (Table 1
). Counts performed on cauda epididymal spermatozoa were also similar between WT and Tekt4-null males. However, the null males showed a drastic reduction in the percentage of motile spermatozoa in the cauda (Table 1)
, whereas WT males had 82.3 ± 2.0% motile spermatozoa, only 35.6 ± 2.3% of actively motile spermatozoa were present in knockout males (P<0.0001). Since older males appeared to have reduced fertility, we examined 129S5-Tekt4–/– males at 5 months of age and observed comparable testis weights and sperm counts but with a further reduction in the motile percentage of caudal spermatozoa (Table 1)
. Testis histology was normal in 5 month old males (data not shown).
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We analyzed the deficit in progressive motility of Tekt4-null spermatozoa by using video microscopy. Manual reconstruction of sperm tracks was performed by using a frame-by-frame tracing of the sperm head and allowed us to measure the VSL of spermatozoa. WT males had a mean VSL of 52.13 ± 4.6 µm/s (±SE) and had several progressively motile spermatozoa in all fields tested (Supplemental Video S1). However, very few Tekt4-null spermatozoa had progressive motility, and those that did were extremely sluggish with a VSL of 11.61 ± 1.04 µm/s (P=0.0002), suggesting that the Tekt4-null spermatozoa had severely reduced forward progressive motility (Supplemental Video S2).
Tekt4-null spermatozoa also had an obvious defect in flagellar bending in the midpiece with a characteristic flagellar bend restricted only to the end piece. The principal piece of most spermatozoa had a characteristic twitching motion without any waveform propagation along the flagellum suggesting a defect in flagellar beat generation and/or propagation. This caused motile null spermatozoa to stall in their path or to undertake a slow circular trajectory (Supplemental Video S3). In contrast, WT spermatozoa had symmetric waveform generation and propagation throughout the principal and the end-piece, which resulted in a greater propulsive force across the field.
Tekt4-null spermatozoa have subtle disorganization of flagellar ultrastructure
Since tektins are a group of flagellar proteins that are believed to be important for axonemal architecture, we determined flagellar ultrastructure of 8-wk-old 129S5Tekt4-null spermatozoa by TEM. Cross-sections through the midpiece and the principal piece of the null spermatozoa did not reveal any obvious deficits (Fig. 5
). All nine outer doublets and the central singlets with clearly visible outer and inner dynein arms and radial spokes were intact in Tekt4 null flagella. All nine ODFs were also present juxtaposed to the outer doublets. In the principal piece, the longitudinal columns of the FS were present in the correct plane and were interconnected by circumferential ribs. However, on closer analysis, subtle alterations were apparent in several null sperm. The intervening space that is immediately peripheral to the ODFs and delimited by the mitochondrial sheath in the midpiece and the corresponding space in the principal piece appeared to have expanded in null flagella (Fig. 5B, F
). In the midpiece of mammalian WT sperm, this space is compactly organized by the submitochondrial reticulum (SMR), an electron-dense lattice (Fig. 5A
) of interconnected longitudinal bands that is attached to the overlying mitochondrial sheath (19)
. In midpiece cross-sections of null sperm, the SMR appeared to be diminished or disorganized (Fig. 5B
); although a structure analogous to the SMR has not been identified adjacent to the FS, similar electron-dense deposits were also absent in the principal piece of null flagella (Fig. 5F
). In addition, the circumferential ribs connecting the longitudinal columns of the FS showed greater variation in their spacing (Fig. 5D
) and frequently breaks were observed in their continuity (Fig. 5G
). Thus, although the molecular components of the axoneme and the accessory structures were intact in the Tekt4-null spermatozoa, we observed subtle disorganization of the ultrastructure that possibly contributes to the motility defect in the null spermatozoa.
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Sperm ATP levels are depleted 10-fold in Tekt4-null spermatozoa
Tekt4-null spermatozoa underwent a gradual decline in motility when incubated in M16 medium at 37°C in 5% CO2 and air (Fig. 6A
). Immediately after collection from the cauda epididymis, 35.6 ± 2.3% of Tekt4-null spermatozoa had progressive motility that declined further to 14.5 ± 1.27% after 2 h and to 4.5 ± 0.64% after 3 h. In contrast, WT sperm maintained vigorous progressive motility through 2 and 3 h of incubation. To investigate whether the ineffective flagellar strokes in the null spermatozoa might cause over-consumption of intracellular energy stores and lead to the gradual decline in progressive motility, we measured sperm ATP levels from age-matched Tekt4-null males and littermate controls after incubation for 0.25, 1.5, and 3 h in M16 medium. At 0.25 h, null sperm had a modest reduction in ATP levels to 75.8% of WT levels. However, after 1.5 and 3 h in M16 medium, ATP levels in Tekt4-null sperm were drastically reduced to levels that were >10-fold and >20-fold lower, respectively, relative to WT sperm (Fig. 6B
), reflecting a significant reduction in intracellular ATP at both time-points (P<0.001). Since the initial difference in ATP levels between Tekt4-null sperm and WT sperm (at t=0.25 h) was modest, this suggests that the dramatic reduction in ATP levels after increasing incubation is due to over-consumption caused by ineffective flagellar beating.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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21
22)
and is believed to contain >250 proteins. Several proteins that constitute the axoneme and its appendages are essential for flagellar motion and are mutated in several human disorders (23
, 24)
. Tektins are a highly conserved family of flagellar and ciliary proteins in species as diverse as Chlamydomonas, sea urchins, pufferfish, rodents, and humans (10
, 25
26
27)
. In sea urchins, tektins A and B have been found to form axially continuous heterodimers that exist with an inherent structural periodicity of 16 nm that is exactly twice that of the 8 nm tubulin repeat (8)
and matches the 96 nm longitudinal repeat of axonemes (28)
. This led to the hypothesis that the axially repeating tektin filaments are required for the periodic organization of the axonemal appendages along the flagella. Mice lacking TEKT2, the mouse homologue of tektin B, reveal defects in the organization of the inner dynein arms on doublets 2 and 6 (9)
, supporting the notion that axonemal tektins play an essential role in the organization of the appendages. Indirect support also came from studies in Chlamydomonas, in which a slow-swimming mutant lacking inner dynein arms was found to have decreased levels of a tektin homologue (27)
.
However, in contrast to the prototypical tektins that were originally purified from detergent-resistant protofilaments of sea urchin axonemes, tektins in other species have also been found at various extra-axonemal structures in the flagella. Recent proteomic studies by Cao et al. (14)
indicates that the accessory structures of mouse flagella contain at least five tektins. In previous studies, our group and others have reported five members of the tektin family to be expressed in the testis in mice and humans. Whereas Tekt1 and Tekt2 are also expressed in sensory and respiratory cilia (9
, 29)
, Tekt3 is a germ cell-enriched member of the family (12)
. Here, we analyzed the expression pattern of mouse Tekt4 and found it to be exclusively expressed in testis. In mouse testis, our polyclonal antiserum localized TEKT4 to the adluminal compartment of stages X-XVI tubules and to the flagella of elongated spermatids in stages XV-XVI tubules. Since the message is transcribed in Stages VII-VIII tubules, this suggests a transient post-transcriptional repression of the Tekt4 message that is characteristic of several testicular transcripts (30)
. By immunofluorescence, TEKT4 was localized to the principal piece with a weaker signal in the midpiece. This correlates well with recent studies by Iida et al. (31)
, demonstrating TEKT4 staining throughout the midpiece and the principal piece in mouse and rat flagella by using a paraformaldehyde fixation method. With the use of immuno-gold EM, rat TEKT4 was localized to the cortical layer of the convex surface of ODFs that is adjacent to the mitochondrial sheath in the midpiece and to the FS in the principal piece but was excluded from the axoneme. Together with the proteomic studies of Cao et al. (14)
, these results confirm that TEKT4 is the first tektin family member to be associated with the peri-axonemal ODFs. In contrast to other studies, we also observed moderately strong staining of the acrosomal region; although other tektin family members are present in basal bodies, tektins have not previously been reported in the sperm head. The unique testis-specific expression pattern of Tekt4 prompted us to examine its physiological role during spermatogenesis by generating mutant mice lacking TEKT4.
Based on its high sequence identity with the human and pufferfish orthologs, we hypothesized mouse TEKT4 to serve an important evolutionarily-conserved role in the regulation of flagellar motility. Consistent with this, we see a significant decrease in the number of motile spermatozoa from mice lacking TEKT4. Caudal spermatozoa from Tekt4-null mice showed a drastic reduction in percent motility with the majority of sperm incapable of progressive motion. The motile population had extremely sluggish progressive motility and often demonstrated slow circular motion that was due to asymmetric low-amplitude beating of the flagella. Most spermatozoa were devoid of any normal flagellar bend in the midpiece and lacked waveform progression along the flagella, and nearly all spermatozoa were paralyzed after incubation for 3 h. This suggested a defect in energy metabolism in the null sperm that could potentially be due to a deficit in energy production or inefficient utilization. Although several constitutive members of the ODFs in mammals have been cloned previously (ODF1 and ODF2) (32
33
34)
, the physiological role of these proteins and indeed that of the ODFs in flagellar function has remained unclear. While ODFs are believed to increase the tensile strength of flagella (4)
, the "geometric clutch" model (3)
proposes that anchoring of the ODFs to the outer doublets permits amplification of the bending torque generated by the interdoublet sliding force. This in turn leads to efficient energy usage by transmitting the force to the flagellar base. Since TEKT4 has been localized to the ODF surface, it is an attractive candidate for serving as an anchor, either singly or in combination with SPETEX1, another ODF-associated protein that interacts with TEKT4 in yeast (13
, 35)
.
In this study, we have carefully determined whether loss of TEKT4 leads to a deficiency in ATP production or over-consumption of intracellular ATP. Whereas initial ATP levels were modestly reduced in Tekt4-null sperm, we observed a dramatic >10-fold reduction in ATP levels in null sperm relative to WT sperm with prolonged incubation. This finding strongly supports the "clutch" model that can explain the time-dependent decrease in intracellular ATP levels in Tekt4-null sperm, since ineffective flagellar strokes that fail to transmit the propulsive force through the ODFs would utilize enormous amounts of ATP leading to steady depletion of energy stores. If TEKT4 is indeed involved in anchoring the ODF to the axoneme, this indicates that when uncoupled from the axoneme, the cost of work involved in moving the ODFs is excessive and unfavorable for the cell.
The gradual reduction in progressive motility after incubation in M16 is presumably in turn due to the depletion in ATP levels in Tekt4-null spermatozoa. Interestingly, null mutants on both the inbred and the mixed genetic backgrounds demonstrate similar depletion of ATP levels postincubation (Fig. 6B
and data not shown). Also, on both genetic backgrounds, Tekt4-null males have reduced sperm motility although mixed background males have a higher proportion with progressive motility (Table 1)
. This suggests that the sperm motility defect seen in Tekt4 null males is completely penetrant independent of genetic modifiers. Despite these similarities in motility parameters, however, Tekt4-null males on different genetic backgrounds differ in fertility. Tekt4-null males on a 129-inbred background are subfertile, and one male was infertile during 5 months of breeding, despite testis weights and sperm counts at 8 wk of age being comparable to control littermates (Table 1)
. In contrast, Tekt4–/– males on a mixed genetic background appear to have nearly normal fertility. This suggests that by itself reduction in sperm motility is not sufficient to completely explain the fertility deficit and that regulation of the Tekt4–/– mutation by as yet unknown modifiers on the 129-inbred background lead to the decline in fertility. Male infertility is a complex disorder with multifactorial inheritance in which gene-gene and gene-environment interactions modulate the overall phenotype of a disease allele. The diagnosis of male infertility is the extreme end of a spectrum of disorders that span subtle defects in spermiograms, subfertility, and sterility. Not surprisingly, the effect of genetic backgrounds on highly variable penetrance of the male infertility phenotype has been previously reported in mice for several genes with targeted null mutations (36
37
38
39)
. Additional studies are needed to investigate the role of background-specific genetic modifiers of TEKT4.
The fertility of 129S5-Tekt4–/– males declined gradually with increasing age. While four out of five males tested sired at least one litter, by the fourth month or earlier, all of them stopped breeding and were infertile for at least 1 month, when scarificed. Significantly, older 129S5-Tekt4–/– males at 5 months of age demonstrate reduced fertility despite normal testis weights and sperm counts comparable to 8-wk-old males and WT littermates (Table 1)
and normal testis histology (data not shown). Older null males have a further reduction in the progressively motile population of spermatozoa compared to younger null males; however, we have not looked for defects in hyperactivated motility in older males, which might explain the gradual decline in fertility. In mice and humans, hyperactivated motility is absolutely essential for sperm to penetrate the egg, as demonstrated by the infertility of mice lacking Catsper2, which have normal progressive motility but are unable to undergo hyperactivation (40)
. One possibility could be that additional ultrastructural alterations in the ODFs in older males cause defective hyperactivation. The ODFs contain 93–97% of Zn2+ present in the sperm. Zn2+ is added to the ODFs during spermiogenesis and stabilizes cysteine-containing proteins by forming Zn2+-mercaptide complexes. In the epididymis, removal of Zn2+ is critical before the sulfhydryl groups in cysteine can form disulfide linkages that impart elasticity. Failure to remove Zn2+ leads to decreased sperm motility. Significantly, at least two studies (41
, 42)
have shown increased Zn2+ concentrations in epididymal sperm of older men and consequently decreased motility. It would be interesting to study the effects of Zn2+ on the ODFs of older Tekt4-null sperm, and it is tempting to speculate that there might be additional ultrastructural alterations in the sperm flagella in older mice that prevent efficient generation of hyperactivated motility.
To examine if loss of TEKT4 leads to an ultrastructural defect in the ODF or the axoneme, we performed TEM on sperm from 8-wk-old Tekt4-null male. To our surprise, we did not find any gross ultrastructural anomalies in the null sperm. All ODFs and the axonemal appendages as well as the axoneme were intact in the flagella from Tekt4-null mice. However, we observed subtle defects in the space peripheral to the ODF-axoneme complex, both in the midpiece and principal piece of the mutant sperm. Most frequently, we found moderate expansion of this peri-ODF space in null sperm compared to WT and an accompanying diminution of the submitochondrial reticulum (SMR), an electron-dense matrix of unknown function that extends in the midpiece from the connecting piece to the annulus, and is attached to the overlying outer mitochondrial membrane (43)
. The plaques of the SMR are normally positioned between and adjacent to the indentations on the abaxial (convex) surface of ODFs (43)
in close proximity to the observed localization of TEKT4 (31)
. It is possible, therefore, that TEKT4 interacts with the proteins of the SMR to tether the ODF-axoneme complex indirectly to the mitochondrial sheath and to the segmented columns of the connecting piece at the base of the flagellum. In addition, the principal piece of several null sperm showed moderate enlargement and frequently breaks were observed in the circumferential ribs of the FS, suggesting that the lack of TEKT4 might have caused a decrease in the tensile strength of flagella. Formal verification of these possibilities would require identification of novel TEKT4-interacting partners that are present in the SMR or in the FS. However, it is unlikely that these subtle abnormalities directly cause the severe motility defects in these mutants.
In conclusion, our studies indicate that members of the expanded tektin family in higher vertebrates may have acquired novel functions in accordance with their localization in discrete peri-axonemal locations. This is the first study highlighting the physiological role of a tektin family member that is present in an accessory peri-axonemal structure. It is worth recalling that most flagellated lower organisms (including sea urchins) do not contain either ODFs or the FS, suggesting that the evolution of these structures and expansion of the tektin family might have proceeded simultaneously. Based on the ultrastructural localization of TEKT4 (31)
and the phenotype described in this report, TEKT4 likely functions in the transmission of the bending force from the axoneme through the ODF to the connecting piece at the base of the flagella. Isolation of ODFs from sperm flagella of Tekt4–/– mice would be needed to analyze the structural integrity of these elements in the null sperm. Identification of novel interaction partners and additional studies addressing the question of energy metabolism and detailed analysis of the kinematic parameters in the null sperm are also likely to provide greater insight into the physiological role of TEKT4 in male reproduction.
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ACKNOWLEDGMENTS |
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Received for publication July 31, 2006. Accepted for publication October 31, 2006.
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