Construction of a Catsper1 DNA Vaccine and Its Antifertility Effect on Male Mice

Cation channel of sperm 1 (CATSPER1) is a unique sperm cation channel protein, and essential for sperm function and male fertility. CATSPER1 exclusively expresses in meiotic and postmeiotic spermatogenic cells, thus belongs to the spermatogenesis-specific antigen that escape central tolerance. We have previously demonstrated the immunocontraceptive potential of its transmembrane domains and pore region, and reported the antifertility effects of its B-cell epitopes on male mice. Aiming to develop DNA vaccine targeting CATSPER1 for male contraception, here the whole open reading frame of mouse Catsper1 was cloned into the plasmid pEGFP-N1 to obtain a DNA vaccine pEGFP-N1-Catsper1. The vaccine was confirmed to be transcribed and translated in mouse N2a cell in vitro and mouse muscle tissue in vivo. Intramuscular injection with the vaccine on male mice induced specific immune reaction and caused significant inhibition on sperm hyperactivated motility and progressive motility (P<0.001 for both), and consequently reduced male fertility. The fertility rate of experimental group was 40.9%, which was significant lower (P=0.012) than control group (81.8%). No significant change in mating behavior, sperm production and histology of testis/epididymis was observed. Given that Catsper1 exhibits a high degree of homology among different species, Catsper1 DNA vaccine might be a good strategy for developing an immunocontraceptive vaccine for human and animal use.     

Catsper3 and catsper4 encode two cation channel-like proteins exclusively expressed in the testis

CATSPER1 and CATSPER2 are two cation channel-like proteins exclusively expressed in the testis and essential for normal sperm motility and male fertility. Using in silico subtraction and database mining, we identified expressed sequence tags encoding two previously uncharacterized cation channel-like proteins structurally homologous to CATSPER1 and CATSPER2. Similar to CATSPER1 and CATSPER2, these two proteins contain a single-ion transport domain comprised of six transmembrane spanning regions, in which the fourth transmembrane region resembles a voltage sensor and a pore-forming region lies between transmembrane regions 5 and 6. The pore contains the consensus sequence T x D x W, which is indicative of a potential calcium-selective channel. The mRNAs for Catsper3 and Catsper4 were detected exclusively in the testis using multitissue Northern blot and RT-PCR analyses. The onsets of both genes coincide with the first appearance of spermatids during testicular development. In situ hybridization analyses revealed that Catsper3 and Catsper4 mRNAs displayed identical localization patterns and were confined to spermatids of steps 1-8. Immunofluorescence and immunohistochemistry analyses demonstrated that these two proteins were expressed within the acrosome of late spermatids and spermatozoa. Our data suggest that CATSPER3 and CATSPER4 are two cation-channel proteins and have roles in acrosome reaction and male fertility.     

CATSPER channel-mediated Ca2+ entry into mouse sperm triggers a tail-to-head propagation

Many Ca(2+) channel proteins have been detected in mammalian sperm, but only the four CATSPER channels have been clearly shown to be required for male fertility. Ca(2+) entry through the principal piece-localized CATSPER channels has been implicated in the activation of hyperactivated motility. In the present study, we show that the Ca(2+) entry also triggers a tail-to-head Ca(2+) propagation in the mouse sperm. When activated with 8-Br-cAMP, 8-Br-cGMP, or alkaline depolarization, a CATSPER-dependent increase in intracellular Ca(2+) concentration starts in the principal piece, propagates through the midpiece, and reaches the head in a few seconds. The Ca(2+) propagation through the midpiece leads to a Ca(2+)-dependent increase in NADH fluorescence. In addition, CatSper1-mutant sperm have lower intracellular ATP levels than wild-type sperm. Thus, a Ca(2+) influx in the principal piece through CATSPER channels can not only initiate hyperactivated motility, but can also trigger a tail-to-head Ca(2+) propagation that leads to an increase in [NADH] and may regulate ATP homeostasis.     

Targeted ablation of plasma membrane Ca2+-ATPase (PMCA) 1 and 4 indicates a major housekeeping function for PMCA1 and a critical role in hyperactivated sperm motility and male fertility for PMCA4

The relative importance of plasma membrane Ca2+-ATPase (PMCA) 1 and PMCA4 was assessed in mice carrying null mutations in their genes (Atp2b1 and Atp2b4). Loss of both copies of the gene encoding PMCA1 caused embryolethality, whereas heterozygous mutants had no overt disease phenotype. Despite widespread and abundant expression of PMCA4, PMCA4 null (Pmca4-/-) mutants exhibited no embryolethality and appeared outwardly normal. Loss of PMCA4 impaired phasic contractions and caused apoptosis in portal vein smooth muscle in vitro; however, this phenotype was dependent on the mouse strain being employed. Pmca4-/- mice on a Black Swiss background did not exhibit the phenotype unless they also carried a null mutation in one copy of the Pmca1 gene. Pmca4-/- male mice were infertile but had normal spermatogenesis and mating behavior. Pmca4-/- sperm that had not undergone capacitation exhibited normal motility but could not achieve hyperactivated motility needed to traverse the female genital tract. Ultrastructure of the motility apparatus in Pmca4-/- sperm tails was normal, but an increased incidence of mitochondrial condensation indicated Ca2+ overload. Immunoblotting and immunohistochemistry showed that PMCA4 is the most abundant isoform in testis and sperm and that it is localized to the principle piece of the sperm tail, which is also the location of the major Ca2+ channel (CatSper) required for sperm motility. These results are consistent with an essential housekeeping or developmental function for PMCA1, but not PMCA4, and show that PMCA4 expression in the principle piece of the sperm tail is essential for hyperactivated motility and male fertility.     

Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa

During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca²⁺. The elevation of Ca²⁺ is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K⁺ current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca²⁺ current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1–4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K⁺ current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K⁺ current present in the Slo3 ^−/− sperm arise from CATSPER? Second, can any additional membrane K⁺ currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 ^−/− sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca²⁺ influx in response to alkalization.     

Positive selection for indel substitutions in the rodent sperm protein catsper1

Catsper1 is a voltage-gated calcium channel located in the plasma membrane of the sperm tail and is necessary for sperm motility and fertility in mice. We here examine the evolutionary pattern of Catsper1 from nine species of the rodent subfamily Murinae of family Muridae. We show that the rate of insertion/deletion (indel) substitutions in exon 1 of the gene is 4-15 times that in introns or neutral genomic regions, suggesting the presence of strong positive selection that promotes fixations of indel mutations in exon 1. The number of indel polymorphisms within species appears higher than expected from interspecific comparisons, although there are too little data to provide a statistically significant conclusion. These results, together with an earlier report in primates, indicate that positive selection promoting length variation in Catsper1 may be widespread in mammals. A structural model of Catsper1 suggested the importance of the exon 1-encoded region in regulating channel inactivation, which may affect sperm mobility and sperm competition. Our findings provide a necessary foundation for future experimental investigations of Catsper1's function in sperm physiology and role in sperm competition using rodent models.     

Expression of the gene for mouse lactate dehydrogenase C (Ldhc) is required for male fertility

The lactate dehydrogenase (LDH) protein family members characteristically are distributed in tissue- and cell type-specific patterns and serve as the terminal enzyme of glycolysis, catalyzing reversible oxidation reduction between pyruvate and lactate. They are present as tetramers, and one family member, LDHC, is abundant in spermatocytes, spermatids, and sperm, but also is found in modest amounts in oocytes. We disrupted the Ldhc gene to determine whether LDHC is required for spermatogenesis, oogenesis, and/or sperm and egg function. The targeted disruption of Ldhc severely impaired fertility in male Ldhc(-/-) mice but not in female Ldhc(-/-) mice. Testis and sperm morphology and sperm production appeared to be normal. However, total LDH enzymatic activity was considerably lower in Ldhc(-/-) sperm than in wild type sperm, indicating that the LDHC homotetramer (LDH-C(4)) is responsible for most of the LDH activity in sperm. Although initially motile when isolated, there was a more rapid reduction in the level of ATP and in motility in Ldhc(-)(/-) sperm than in wild-type sperm. Moreover, Ldhc(-/-) sperm did not acquire hyperactivated motility, were unable to penetrate the zona pellucida in vitro, and failed to undergo the phosphorylation events characteristic of capacitation. These studies showed that LDHC plays an essential role in maintenance of the processes of glycolysis and ATP production in the flagellum that are required for male fertility and sperm function.     

Tektin 3 is required for progressive sperm motility in mice

Tektins are evolutionarily conserved flagellar (and ciliary) filamentous proteins present in the axoneme and peri-axonemal structures in diverse metazoan species. We have previously shown that tektin 3 (TEKT3) and tektin 4 (TEKT4) are male germ cell-enriched proteins, and that TEKT4 is essential for coordinated and progressive sperm motility in mice. Here we report that male mice null for TEKT3 produce sperm with reduced motility (47.2% motility) and forward progression, and increased flagellar structural bending defects. Male TEKT3-null mice however maintain normal fertility in two different genetic backgrounds tested, in contrast to TEKT4-null mice. Furthermore, male mice null for both TEKT3 and TEKT4 show subfertility on a mixed B6;129 genetic background, significantly different from either single knockouts, suggesting partial nonredundant roles for these two proteins in sperm physiology. Our results suggest that tektins are potential candidate genes for nonsyndromic asthenozoospermia in humans.     

Up-regulation of CatSper genes family by selenium

Background  

CatSper1-4 are a unique family of sperm cation channels, which are exclusively expressed in the testis and play an important role in sperm motility and male fertility. Despite their vital role in male fertility, almost nothing is known about the factors regulating their expression. Here, we investigated the effects of selenium (Se) on the expression of CatSper genes and sperm parameters in aging versus young male mice.     

Pharmacological Targeting of Native CatSper Channels Reveals a Required Role in Maintenance of Sperm Hyperactivation

The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca²⁺ entry evoked by alkaline depolarization. In the absence of external Ca²⁺, Na⁺ carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na⁺]_i-reporting probe SBFI in populations of intact sperm. Removal of external Ca²⁺ increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na⁺]_i is greater for sperm alkalinized with NH₄Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na⁺]_i rise is slowed by candidate CatSper blocker HC-056456 (IC₅₀ ∼3 µM). HC-056456 similarly slows the rise of [Ca²⁺]_i that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO₃ ⁻ but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca²⁺ entry through the CatSper channel is terminated by removal of external Ca²⁺. Thus, open CatSper channels and entry of external Ca²⁺ through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception.     

Deficiency in fertilization by morphologically abnormal sperm produced by Azh mutant mice

Male mice homozygous for the azh mutation produce spermatozoa with abnormal head shapes and have significantly reduced fecundity, to between 5% and 10% that of wild-type or heterozygous mice. Several possible causes of this infertility were investigated. No gross endocrine disorders in azh/azh male mice were observed, and they exhibited apparently normal mating behavior. In addition, their sperm were motile, were capable of hyperactivated motility, and did not show premature acrosome reactions. However, quantitative analysis revealed slight but significant reductions in several motility parameters. Analysis of embryos following mating of azh/azh males with superovulated females indicated a reduction in the number of fertilized eggs compared to control matings. In vitro, spermatozoa from azh/azh mice failed to fertilize cumulus-intact/zona-intact and cumulus-free/zona-intact ova, although they successfully fertilized zonafree ova. These results indicate that the primary defect in fertility of azh/azh male mice is a result of sperm quality, likely, in sperm morphology, and is manifest at the level of interaction with the zona pellucida. © 1994 Wiley-Liss, Inc.     

Two distinct Ca(2+) signaling pathways modulate sperm flagellar beating patterns in mice

Hyperactivation, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization. It is characterized by asymmetrical flagellar beating and an increase of cytoplasmic Ca(2+). We observed that some mouse sperm swimming in the oviduct produce high-amplitude pro-hook bends (bends in the direction of the hook on the head), whereas other sperm produce high-amplitude anti-hook bends. Switching direction of the major bends could serve to redirect sperm toward oocytes. We hypothesized that different Ca(2+) signaling pathways produce high-amplitude pro-hook and anti-hook bends. In vitro, sperm that hyperactivated during capacitation (because of activation of CATSPER plasma membrane Ca(2+) channels) developed high-amplitude pro-hook bends. The CATSPER activators procaine and 4-aminopyridine (4-AP) also induced high-amplitude pro-hook bends. Thimerosal, which triggers a Ca(2+) release from internal stores, induced high-amplitude anti-hook bends. Activation of CATSPER channels is facilitated by a pH rise, so both Ca(2+) and pH responses to treatments with 4-AP and thimerosal were monitored. Thimerosal triggered a Ca(2+) increase that initiated at the base of the flagellum, whereas 4-AP initiated a rise in the proximal principal piece. Only 4-AP triggered a flagellar pH rise. Proteins were extracted from sperm for examination of phosphorylation patterns induced by Ca(2+) signaling. Procaine and 4-AP induced phosphorylation of proteins on threonine and serine, whereas thimerosal primarily induced dephosphorylation of proteins. Tyrosine phosphorylation was unaffected. We concluded that hyperactivation, which is associated with capacitation, can be modulated by release of Ca(2+) from intracellular stores to reverse the direction of the dominant flagellar bend and, thus, redirect sperm.     

Classification of mouse sperm motility patterns using an automated multiclass support vector machines model

Vigorous sperm motility, including the transition from progressive to hyperactivated motility that occurs in the female reproductive tract, is required for normal fertilization in mammals. We developed an automated, quantitative method that objectively classifies five distinct motility patterns of mouse sperm using Support Vector Machines (SVM), a common method in supervised machine learning. This multiclass SVM model is based on more than 2000 sperm tracks that were captured by computer-assisted sperm analysis (CASA) during in vitro capacitation and visually classified as progressive, intermediate, hyperactivated, slow, or weakly motile. Parameters associated with the classified tracks were incorporated into established SVM algorithms to generate a series of equations. These equations were integrated into a binary decision tree that sequentially sorts uncharacterized tracks into distinct categories. The first equation sorts CASA tracks into vigorous and nonvigorous categories. Additional equations classify vigorous tracks as progressive, intermediate, or hyperactivated and nonvigorous tracks as slow or weakly motile. Our CASAnova software uses these SVM equations to classify individual sperm motility patterns automatically. Comparisons of motility profiles from sperm incubated with and without bicarbonate confirmed the ability of the model to distinguish hyperactivated patterns of motility that develop during in vitro capacitation. The model accurately classifies motility profiles of sperm from a mutant mouse model with severe motility defects. Application of the model to sperm from multiple inbred strains reveals strain-dependent differences in sperm motility profiles. CASAnova provides a rapid and reproducible platform for quantitative comparisons of motility in large, heterogeneous populations of mouse sperm.     

Diet-Induced Obesity in Male C57BL/6 Mice Decreases Fertility as a Consequence of Disrupted Blood-Testis Barrier

Obesity is a complex metabolic disease that is a serious detriment to both children and adult health, which induces a variety of diseases, such as cardiovascular disease, type II diabetes, hypertension and cancer. Although adverse effects of obesity on female reproduction or oocyte development have been well recognized, its harmfulness to male fertility is still unclear because of reported conflicting results. The aim of this study was to determine whether diet-induced obesity impairs male fertility and furthermore to uncover its underlying mechanisms. Thus, male C57BL/6 mice fed a high-fat diet (HFD) for 10 weeks served as a model of diet-induced obesity. The results clearly show that the percentage of sperm motility and progressive motility significantly decreased, whereas the proportion of teratozoospermia dramatically increased in HFD mice compared to those in normal diet fed controls. Besides, the sperm acrosome reaction fell accompanied by a decline in testosterone level and an increase in estradiol level in the HFD group. This alteration of sperm function parameters strongly indicated that the fertility of HFD mice was indeed impaired, which was also validated by a low pregnancy rate in their mated normal female. Moreover, testicular morphological analyses revealed that seminiferous epithelia were severely atrophic, and cell adhesions between spermatogenic cells and Sertoli cells were loosely arranged in HFD mice. Meanwhile, the integrity of the blood-testis barrier was severely interrupted consistent with declines in the tight junction related proteins, occludin, ZO-1 and androgen receptor, but instead endocytic vesicle-associated protein, clathrin rose. Taken together, obesity can impair male fertility through declines in the sperm function parameters, sex hormone level, whereas during spermatogenesis damage to the blood-testis barrier (BTB) integrity may be one of the crucial underlying factors accounting for this change.     

The small heat shock protein ODF1/HSPB10 is essential for tight linkage of sperm head to tail and male fertility in mice

Sperm motility and hence male fertility strictly depends on proper development of the sperm tail and its tight anchorage to the head. The main protein of sperm tail outer dense fibers, ODF1/HSPB10, belongs to the family of small heat shock proteins that function as molecular chaperones. However, the impact of ODF1 on sperm tail formation and motility and on male fecundity is unknown. We therefore generated mutant mice in which the Odf1 gene was disrupted. Heterozygous mutant male mice are fertile while sperm motility is reduced, but Odf1-deficient male mice are infertile due to the detachment of the sperm head. Although headless tails are somehow motile, transmission electron microscopy revealed disturbed organization of the mitochondrial sheath, as well as of the outer dense fibers. Our results thus suggest that ODF1, besides being involved in the correct arrangement of mitochondrial sheath and outer dense fibers, is essential for rigid junction of sperm head and tail. Loss of function of ODF1, therefore, might account for some of the cases of human infertility with decapitated sperm heads. In addition, since sperm motility is already affected in heterozygous mice, impairment of ODF1 might even account for some cases of reduced fertility in male patients.     

Coupling biochemistry and hydrodynamics captures hyperactivated sperm motility in a simple flagellar model

Hyperactivation in mammalian sperm is characterized by highly asymmetrical waveforms and an increase in the amplitude of flagellar bends. It is important for the sperm to be able to achieve hyperactivated motility in order to reach and fertilize the egg. Calcium (Ca²⁺) dynamics are known to play a large role in the initiation and maintenance of hyperactivated motility. Here we present an integrative model that couples the CatSper channel mediated Ca²⁺ dynamics of hyperactivation to a mechanical model of an idealized sperm flagellum in a 3-d viscous, incompressible fluid. The mechanical forces are due to passive stiffness properties and active bending moments that are a function of the local Ca²⁺ concentration along the length of the flagellum. By including an asymmetry in bending moments to reflect an asymmetry in the axoneme's response to Ca²⁺, we capture the transition from activated motility to hyperactivated motility. We examine the effects of elastic properties of the flagellum and the Ca²⁺ dynamics on the overall swimming patterns. The swimming velocities of the model flagellum compare well with data for hyperactivated mouse sperm.     

Theoretical aspects of canine cryopreserved semen evaluation

Evaluation of canine cryopreserved semen has the ultimate goal of determining if an individual frozen ejaculate will have acceptable fertility. This is difficult in that there is no accepted normal fertility for the dog. The fertility of the female also plays a crucial role in estimating the fertility of the male. Poor female fertility can make a fertile male appear less fertile. Variability of animals, breeding technique, breeding timing, and number of cells inseminated make comparisons in canine fertility difficult to truly measure. Many more animals are needed to provide meaningful statistical results than are usually used. Several tests, including motility in bright field and phase contrast microscopy, computer analysis of motility, sperm morphology, sperm membrane integrity, capacitation and sperm function tests have been investigated to predict fertility, however few of these tests have actually been correlated with fertility. More work is needed to create one or more tests that accurately predict fertility of cryopreserved canine semen.     

Hamster sperm motility transformation during development of hyperactivation in vitro and epididymal maturation

The transformation of hamster sperm motility during capacitation in vitro and during maturation in the caudal epididymis was analyzed and compared using videomicrography. Sperm recovered from the distal portion of the caudal epididymis, as well as ejaculated sperm recovered from the uterus exhibited low amplitude, planar flagellar beating. By 3 hr of incubation under capacitating conditions, the caudal epididymal sperm were swimming in helical patterns apparently produced by significantly increased acuteness of flagellar bending and by torsion seen as abrupt, periodic turning of the head. By 4 hr, most sperm were hyperactivated, swimming in circles resulting from asymmetrical, planar flagellar bending that was significantly more acute than the preceding patterns. When motility parameters of fresh sperm were compared with those of sperm swimming in the transitional helical pattern and with hyperactivated sperm, transitional sperm had significantly higher net and average path velocities than the others, indicating that they covered space at the greatest rate. This suggests that the transitional phase plays an important role in sperm transport. Sperm recovered from the proximal region of the caudal epididymis, near the corpus, swam in either the helical or hyperactivated patterns, or a mixture of the two. The means of their flagellar curvature ratios and linear indices were intermediate between helical and hyperactivated mean values. Thus, sperm undergoing final maturation in the caudal epididymis reverse the pattern of development of hyperactivation. Also, the development of hyperactivated motility must therefore entail induction of a preexisting potential for flagellar movement, rather than a maturational process.     

The motor domain of testis-enriched kinesin KIF9 is essential for its localization in the mouse flagellum

Kinesin is a molecular motor that moves along microtubules. Testis-enriched kinesin KIF9 (Kinesin family member 9) is localized in the mouse sperm flagellum and is important for normal sperm motility and male fertility; however, it is unclear if the motor domain of KIF9 is involved in these processes. In this study, we substituted threonine of the ATP binding motif in the KIF9 motor domain to asparagine (T100N) in mice using the CRISPR/Cas9 system, which is known to impair kinesin motor activity. T100N mutant mice exhibit reduced sperm motility and male fertility consistent with Kif9 knockout mice. Further, KIF9 was depleted in the spermatozoa of T100N mutant mice although the amounts of KIF9 were comparable between wild-type and T100N mutant testes. These results indicate that the motor domain of KIF9 is essential for its localization in the sperm flagellum.