Table of Contents/2000/sjbr

Calcium Ion Channels in Spermatozoa and Their Potential as a Target for the Development of a Male Contraceptive

Darren Schwede

May 23, 2003

As a result of viewing a Public Broadcasting Station (PBS) special on the history of the "pill", I became interested in current efforts towards development of a male, or possibly unisex, contraceptive. If such a pill were available, more responsibility for the prevention of unwanted pregnancies would be placed on the male. This responsibility would lead to a proactive approach on the part of the male population to prevent unwanted pregnancies. A decline in unwanted pregnancies would reduce the social problems created by fatherless homes throughout this country.

Initial investigation uncovered three prominent paths towards this goal. Dr. Joseph Hall and company are in the process of developing a polysaccharide antagonist to inhibit the proper functioning of egg recognition receptors in the sperm plasma membrane. They believe this antagonist would lead to the sperm not being able to recognizing the egg or the receptor not being able to signal further cell reactions needed to enzymatically attack the matrix of proteins and polysaccharides called the zona pellucida¹ that coats the outside of the egg’s cell membrane. A second line of attack is the development of a contraceptive vaccine based on known antigens that currently account for much of the natural infertility problems in otherwise normal individuals^2,3. The final approach to a male contraceptive, investigated in this report, is the structural analysis of a specific calcium ion channel found in the flagella of mature sperm and the design of an antagonist specific for this channel. Of further note, hormones have been studied in the past for their possible use in this area, but the multiple metabolic pathways affected resulted in side effects that could not be overcome and this line of research has become very limited⁴.

In October of 2000, David Clapham and associates reported on the discovery of a sperm cation channel, CatSper, that is required for sperm motility and fertility⁵. Two key points about this channel appear to make it a strong candidate for a contraceptive and thus motivated this report. First, it was determined that this particular calcium ion channel is only present in the principal piece of the sperm flagella and in no other tissues tested. Second, disruption of the gene responsible for this channel resulted in sperm unable to fertilize intact eggs. Further exploration of the subject, indicates that it will not be easy to turn this channel off. This report explores some of the signaling processes that go into creating a mature sperm capable of fertilizing an egg, how the CatSper channel is believed to be involved in fertilization, and what tactics are underway to use this channel as a target for a male contraceptive.

Sperm must go through a two-step process in order to fertilize an egg: a motility development process and a maturation process called capacitation which includes the acrosome reaction (AR) and hyperactivation of motility. The "potential" for motility is triggered as the sperm cell passes through either the vas deferens or epididymis, but actual progressive motility (forward movement) does not occur until the sperm is in the isthmus of the female reproductive tract or the "reproductive ground" in the case of organisms such as sea urchins^6,7,8. The AR is a reaction that occurs when the sperm and egg are in close proximity. The head of the sperm cell contains a vesicle, the acrosome, which releases enzymes through the process of exocytosis. The release of enzymes and/or the hyperactivation motion of the sperm cell enables it to penetrate the thick zona pellucida coating the egg, leading to cell membrane fusion. Hyperactivation is a motility pattern characterized by increased amplitude and asymmetry of flagella oscillatory movement which leads to a circular motion rather than straight-line motion^9,10. Hyperactivation and the AR usually occur in unison to achieve egg fertilization, but are not controlled by the same signal pathways. Intensive research has been done on the signal paths involved in the AR and hyperactivation; in all cases, signal transduction is believed to be mediated by Ca²⁺ or K⁺ channels.

Without exception, the research acknowledges the difficulty in characterizing the Ca²⁺ channel mechanisms of action. There are three reasons for this. One is that standard patch-clamp methods do not work because of the small size of mature sperm. The second reason is that when studying the developmental precursors of the sperm, spermatogenic cells, which are larger and spherical and lend themselves to electrophysiological study, one ends up studying cells that undergo substantial redistribution and reorganization of protein structures before they are true sperm cells. Finally, little is known of the modifications in protein distribution that takes place during the capacitation process and its effect on ion channel form and function^11,12,13.

With that said, these same papers suggest that there are many different voltage-dependent/operated Ca²⁺ channels (VDCC) / (VOCC) present in sperm cells. The channels fall into one of two categories based on voltage-activation thresholds: low voltage- or high voltage-activated channels (LVA or HVA respectively). Four HVA channels (L, N, R, and P/Q types) and one LVA channel (T type) have been identified. It generally appears that these channels work in a choreographed way to respond to the external environment. The T-type channels are interesting in that they require less depolarization to function and therefore are the first to open. They, along with possible K⁺channel function¹⁴ are believed to initiate most responses involved in motility and capacitation. Figure 1., below, is a proposed model for the signal processes involved in the AR and indicates that the signal is initiated when (ZP3), a zona pellucida glycoprotein, triggers a transient voltage drop (ΔV_M) via the ZP3 receptor (R). This activates the influx of Ca²⁺ (Ca_LVA) through a T-type channel (LVA) and also activates phospholipase C (PLC) to produce inositol 1,4,5-trisphospate, (IP₃). These are the early events of signaling. The IP₃ acts as a secondary messenger to open IP₃ gated Ca²⁺ channels (IP₃R) in the internal Ca²⁺ stores (Ca_s). This is considered the intermediate event of the signaling process. In response to the depletion of these reserves of Ca²⁺, a gated store-operated Ca²⁺ channel (SOC), located on the cell membrane, opens to allow for a continuous influx of Ca²⁺ ions from the external environment. These late events allow for continuous ion influx, which drives the AR.^15,16. These signals are likely important for the AR because the signals affect the cAMP levels that affect the hyperactivity needed for the AR and penetration of the sperm into the egg. Finally, Cl^-, bicarbonate, and pH are believed to play a part in many of these signal interactions.

Figure 1.

Many different toxins, antagonists, pH and Ca²⁺ levels, inhibitors and chemical environments have been used to distinguish between the different channels and signal pathways described above.

Clapham and associates discovered the CatSper channel while they were looking for Ca²⁺ channels in sperm cells of mice. In August of 2001, they reported the discovery of a second Ca²⁺ channel with similar characteristics and designated it the CatSper2 channel¹⁷. In the literature, they continually referred to this channel as the putative channel and one must approach the literature from the point of view that much is still unclear.

Through gene sequence analysis using polymerase chain reaction (PCR) to amplify the quantity of DNA, the full-length sequence of the cDNA for CatSper was determined. This sequence predicts a primary protein structure consistent with a hydropathy plot that indicates the presence of a six-transmembrane (6TM)-spanning repeat integral protein. This protein should be similar in structure to the six-transmembrane-spanning repeats in monomers of voltage-dependent K⁺ (K_v) channels, cyclic-nucleotide-gated (CNG) channels or transient receptor potential (TRP) proteins, but its functional pore region is more reminiscent of a single domain of the four-repeat VDCC’s. Its primary structure indicates the presence of a voltage sensitive (S4) region containing positively charged amino acids (lysine/arginine) every three amino acids in this region, and an abundance of histidine residues in its internal amino terminus indicating possible pH response. pH response, whether from this ion channel or not, has been seen in sperm cells as a variation in motility. Human and mouse sperm display a great deal of conservation in peptide sequence in the pore, terminal and voltage sensitive (S4) regions of the protein and thus it is assumed the mouse model is a good one to study.

Northern blot analysis indicates that the CatSper protein shows up only in the testis and none of the other 15 organs analyzed. Through immunofluorescence and immunogold electron microscopy, it was determined that this protein is located almost solely in what is termed the principal piece on the flagella. Figure 2., below, shows a simple diagram identifying the major components of a sperm cell.

Figure 2.

The CatSper gene was removed from the genome of the experimental mice by homologous recombination in which a noncoding sequence was inserted in place of the CatSper encoding sequence. CatSper (-/-) mutant specimens were indistinguishable from wild-type (+/+) littermates in size, behavior and appearance. Homozygous (-/-) females mated to heterozygous (+/-) or wild-type males were fertile. Mutant male specimens were unable to impregnate wild-type females for up to a nine-month period. In contrast, the wild-type males were 100% fertile.

In order to find the cause of infertility, the team examined live mutant sperm under a light microscope and through computer-assisted sperm analysis verified that the progressive motility was severely decreased. Further study revealed that in vitro these mutant sperm could not fertilize intact eggs, but could fertilize eggs with their zona pellucida removed. Their studies also indicated that CatSper is required for the cAMP- and cGMP-stimulated Ca²⁺ influx in sperm, even though the primary structure of the protein indicates no cyclic-nucleotide-binding regions. The research is inconclusive in determining what the associated structures of this channel are, but it is believed that the CatSper protein is one subunit of a Ca²⁺-permeant channel that is composed of four total subunits; whether these subunits are identical or not is unknown. Because it is gated directly or indirectly by cyclic nucleotides and there is no ligand-gating site on the CatSper monomer, there is probably another associated structure different from the CatSper protein structure associated with, but not detected by the experimental methods employed. The reason for the lack of information is that, although they were able to express the CatSper protein in a heterologous (in vitro) system, cells other than sperm cells where patch-clamp techniques are feasible, they were unable to show that the putative channel formed a functional channel, even in the presence of cAMP or cGMP and the CNG b -subunits known to be associated with and gate these types of channels. The researchers believe this shows that there are other associated proteins required for CatSper channel functionality and that they either did not include these in the heterologous system or they had the required proteins but other structural features present in the principal piece of the flagella were not present in the heterologous system. The heterologous system in which the CatSper protein could be expressed and in which patch-clamp techniques worked but gave no results were Xenopus oocytes, HEK-293 and CHO-K1 cells. It is hypothesized that this channel is an evolutionary transition that occurred before gene duplication resulted in the four-repeat structures of the Ca_v seen in more modern, evolutionary wise, organisms.

How the CatSper-controlled influx of Ca²⁺ then regulates motility is also conjecture. It is possible that the [Ca²⁺]_i triggers Ca²⁺-sensitive adenylyl cyclases to increase cAMP in the cell that is used by motor proteins in the tail. It is theorized that this channel might be part of a feedback loop important to the frequency and asymmetry of the flagellar beating motion during hyperactivation.

Ho and Suarez⁹ support this last contention. Their research focuses on the axoneme, which is the core of the flagella surrounded by outer structures of dense fibers and mitochondria in the midpiece and by a fibrous sheath in the principal piece. By treating sperm with Triton-X100 (a nonionic detergent), they were able to remove all structures except the axoneme and support structures. If ATP and cAMP were present, these spermatozoa structures began swimming and became hyperactivated only on the addition of Ca²⁺. How this signal gets to the axoneme in whole sperm cells is not known. They also concede that there may be Ca²⁺ stores present to activate SOCs that then promote hyperactivation. They have shown that sperm in a Ca²⁺ free environment revert to simple activated motion, which promotes the idea, by others, that it is not the internal Ca²⁺ stores themselves that maintain hyperactivation. Their research shows that proper Ca²⁺ levels are responsible for increasing flagellar asymmetry, characteristic of hyperactivated motility, and cAMP is associated more with the initiation and maintenance of flagellar beats. Figure 3., below, indicates the hypothesized signal paths involved in hyperactivation. This picture places emphasis on a cAMP and cyclic nucleotide-gated (CNG) Ca²⁺ channel that allows external Ca²⁺ in. This signal pathway was not discussed by O’Toole and associates (figure 1). In this diagram, the early and intermediate events of figure 1. signal adenylyl cyclase (AC), located in the membrane, to produce cAMP, which then opens the CNG Ca²⁺ channel. The cAMP also activates protein kinase A (PKA) to phosphorylate structures in the flagella, resulting in flagellar beating. The Ca²⁺-calmodulin complex works to induce the asymmetric beating seen in hyperactivation. The central CNG may be the putative CatSper channel identified by Clapham and associates.

Figure 3.

Aoki and associates studied the relationship between Ca²⁺ and cAMP and found that Ca²⁺ concentrations both inside and outside affect the symmetric / asymmetric motion. Their results show that the external Ca²⁺ concentration has its affect on the flagellar motion via a cAMP-dependant Ca²⁺ pump, which may be the pump that Clapham is looking at.

Initially Clapham and associates were interested in finding a CatSper homolog for functional analysis, as the heterologous system did not yield a functioning channel. They discovered a voltage-gated sodium channel (Na_v) of the bacteria Bacillus halodurans (NaChBac).¹⁸ This channel is a 6TM structure with a pore domain and overall primary structure very similar to a Ca_v channel even though it is selective for Na⁺. It is a good candidate for study because it can be expressed in a heterologous system, nonsperm mammalian cells, and its functional properties studied in detail.¹⁹ The researchers were able to isolate a six-residue (residues 190-195 -- LESWAS) primary structure in the pore region of this channel that imparted ion-selectivity to the channel. By altering residues in this segment using a Quickchange^TM site directed mutagenesis kit, they were able to create both nonselective ion channels and ion channels selective for Ca²⁺. The sequence that created the greatest selectivity for Ca²⁺ was (LDDWAD). This altered channel represents a working model of a single 6TM voltage-gated structure that has not been discovered in mammalian cells. The researchers are hoping that this channel may be a very close homolog of the CatSper channel. Figure 4., below, indicates the primary and tertiary structure of the putative pore and the area where mutations determine pore selectivity.

Figure 4.

So far, there are no antagonists for these channels, but it is assumed that any drug candidates will first be tested on the altered NaChBac channel called CaChBac_m. If the CatSper is as simple as a single 6TM voltage gated protein then it would seem that the antagonist would also have to be simple in structure. It seems that the bad news would be if this channel were not signaled directly by the CatSper protein, but was signaled or gated by some associated protein not confined solely to the sperm cell. Such a situation would make it very difficult to create an antagonist that would not affect other signaling pathways. It appears that more research should be done to determine the identity and function of these associated protein structures. The road to a contraceptive will be a slow cautious process as the scientists involved take the slow steps needed for better understanding of the basic form and functions of the ion channels involved.

Bibliography

1. http://www.s-t.com/daily/10-98/10-02-98/c02he074.htm

2. Aitken, R. J., Immunocontraceptive vaccines for human use, Journal of Reproductive Immunology, 57, 2002, 273-287.

3. Naz, R. K., Application of Sperm Antigens in Immunocontraception, Frontiers in Bioscience 1, e87-95, 1 September 1996.

4. www.malecontraceptives.org/methods/articles/hall_cbs.htm

5. Ren, D., Navarro, B., Perez, G., Jackson, A. C., Hsu, S. Shi, Q., Tilly, J. L., Clapham, D. E., A sperm ion channel required for sperm motility and male fertility, Nature, 413, 603-609, 2001.

6. Darszon, A., Beltran, C., Felix, R., Nishigaki, T., Travino, C., Review Ion Transport in Sperm Signaling, Developmental Biology, 240, 1-14, 2000.

7. Garbers, D. L., Swimming with sperm, Nature, 413, 579-582, 2001.

8. Trevino, C., Serrano, C. J., Beltran, C., Felix, R., Darszon, A., Identification of mouse trp homologs and lipid rafts from spermatogenic cells and sperm, FEBS Letters, 509, Issue 1, 2001, 119-125.

9. Ho, H., Suarez, S., Hyperactivation of mammalian spermatozoa: function and regulation, Reproduction, 2001, 122, 519-26.

10. Aoki, F., Sakai, S., Kohmoto, K., Regulation of Flagellar Bending by camp and Ca²⁺ in Hamster Sperm, Molecular Reproduction and Development 53:77-83, 1999.

11. Serrano, C. J. Trevino, C.L., Felix, R., Darszon, A., Voltage-dependent Ca²⁺ channel subunit expression and immunolocalization in mouse spermatogenic cells and sperm, FEBS Letters 462, 1999, 171-176.

12. Wennemuth, G., Westenbroek, R. E., Xu, T., Hille, B., Babcock, D. F., Ca_v2.2 and Ca_v2.3 (N- and R-type) Ca²⁺ Channels in Depolarization-evoked Entry of Ca²⁺ into Mouse Sperm, The Journal of Biological Chemistry, 275, No. 28, 21210-21217, 2000.

13. Publicover, S. J., Barratt, C.L.R., Opinion – Voltage-operated Ca²⁺ channels and the acrosome reaction: which channels are present and what do they do?, Human Reproduction, 14, no 4, 873-879, 1999.

14. Felix, R., Serrano, C. J., Trevino, C. L., Munoz-Garay, C., Bravo, A., Navarro, A., Pacheco, J., Tsutsumi, V., Darszon, A., Identification of distinct K⁺ channels in mouse spermatogenic cells and sperm, Zygote, 10 (May), 183-188, 2002.

15. O’Toole, C. M. B., Arnoult, C., Darszon, A., Steinhardt, R. A., Florman, H. M., Ca²⁺ Entry through Store-operated Channels in Mouse sperm Is Initiated by Egg ZP3 and Drives the Acrosome Reaction, MBC, 11, issue 5, 1571-1584, 2000.

16. Jungnickel, M. K., Marrero, J., Birnbaumer, L., Lemos, J. R., Florman, H. M., Trp2 regulates entry of Ca²⁺ into mouse sperm triggered by egg ZP3, Nature Cell Biology, 3, 2001 499-502.

17. Quill, T. A., Ren, D., Clapham, D. E., Farbers, D. L., A voltage-gated ion channel expressed specifically in spermatozoa, PNAS, 98, no. 22, 12527-12531, 2001.

18. Ren, D., Navarro, B., Xu, H., Yue, L., Shi, Q., Clapham, D. E., A Prokaryotic Voltage-Gated Sodium Channel, Science, 294, 2373-2375, 2001.

19. Yue, L., Navarro, B., Ren, D., Ramos, A., Clapham, D. E., The Cation Selectivity Filter of the Bacterial Sodium Channel, NaChBac, Journal of General Physiology -http://Clapham.

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