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The human voltage-gated proton channel H_v1 plays an important physiological role in male fertility in humans. Specifically it is integral for regulation of the internal pH of the sperm cell. The pH of the sperm cytoplasm is a major regulator of sperm cell motility, capacitation, hyperctivation and the acrosome reaction. Each of these processes are required for fertilization of the egg.

In the epididymis (where the sperm are stored pre-ejaculation in the male reproductive tract) the sperm cells are quiescent and their cytoplasmic pH is low (around 6.8) (Hamamah et al., 1996). This makes sense, unnecessary metabolic activity would waste energy and damage the cells physically since they would be thrashing around and bumping into each other. Also if they were active they would require lots of energy and this metabolic output from the mitochondria would generate reactive oxygen species (ROSs) that would damage the precious DNA. Remember that sperm are in essence DNA delivery cells and damage of this precious genetic material from poor ROS regulation is one of the leading causes of male infertility (Aitken & Curry, 2011). For these reasons the sperm cells remain quiescent in the male reproductive system, however in order to find the egg the sperm cells have to activate and start swimming. How does this process take place? What is the signal for activation?

In most cells, signalling takes place by a ligand binding to a receptor and through a chain of events altering the internal state of the cell (gene expression) or the electronic state of the membrane (ligand gated ion channels). However, most cells in the body don’t move around much or at all, they stay in their tissue, or if they do circulate they don’t leave the body. Sperms cells, of course, are different. Their normal physiological role involves the powerful ejection from the body into the completely foreign environment of the female reproductive system. Conceptually it is equivalent to taking a cell swimming in the ocean and throwing it into fresh water (but not quite so traumatic). This in and of itself is a powerful signal, exposing the cells to a vast new array of ligands and instantaneously changing the electrical state of the membrane, without even having to open any ion channels. The sperm cell takes advantage of this signal to quickly snap out of its quiescence and start swimming.

In human sperm cells, one of the first responses occurs just before ejaculation when the quiescent sperm cells from the epididymis are mixed with the seminal plasma. The seminal plasma has a much higher pH than that of the epididymis and this causes the first proton dump from the sperm cell cytoplasm through the opening of H_v channels, raising the cytoplasmic pH of the sperm cell to >;7.0 (Hamamah et al., 1996). In this way the sperm cells are primed to start swimming as they enter the female reproductive system.

Now inside the vaginal canal the sperm cells begin to under go the process of capacitation and hyperactivation. It was known for a long time that one of the first steps in the capacitation process was the dumping of protons out of the sperm cytoplasm a.k.a. alkalinization (Hamamah & Gatti, 1998; Giroux-Widemann et al., 1991). However, it wasn’t know until the ground breaking work of Lishko et al. that this proton dump in human sperm cells was facilitated by H_v. This role of H_v seems to be specific to human sperm cells. In past studies, when the electrical conductance of mouse sperm cells were investigated by whole-cell patch-clamping no voltage-gated proton current was observed (Kirichok et al., 2006). Further research needs to be carried out to examine whether H_v may play an important role in the fertility of other non-human species. As early as 1983 a voltage-gated proton channel was postulated in the activation of bovine spermatozoa, but to my knowledge this hypothesis has not been followed up by direct electrophysiological measurement (Babcock et al., 1983).

The technical break through that allowed the role of H_v currents to be discovered in human sperm was that Lishko et al. were the first ever able to patch onto the membrane of the human sperm cell, giving them complete electrical control. Beyond the hitherto unexpected role of H_v, what they discovered shed light onto many aspects of human sperm cell physiology and contrasted significantly from what we thought we knew from mouse spermatozoa electrophysiology.

One important discovery was a potential mechanisms for the role Zn²⁺ in sperm cell activation. Since the very early characterization of H_v current it was show that Zn²⁺ is a a potent inhibitor of H_v channels (Mahaut-Smith, 1989). It is telling that the highest concentration of Zn²⁺ in the human body is found in the seminal fluid (Saaranen et al., 1987). High Zn²⁺ would prevent premature capacitation and removal of Zn²⁺ would promote proton dumping from the sperm cytoplasm. It has been demonstrated in rats that once entering the female reproductive tract the Zn²⁺ is rapidly diluted (Gunn & Gould, 1958). Furthermore, the Zn²⁺ would be sequestered by the high concentration of albumin (a protein that is a strong Zn²⁺ chelator) in the vaginal mucus (Lu et al., 2008). This disinhibition of H_v allows for the changes in cytoplasmic pH required for the proper activity of the sperm cells.

There is also evidence that a lipid derived hormone, anandamide present in the human reproductive system (Scheul & Burkman, 2005) may have a direct activating influence on H_v channels (Lishko et al., 2010). Although this finding needs to be followed up experimentally the possibility of a pharmacological agent acting directly on the H_v channel is a very exciting prospect that could have major impact on future electrophysiological and biophysical work.

As the sperm travels up the female reproductive tract first through the cervix then into the fillopian tube the pH of the surroundings steadily increases (Eggert-Kruse et al., 1993). This results in further dumping of protons and hyperactivation of the sperm cell, characterized by a more powerful ‘whip-like’ beating of the sperm cell’s tail that generates greater swimming force (Suarez, 2008). This allows the sperms cells to penetrate the thicker mucus of the zona pelicula that surrounds the egg cell.

Once the sperm’s long journey comes to an end and it arrives at the egg the acrosome reaction is triggered. The acrosome is a large golgi derived vacuole that is located at the very tip of the sperm cell’s head. This reaction results in the fusing of the membranes of the two cells and the release of the acrosome’s contents. The enzymes in the acrosome help break down the tough coating of the egg and allow for fetillization. It is know that sperm cell cytoplasmic pH is central to, but may not be solely responsible for, initiating the acrosome reaction and it is not difficult to imagine that in human sperm cells H_v also provides the main proton dump required (Cross & Razy-Faulkner, 1997).

As you can imagine with H_v playing such an important role in sperm cell activation, many people have begun to suggest the channel as a potential target for contraceptive agents. Essentially, by blocking the current of these channels it may be possible to prevent the proper activation of the spermatozoa and hence abrogate fertilization of the egg. In addition, genetic defects in H_v have been suggested as a possible cause for some types of male infertility. A human genetic study of infertile individuals could greatly enhance our understanding of H_v‘s role in the sperm cell.

Of course many other ion channels and pumps are important in these processes. Interestingly, a number of these membrane proteins are expressed only in the spermatozoa, like CatSper and Slo3 for example. The levels of intracellular Ca²⁺ and cyclic-AMP, as well as protein phosphorylation and membrane cholesterol levels, also play very important regulatory roles. However, I decided to focus solely on the H_v channel for this post since cytoplasmic pH is a global regulator of sperm cell activation. Also H_v is the channel that I work on and thus it is close to my heart. If you would like to read more about the role of ion channels in sperm cell activation I recommend the excellent recent review by Lishko et al. entitled “The Control of male fertility by spermatozoan ion channels.”

In future blogs I intent to revisit this review article to express my opinions on the state of our understanding of this important physiological process.

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Work Cited in this Post

Aitken, R. J., & Curry, B. J. (2011). Redox regulation of human sperm function: from the physiological control of sperm capacitation to the etiology of infertility and DNA damage in the germ line. Antioxidants & redox signaling, 14(3), 367–381. doi:10.1089/ars.2010.3186

Babcock, D. F., Rufo, G. A., & Lardy, H. A. (1983). Potassium-dependent increases in cytosolic pH stimulate metabolism and motility of mammalian sperm. Proceedings of the National Academy of Sciences, 80(5), 1327–1331.

Cross, N. L., & Razy-Faulkner, P. (1997). Control of human sperm intracellular pH by cholesterol and its relationship to the response of the acrosome to progesterone. Biology of reproduction, 56(5), 1169–1174.

Eggert-Kruse, W., Köhler, A., Rohr, G., & Runnebaum, B. (1993). The pH as an important determinant of sperm-mucus interaction. Fertility and sterility, 59(3), 617–628.

GUNN, S. A., & GOULD, T. C. (1958). Role of zinc in fertility and fecundity in the rat. The American journal of physiology, 193(3), 505–508.

Hamamah, S., & Gatti, J. L. (1998). Role of the ionic environment and internal pH on sperm activity. Human Reproduction, 13(suppl 4), 20–30. doi:10.1093/humrep/13.suppl_4.20

Hamamah, S., Magnoux, E., Royere, D., Barthelemy, C., Dacheux, J. L., & Gatti, J. L. (1996). Internal pH of human spermatozoa: effect of ions, human follicular fluid and progesterone. Molecular human reproduction, 2(4), 219–224.

Kirichok, Y., Navarro, B., & Clapham, D. E. (2006). Whole-cell patch-clamp measurements of spermatozoa reveal an alkaline-activated Ca2+ channel. Nature, 439(7077), 737–740. doi:10.1038/nature04417

Lishko, P. V., Botchkina, I. L., Fedorenko, A., & Kirichok, Y. (2010). Acid extrusion from human spermatozoa is mediated by flagellar voltage-gated proton channel. Cell, 140(3), 327–337. doi:10.1016/j.cell.2009.12.053

Lishko, P. V., Kirichok, Y., Ren, D., Navarro, B., Chung, J.-J., & Clapham, D. E. (2012). The control of male fertility by spermatozoan ion channels. Annual review of physiology, 74, 453–475. doi:10.1146/annurev-physiol-020911-153258

Lu, J., Stewart, A. J., Sadler, P. J., Pinheiro, T. J. T., & Blindauer, C. A. (2008). Albumin as a zinc carrier: properties of its high-affinity zinc-binding site. Biochemical Society transactions, 36(Pt 6), 1317–1321. doi:10.1042/BST0361317

Mahaut-Smith, M. (1989). The effect of zinc on calcium and hydrogen ion currents in intact snail neurones. Journal of Experimental Biology.

Saaranen, M., Suistomaa, U., Kantola, M., Saarikoski, S., & Vanha-Perttula, T. (1987). Lead, magnesium, selenium and zinc in human seminal fluid: comparison with semen parameters and fertility. Human reproduction (Oxford, England), 2(6), 475–479.

Schuel, H., & Burkman, L. J. (2005). A tale of two cells: endocannabinoid-signaling regulates functions of neurons and sperm. Biology of reproduction, 73(6), 1078–1086. doi:10.1095/biolreprod.105.043273

Suarez, S. S. (2008). Control of hyperactivation in sperm. Human Reproduction Update, 14(6), 647–657. doi:10.1093/humupd/dmn029

Widemann, V. G., & Jouannet, P. (1991). Effects of pH on the reactivation of human spermatozoa demembranated with triton X-100 – Giroux-Widemann – 2005 – Molecular Reproduction and Development – Wiley Online Library. Molecular ….

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