Dual Sensing of Physiologic pH and Calcium by EFCAB9 Regulates Sperm Motility.

Varying pH of luminal fluid along the female reproductive tract is a physiological cue that modulates sperm motility. CatSper is a sperm-specific, pH-sensitive calcium channel essential for hyperactivated motility and male fertility. Multi-subunit CatSper channel complexes organize linear Ca2+ signaling nanodomains along the sperm tail. Here, we identify EF-hand calcium-binding domain-containing protein 9 (EFCAB9) as a bifunctional, cytoplasmic machine modulating the channel activity and the domain organization of CatSper. Knockout mice studies demonstrate that EFCAB9, in complex with the CatSper subunit, CATSPERζ, is essential for pH-dependent and Ca2+-sensitive activation of the CatSper channel. In the absence of EFCAB9, sperm motility and fertility is compromised, and the linear arrangement of the Ca2+ signaling domains is disrupted. EFCAB9 interacts directly with CATSPERζ in a Ca2+-dependent manner and dissociates at elevated pH. These observations suggest that EFCAB9 is a long-sought, intracellular, pH-dependent Ca2+ sensor that triggers changes in sperm motility.

Single-cell Motility Analysis of Tethered Human Spermatozoa.

Vigorous sperm flagellar motility is essential for fertilization, and so the quantitative measurement of motility is a useful tool to assess the intrinsic fertility potential of sperm cells and explore how various factors can alter sperm's ability to reach the egg and penetrate its protective layers. Human sperm beat their flagella many times each second, and so recording and accurately quantifying this movement requires a high-speed camera. The aim of this protocol is to provide a detailed description of the tools required for quantitative beat frequency measurement of tethered human sperm at the single-cell level and to describe methods for investigating the effects of intracellular or extracellular factors on flagellar motion. This assay complements bulk measurements of sperm parameters using commercially-available systems for computer-assisted sperm analysis (CASA).

Asymmetrically Positioned Flagellar Control Units Regulate Human Sperm Rotation.

Ion channels control sperm navigation within the female reproductive tract and, thus, are critical for their ability to find and fertilize an egg. The flagellar calcium channel CatSper controls sperm hyperactivated motility and is dependent on an alkaline cytoplasmic pH. The latter is accomplished by either proton transporters or, in human sperm, via the voltage-gated proton channel Hv1. To provide concerted regulation, ion channels and their regulatory proteins must be compartmentalized. Here, we describe flagellar regulatory nanodomains comprised of Hv1, CatSper, and its regulatory protein ABHD2. Super-resolution microscopy revealed that Hv1 is distributed asymmetrically within bilateral longitudinal lines and that inhibition of this channel leads to a decrease in sperm rotation along the long axis. We suggest that specific distribution of flagellar nanodomains provides a structural basis for the selective activation of CatSper and subsequent flagellar rotation. The latter, together with hyperactivated motility, enhances the fertility of sperm.

CatSper: A Unique Calcium Channel of the Sperm Flagellum.

To overcome egg protective vestments and ensure successful fertilization, mammalian spermatozoa switch symmetrical progressive motility to a powerful, whip-like flagellar motion, known as hyperactivation. The latter is triggered by a calcium influx through the sperm-specific, voltage-dependent, and alkalization-activated calcium channel of sperm - CatSper. The channel comprises nine subunits which together form a heteromeric complex. CatSper-deficient male mice and men with mutations in CatSper genes are infertile. This calcium channel is regulated by various endogenous compounds, such as steroids, prostaglandins, endocannabinoids, and intracellular pH. Being a sperm-specific ion channel that is not expressed anywhere else in the body, CatSper represents an ideal target for the development of female and even male contraceptives. In this review, we discuss the recent advances in studying CatSper functional properties and discuss future steps that are required to take in order to achieve a deep understanding of the molecular basis of CatSper function.

Regulation of the sperm calcium channel CatSper by endogenous steroids and plant triterpenoids.

The calcium channel of sperm (CatSper) is essential for sperm hyperactivated motility and fertility. The steroid hormone progesterone activates CatSper of human sperm via binding to the serine hydrolase ABHD2. However, steroid specificity of ABHD2 has not been evaluated. Here, we explored whether steroid hormones to which human spermatozoa are exposed in the male and female genital tract influence CatSper activation via modulation of ABHD2. The results show that testosterone, estrogen, and hydrocortisone did not alter basal CatSper currents, whereas the neurosteroid pregnenolone sulfate exerted similar effects as progesterone, likely binding to the same site. However, physiological concentrations of testosterone and hydrocortisone inhibited CatSper activation by progesterone. Additionally, testosterone antagonized the effect of pregnenolone sulfate. We have also explored whether steroid-like molecules, such as the plant triterpenoids pristimerin and lupeol, affect sperm fertility. Interestingly, both compounds competed with progesterone and pregnenolone sulfate and significantly reduced CatSper activation by either steroid. Furthermore, pristimerin and lupeol considerably diminished hyperactivation of capacitated spermatozoa. These results indicate that (i) pregnenolone sulfate together with progesterone are the main steroids that activate CatSper and (ii) pristimerin and lupeol can act as contraceptive compounds by averting sperm hyperactivation, thus preventing fertilization.

Unconventional endocannabinoid signaling governs sperm activation via the sex hormone progesterone.

Steroids regulate cell proliferation, tissue development, and cell signaling via two pathways: a nuclear receptor mechanism and genome-independent signaling. Sperm activation, egg maturation, and steroid-induced anesthesia are executed via the latter pathway, the key components of which remain unknown. Here, we present characterization of the human sperm progesterone receptor that is conveyed by the orphan enzyme α/ß hydrolase domain-containing protein 2 (ABHD2). We show that ABHD2 is highly expressed in spermatozoa, binds progesterone, and acts as a progesterone-dependent lipid hydrolase by depleting the endocannabinoid 2-arachidonoylglycerol (2AG) from plasma membrane. The 2AG inhibits the sperm calcium channel (CatSper), and its removal leads to calcium influx via CatSper and ensures sperm activation. This study reveals that progesterone-activated endocannabinoid depletion by ABHD2 is a general mechanism by which progesterone exerts its genome-independent action and primes sperm for fertilization.

Normal Fertility Requires the Expression of Carbonic Anhydrases II and IV in Sperm.

HCO3 (-) is a key factor in the regulation of sperm motility. High concentrations of HCO3 (-) in the female genital tract induce an increase in sperm beat frequency, which speeds progress of the sperm through the female reproductive tract. Carbonic anhydrases (CA), which catalyze the reversible hydration of CO2 to HCO3 (-), represent potential candidates in the regulation of the HCO3 (-) homeostasis in sperm and the composition of the male and female genital tract fluids. We show that two CA isoforms, CAII and CAIV, are distributed along the epididymal epithelium and appear with the onset of puberty. Expression analyses reveal an up-regulation of CAII and CAIV in the different epididymal sections of the knockout lines. In sperm, we find that CAII is located in the principal piece, whereas CAIV is present in the plasma membrane of the entire sperm tail. CAII and CAIV single knockout animals display an imbalanced HCO3 (-) homeostasis, resulting in substantially reduced sperm motility, swimming speed, and HCO3 (-)-enhanced beat frequency. The CA activity remaining in the sperm of CAII- and CAIV-null mutants is 35% and 68% of that found in WT mice. Sperm of the double knockout mutant mice show responses to stimulus by HCO3 (-) or CO2 that were delayed in onset and reduced in magnitude. In comparison with sperm from CAII and CAIV double knockout animals, pharmacological loss of CAIV in sperm from CAII knockout animals, show an even lower response to HCO3 (-). These results suggest that CAII and CAIV are required for optimal fertilization.

Slo1 is the principal potassium channel of human spermatozoa.

Mammalian spermatozoa gain competence to fertilize an oocyte as they travel through the female reproductive tract. This process is accompanied by an elevation of sperm intracellular calcium and a membrane hyperpolarization. The latter is evoked by K(+) efflux; however, the molecular identity of the potassium channel of human spermatozoa (hKSper) is unknown. Here, we characterize hKSper, reporting that it is regulated by intracellular calcium but is insensitive to intracellular alkalinization. We also show that human KSper is inhibited by charybdotoxin, iberiotoxin, and paxilline, while mouse KSper is insensitive to these compounds. Such unique properties suggest that the Slo1 ion channel is the molecular determinant for hKSper. We show that Slo1 is localized to the sperm flagellum and is inhibited by progesterone. Inhibition of hKSper by progesterone may depolarize the spermatozoon to open the calcium channel CatSper, thus raising [Ca(2+)] to produce hyperactivation and allowing sperm to fertilize an oocyte. DOI:http://dx.doi.org/10.7554/eLife.01009.001.

Disruption of the principal, progesterone-activated sperm Ca2+ channel in a CatSper2-deficient infertile patient.

The female steroid hormone progesterone regulates ovulation and supports pregnancy, but also controls human sperm function within the female reproductive tract. Progesterone causes elevation of sperm intracellular Ca(2+) leading to sperm hyperactivation, acrosome reaction, and perhaps chemotaxis toward the egg. Although it has been suggested that progesterone-dependent Ca(2+) influx into human spermatozoa is primarily mediated by cationic channel of sperm (CatSper), the principal flagellar Ca(2+) channel of sperm, conclusive loss-of-function genetic evidence for activation of CatSper by progesterone has yet to be provided. Moreover, it is not clear whether the responsiveness of CatSper to progesterone is an innate property of human spermatozoa or is acquired as the result of exposure to the seminal plasma. Here, by recording ionic currents from spermatozoa of an infertile CatSper-deficient patient, we demonstrate that CatSper is indeed the principal Ca(2+) channel of human spermatozoa, and that it is strongly potentiated by progesterone. In addition, by recording CatSper currents from human epididymal and testicular spermatozoa, we show that CatSper sensitivity to progesterone arises early in sperm development and increases gradually to a peak when spermatozoa are ejaculated. These results unambiguously establish an important role of CatSper channel in human sperm nongenomic progesterone signaling and demonstrate that the molecular mechanism responsible for activation of CatSper by progesterone arises early in sperm development concurrently with the CatSper channel itself.

Glucose is a pH-dependent motor for sperm beat frequency during early activation.

To reach the egg in the ampulla, sperm have to travel along the female genital tract, thereby being dependent on external energy sources and substances to maintain and raise the flagellar beat. The vaginal fluid is rich in lactate, whereas in the uterine fluid glucose is the predominant substrate. This evokes changes in the lactate content of sperm as well as in the intracellular pH (pH(i)) since sperm possess lactate/proton co-transporters. It is well documented that glycolysis yields ATP and that HCO(3)- is a potent factor in the increase of beat frequency. We here show for the first time a pathway that connects both parts. We demonstrate a doubling of beat frequency in the mere presence of glucose. This effect can reversibly be blocked by 2-deoxy-D-glucose, dichloroacetate and aminooxyacetate, strongly suggesting that it requires both glycolysis and mitochondrial oxidation of glycolytic end products. We show that the glucose-mediated acceleration of flagellar beat and ATP production are hastened by a pH(i) ≥7.1, whereas a pH(i) ≤7.1 leaves both parameters unchanged. Since we observed a diminished rise in beat frequency in the presence of specific inhibitors against carbonic anhydrases, soluble adenylyl cyclase and protein kinase, we suggest that the glucose-mediated effect is linked to CO(2) hydration and thus the production of HCO(3)- by intracellular CA isoforms. In summary, we propose that, in sperm, glycolysis is an additional pH(i)-dependent way to produce HCO(3)-, thus enhancing sperm beat frequency and contributing to fertility.

Basigin interacts with both MCT1 and MCT2 in murine spermatozoa.

Lactate is provided to spermatogenic cells by Sertoli cells as an energy substrate and its transport is regulated by H(+)-monocarboxylate co-transporters (MCTs). In the case of several cell types it is known that MCT1 is associated with basigin and MCT2 with embigin. Here we demonstrate co-localization and co-immunoprecipitation of basigin with both MCT1 and MCT2 in sperm, whereas no interaction with embigin was detectable. An investigation of the functional activity of MCT proteins revealed that it was mainly the application of L-lactate which resulted in a decrease in pH(i) . The pH(i) changes were blocked with α-cyano-4-OH cinnamate and the preference for L-lactate-as opposed to D-Lactate-was demonstrated by the determination of ATP after exposure to both lactate isomers. We propose that basigin interacts with MCT1 and MCT2 to locate them properly in the membrane of spermatogenic cells and that this may enable sperm to utilize lactate as an energy substrate contributing to cell survival.

Role of carbonic anhydrase IV in the bicarbonate-mediated activation of murine and human sperm.

HCO(3) (-) is the signal for early activation of sperm motility. In vivo, this occurs when sperm come into contact with the HCO(3) (-) containing fluids in the reproductive tract. The activated motility enables sperm to travel the long distance to the ovum. In spermatozoa HCO(3) (-) stimulates the atypical sperm adenylyl cyclase (sAC) to promote the cAMP-mediated pathway that increases flagellar beat frequency. Stimulation of sAC may occur when HCO(3) (-) enters spermatozoa either directly by anion transport or indirectly via diffusion of CO(2) with subsequent hydration by intracellular carbonic anhydrase (CA). We here show that murine sperm possess extracellular CA IV that is transferred to the sperm surface as the sperm pass through the epididymis. Comparison of CA IV expression by qRT PCR analysis confirms that the transfer takes place in the corpus epididymidis. We demonstrate murine and human sperm respond to CO(2) with an increase in beat frequency, an effect that can be inhibited by ethoxyzolamide. Comparing CA activity in sperm from wild-type and CA IV(-/-) mice we found a 32.13% reduction in total CA activity in the latter. The CA IV(-/-) sperm also have a reduced response to CO(2). While the beat frequency of wild-type sperm increases from 2.86±0.12 Hz to 6.87±0.34 Hz after CO(2) application, beat frequency of CA IV(-/-) sperm only increases from 3.06±0.20 Hz to 5.29±0.47 Hz. We show, for the first time, a physiological role of CA IV that supplies sperm with HCO(3) (-), which is necessary for stimulation of sAC and hence early activation of spermatozoa.

Expression of proteinase-activated receptor-2 (PAR2) is androgen-dependent in stromal cell line (hPCPs) from benign prostatic hyperplasia.

BACKGROUND: Growth properties of the prostate are regulated by a variety of hormones and growth factors. Benign prostatic hyperplasia (BPH) is characterized by abnormal epithelial and stromal proliferation. Varying androgen hormone levels in elderly men are correlated with abnormal proliferations of the prostate. Proteinase-activated receptor-2 (PAR2), a subtype of G-protein-coupled receptors, is known to induce multiple biological processes. It could also play a key role in the proliferation and metastasis of prostate cancer, but its effect on BPH pathogenesis is to a great extent unknown. METHODS: Localization of PAR2 was determined both in pathologically altered and in normal prostate tissues by using immunohistochemical techniques. PAR2 activity was assessed by measuring changes in intracellular calcium [Ca(2+)](i) following stimulation of cultured stromal cells with a PAR2 agonist (trypsin) and a synthetic PAR2-activating peptide (AP). DHT-dependence of PAR2 expression in prostate cancer and prostatic stromal cell lines was examined with semi-quantitative and quantitative PCR. Cultured stromal cells (hPCPs) were stimulated with PAR2 AP and cell proliferation was determined through [(3)H]-thymidine incorporation. RESULTS: In comparison to normal prostate, PAR2 expression was increased in BPH stroma. DHT induced a higher expression of PAR2 when sub-physiological DHT-levels were used. Higher levels of DHT produced reduced PAR2 expression. A mitogenic effect was induced by applying PAR2 AP to hPCPs-cells. CONCLUSIONS: In conclusion, we found that PAR2 expression is hormone-dependent in prostatic stromal cells with a negative correlation and we consider it to be an important factor in mitogenesis in BPH.

Protein kinase CK2 and new binding partners during spermatogenesis.

Protein kinase CK2 is an ubiquitously expressed enzyme that is absolutely necessary for the survival of cells. Besides the holoenzyme consisting of the regulatory ß-subunit and the catalytic α- or α'-subunit, the subunits exist in separate forms. The subunits bind to a number of other cellular proteins. We show the expression of individual subunits as well as interaction with the transitional nuclear protein TNP1 and with the motor neuron protein KIF5C during spermatogenesis. TNP1 is a newly identified binding partner of the α-subunit of CK2. CK2α and KIF5C were found in late spermatogenesis, whereas CK2ß and TNP1 were found in early spermatogenesis. CK2α, CK2α', TNP1, and KIF5C were detected in the acrosome of spermatozoa, while CK2ß was detectable in the mid-piece. Combinations of CK2 subunits might determine interactions with other proteins during spermatogenesis. KIF5C as a kinesin motor neuron protein is probably involved in the redistribution of proteins during spermatogenesis.

ABCG2 is expressed in late spermatogenesis and is associated with the acrosome.

An increasingly exploited strategy for the isolation of stem cells is based on the increased efflux of Hoechst 33342 lipophilic dye mediated by ABCG2, an ATP-binding cassette transporter which is highly expressed in various stem cells. We found ABCG2 expression to be present at later stages of spermatogenesis. Western blot analysis using an anti-ABCG2 antibody revealed expression of a 72kDa band in mature sperm obtained from mice, rats, bulls or humans. Immunocytochemistry studies revealed acrosomal staining pattern of ABCG2 in spermatozoa. Experiments using the Hoechst 33342 ABCG2 substrate and the ABCG2-specific inhibitor FTC demonstrated efflux activity of ABCG2 in mature sperm. Incubation of sperm in capacitating medium in the presence of the ABCG2-inhibitor FTC resulted in decreased cholesterol depletion compared to sperm incubated in the absence of FTC. Our results demonstrate that ABCG2 is expressed at the acrosome in mature sperm. ABCG2 may thus serve to mediate cholesterol removal.