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SummaryPreterm birth is a multifactorial syndrome that is detrimental to the well-being of both the mother and the newborn. During normal gestation, the myometrium is maintained in a quiescent state by the action of progesterone. As a steroid hormone, progesterone is thought to modify uterine and placental morphology by altering gene expression, but a nongenomic mode of action has long been suspected. Here we reveal that progesterone activates both human and murine inwardly rectifying potassium channel Kir7.1, which is expressed in mammalian myometrial smooth muscle and placental pericytes during late gestation. Kir7.1 is also activated by compounds used to prevent premature labor, including the progestogens 17-alpha-hydroxyprogesterone caproate and dydrogesterone, revealing an unexpected mode of action for these drugs. Our results reveal that Kir7.1 is the molecular target of a number of endogenous and synthetic steroids that control uterine excitability and placental function, and is therefore a promising therapeutic target to control utero-placental physiology and support healthy pregnancy.

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm, providing the highest-resolution structural information to date. Our subtomogram averages reveal mammalian sperm-specific protein complexes within the microtubules, the radial spokes and nexin–dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm-specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.

Tmem95 encodes a sperm acrosomal membrane protein, whose knockout has a male-specific sterility phenotype in mice. How TMEM95 plays a role in membrane fusion of sperm and eggs has remained elusive. Here, we utilize a sperm penetration assay as a model system to investigate the function of human TMEM95. We show that human TMEM95 binds to hamster egg membranes, providing evidence for a TMEM95 receptor on eggs. Using X-ray crystallography, we reveal an evolutionarily conserved, positively charged region of TMEM95 as a putative receptor-binding surface. Amino-acid substitutions within this region of TMEM95 ablate egg-binding activity. We identify monoclonal antibodies against TMEM95 that reduce the number of human sperm fused with hamster eggs in sperm penetration assays. Strikingly, these antibodies do not block binding of sperm to eggs. Taken together, these results provide strong evidence for a specific, receptor-mediated interaction of sperm TMEM95 with eggs and suggest that this interaction may have a role in facilitating membrane fusion.Significance statementMembrane fusion of sperm and eggs is pivotal in sexual reproduction. Tmem95 knockout mice show male-specific sterility, but it was unknown how sperm TMEM95 facilitates membrane fusion with eggs. We show here that human TMEM95 binds eggs. Our crystal structure of TMEM95 suggests a region where this binding may occur. We develop monoclonal antibodies against TMEM95 that impair sperm-egg fusion but do not block sperm-egg binding. Thus, we propose that there is a receptor-mediated interaction of sperm TMEM95 with eggs, and that this interaction may have a direct role in membrane fusion. Our work suggests avenues for the identification of the TMEM95 egg receptor and may enable the development of infertility treatments and contraceptives for humans.

Sperm motility is necessary for successful fertilization, but there remains controversy about whether human sperm motility is primarily powered by glycolysis or oxidative phosphorylation. To evaluate the plausibility of reducing human sperm mitochondrial ATP production as an avenue for contraceptive development, we treated human sperm with small-molecule mitochondrial uncouplers, which reduce mitochondrial membrane potential by inducing passive proton flow, and evaluated the effects on a variety of physiological processes that are critical for fertilization. We also sought to clarify the subcellular localization of Adenosine Nucleotide Translocator 4 (ANT4), a gamete-specific protein that has been suggested as a contraceptive target. We determined that ANT4 is mitochondrially localized, that induced mitochondrial uncoupling can be partially mediated by the ANT family, and that two uncouplers, Niclosamide Ethanolamine and BAM15, significantly decreased sperm progressive motility. However, these uncouplers did not reduce sperm ATP content or impair other physiological processes, implying that human sperm can rely on glycolysis for ATP production in the absence of functional mitochondria. Thus, since certain mitochondrial uncouplers impair motility through ATP-independent mechanisms, they could be useful ingredients in on-demand, vaginally-applied contraceptives. However, systemically delivered contraceptives that target sperm mitochondria to reduce their ATP production would need to be paired with sperm-specific glycolysis inhibitors.Significance StatementDevelopment of novel contraceptives is critical, since half of all pregnancies are still unplanned, even in developed countries. This high unplanned pregnancy rate contributes to a wide variety of social, environmental, and ecological problems. Impairing human sperm is a way to develop male and unisex contraceptives, but much remains unknown about these unique cells. Here we settle a long-running debate about human sperm metabolism, finding that human sperm can maintain their ATP levels without mitochondrial oxidative phosphorylation. This finding will help focus future contraceptive development efforts. We also identify the potential use of an FDA-approved compound (Niclosamide) as a motility-impairing ingredient in spermicides and correct the misunderstood subcellular localization of an existing contraceptive target, Adenosine Nucleotide Translocator 4.

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm axonemes, providing the highest resolution structural information to date. Our subtomogram averages reveal mammalian sperm- specific protein complexes within the outer microtubule doublets, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm- specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm, providing the highest-resolution structural information to date. Our subtomogram averages reveal mammalian sperm-specific protein complexes within the microtubules, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm-specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.

Mammalian female fertility is defined by a successful and strictly periodic ovarian cycle, which is under the control of gonadotropins and steroid hormones, particularly progesterone and estrogen. The latter two are produced by the ovaries that are engaged in controlled follicular growth, maturation, and release of the eggs, i.e., ovulation. The steroid hormones regulate ovarian cycles via genomic signaling, by altering gene transcription and protein synthesis. However, despite this well-studied mechanism, steroid hormones can also signal via direct, non-genomic action, by binding to their membrane receptors. Here we show, that the recently discovered membrane progesterone receptor α/β hydrolase domain-containing protein 2 (ABHD2) is highly expressed in mammalian ovaries where the protein plays a novel regulatory role in follicle maturation and the sexual cycle of females. Ablation of Abhd2 caused a dysregulation of the estrous cycle rhythm with females showing shortened luteal stages while remaining in the estrus stage for a longer time. Interestingly, the ovaries of Abhd2 knockout (KO) females resemble polycystic ovary morphology (PCOM) with a high number of atretic antral follicles that could be rescued with injection of gonadotropins. Such a procedure also allowed Abhd2 KO females to ovulate a significantly increased number of mature and fertile eggs in comparison with their wild-type littermates. These results suggest a novel regulatory role of ABHD2 as an important factor in non-genomic steroid regulation of the female reproductive cycle.

The choroid plexus (CP) epithelium secretes cerebrospinal fluid and plays an important role in healthy homeostasis of the brain. CP function can be influenced by sex steroid hormones; however, the precise molecular mechanism of such regulation is not well understood. Here, using whole-cell patch-clamp recordings from male and female murine CP cells, we show that application of progesterone resulted in specific and strong potentiation of the inwardly rectifying potassium channel Kir7.1, an essential protein that is expressed in CP and is required for survival. The potentiation was progesterone specific and independent of other known progesterone receptors expressed in CP. This effect was recapitulated with recombinant Kir7.1, as well as with endogenous Kir7.1 expressed in the retinal pigment epithelium. Current-clamp studies further showed a progesterone-induced hyperpolarization of CP cells. Our results provide evidence of a progesterone-driven control of tissues in which Kir7.1 is present.

The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca2+ gateway into the sperm flagellum. However, CatSper’s complex subunit composition has impeded its reconstitution in vitro and structural elucidation. Here, we applied cryo-electron tomography to visualize the macromolecular organization of the native CatSper channel complex in intact mammalian sperm, as well as identified three additional CatSper-associated proteins. The repeating CatSper units form long zigzag-rows in four nanodomains along the flagella. In both mouse and human sperm, each CatSper repeat consists of a tetrameric pore complex. Murine CatSper contains an additional outwardly directed wing-structure connected to the tetrameric channel. The majority of the extracellular domains form a canopy above each pore-forming channel that interconnects to a zigzag-shaped roof. The intracellular domains link two neighboring channel complexes to a diagonal array. The loss of this intracellular link in Efcab9-/- sperm distorts the longitudinally aligned zigzag pattern and compromises flagellar movement. This work offers unique insights into the mechanisms underlying the assembly and transport of the CatSper complex to generate the nanodomains and provides a long-sought structural basis for understanding CatSper function in the regulation of sperm motility.

The choroid plexus (CP) is an epithelial tissue primarily responsible for the secretion of the cerebrospinal fluid (CSF). Choroid plexuses are found in each of the four brain ventricles: two laterals, third and fourth. They ensure continuous production of CSF to provide nutrients, remove waste products and provide a mechanical buffer to protect the brain. Tight junctions in the CP epithelium form a barrier between the blood plasma and the CSF, which allow channels and transporters in the CP to establish a highly regulated concentration gradient of ions between the two fluids, thereby controlling the composition of CSF. CP plays an important part in healthy brain homeostasis, as its failure to maintain adequate CSF perfusion is implicated in Alzheimer's disease and traumatic brain injury. And yet, the physiology of CP and the mechanism of its age-related functional decline is one of the most understudied areas of neurobiology. Here, we describe a protocol to isolate and identify individual choroid plexus epithelial cells (CPEC) from murine brain for whole-cell patch-clamp recordings and ion channel identification. Using the recording from the inwardly rectifying potassium channel K

The choroid plexus (CP) is an epithelial tissue primarily responsible for the secretion of the cerebrospinal fluid (CSF). Choroid plexuses are found in each of the four brain ventricles: two laterals, third and fourth. They ensure continuous production of CSF to provide nutrients, remove waste products and provide a mechanical buffer to protect the brain. Tight junctions in the CP epithelium form a barrier between the blood plasma and the CSF, which allow channels and transporters in the CP to establish a highly regulated concentration gradient of ions between the two fluids, thereby controlling the composition of CSF. CP plays an important part in healthy brain homeostasis, as its failure to maintain adequate CSF perfusion is implicated in Alzheimer's disease and traumatic brain injury. And yet, the physiology of CP and the mechanism of its age-related functional decline is one of the most understudied areas of neurobiology. Here, we describe a protocol to isolate and identify individual choroid plexus epithelial cells (CPEC) from murine brain for whole-cell patch-clamp recordings and ion channel identification. Using the recording from the inwardly rectifying potassium channel Kir7.1 and TRPM3 that are abundant in CP, we demonstrate a technique to study the regulators of ion channels in the choroid plexus.

The mammalian flagellum is a specific type of motile cilium required for sperm motility and male fertility. Effective flagellar movement is dependent on axonemal function, which in turn relies on proper ion homeostasis within the flagellar compartment. This ion homeostasis is maintained by the concerted function of ion channels and transporters that initiate signal transduction pathways resulting in motility changes. Advances in electrophysiology and super-resolution microscopy have helped to identify and characterize new regulatory modalities of the mammalian flagellum. Here, we discuss what is currently known about the regulation of flagellar ion channels and transporters that maintain sodium, potassium, calcium, and proton homeostasis. Identification of new regulatory elements and their specific roles in sperm motility is imperative for improving diagnostics of male infertility.

Therian female fertility is defined by a successful and strictly periodic ovarian cycle, which is under the control of gonadotropins and steroid hormones, particularly progesterone and estrogen. The latter two are produced by the ovaries that are engaged in controlled follicular growth, maturation and release of the eggs, i.e. ovulation. It is well known that steroid hormones regulate ovarian cycles via genomic signaling, by altering gene transcription and protein synthesis. However, despite this well-studied mechanism, steroid hormones can also signal via direct, non-genomic action, by binding to their membrane receptors. Here we show, that the recently discovered sperm membrane progesterone receptor α/β hydrolase domain-containing protein 2 (ABHD2) is highly expressed in mammalian ovaries where the protein plays a novel regulatory role in follicle maturation and the sexual cycle of females. Ablation ofAbhd2caused a dysregulation of the estrous cycle rhythm with females showing shortened luteal stages while remaining in the estrus stage for a longer time. Interestingly, the ovaries ofAbhd2knockout (KO) females resemble polycystic ovary morphology with a high number of atretic antral follicles that could be rescued with injection of gonadotropins. Such a procedure also allowedAbhd2KO females to ovulate a significantly increased number of mature and fertile eggs in comparison to their wild-type littermates. These results suggest a novel regulatory role of ABHD2 as an important factor in non-genomic steroid regulation of the female reproductive cycle.

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.

Summary: 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. : Miller et al. use super-resolution microscopy, electrophysiology, and electron microscopy to show that the sperm proton channel Hv1 forms bilateral lines positioned asymmetrically down the sperm flagellum. Hv1 inhibition leads to a decrease in sperm rotation, suggesting an important role for this channel in sperm motility. Keywords: hydrogen voltage-gated channel 1, Hv1, CatSper, STORM, super-resolution imaging, ion channels, rotation, sperm flagellum

Author(s): Lishko, Polina V; Mannowetz, Nadja; Mundt, Nadine; Miller, Melissa; Kenny, Samuel; Xu, Ke; Bjoerkgren, Ida T

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).

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/β 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.

SummaryVarying 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 calciumbinding domain-containing protein 9 (EFCAB9) as a dual function, cytoplasmic machine modulating the channel activity and the domain organization of CatSper. Knockout mice studies demonstrate that EFCAB9, in complex with the CatSper subunit, CAT SPERζ, 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.HighlightsEfcab9encodes an evolutionarily conserved, sperm-specific EF-hand domain proteinEfcab9-deficient mice have sperm motility defects and reduced male fertilityEFCAB9 is a pH-tuned Ca2+sensor for flagellar CatSper Ca2+channelEFCAB9 is a dual function machine in gatekeeping and domain organization of CatSper

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.

Author(s): Mannowetz, Nadja; Miller, Melissa R; Kenny, Samuel J; Mansell, Steven A; Wojcik, Michal; Zucker, Robert S; Xu, Ke; Lishko, Polina V

In the letter by Brenker et al. (1), the authors assessed actions of various steroids toward the human calcium channel of sperm (CatSper). The experiments, carried out with calcium imaging, are minimally supplemented by electrophysiology. The authors show that all tested steroids activate CatSper with different efficiencies. However, the plant triterpenoids pristimerin and lupeol fail to interfere with calcium influx. These data contradict our previously published results (2). CatSper is the principal calcium channel of spermatozoa that mediates calcium influx into the sperm tail. When studying CatSper with electrophysiology, inward currents (influx of positive ions into the cell) are of particular interest. However, outward currents (efflux of positive ions) are elicited only under nonphysiological potentials and are not necessarily CatSper-mediated. To study CatSper inward currents, a driving force must be generated experimentally that is chemical and/or … [↵][1]1To whom correspondence should be addressed. Email: lishko{at}berkeley.edu. [1]: #xref-corresp-1-1

STUDY QUESTION Does a man (patient 1) with a previously described deficiency in principle cation channel of sperm (CatSper) function have a mutation in the CatSper-epsilon (CATSPERE) and/or CatSper-zeta (CATSPERZ) gene? SUMMARY ANSWER Patient 1 has a homozygous in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE. WHAT IS KNOWN ALREADY CatSper is the principal calcium channel of mammalian spermatozoa. Spermatozoa from patient 1 had a specific loss of CatSper function and were unable to fertilize at IVF. Loss of CatSper function could not be attributed to genetic abnormalities in coding regions of seven CatSper subunits. Two additional subunits (CatSper-epsilon (CATPSERE) and CatSper-zeta (CATSPERZ)) were recently identified, and are now proposed to contribute to the formation of the mature channel complex. STUDY DESIGN, SIZE, DURATION This was a basic medical research study analysing genomic data from a single patient (patient 1) for defects in CATSPERE and CATSPERZ. PARTICIPANTS/MATERIALS, SETTING, METHODS The original exome sequencing data for patient 1 were analysed for mutations in CATSPERE and CATSPERZ. Sanger sequencing was conducted to confirm the presence of a rare variant. MAIN RESULTS AND THE ROLE OF CHANCE Patient 1 is homozygous for an in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE that is predicted to be highly deleterious. LIMITATIONS, REASONS FOR CAUTION The nature of the molecular deficit caused by the rs761237686 variant and whether it is exclusively responsible for the loss of CatSper function remain to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS Population genetics are available for a significant number of predicted deleterious variants of CatSper subunits. The consequence of homozygous and compound heterozygous forms on sperm fertilization potential could be significant. Selective targeting of CatSper subunit expression maybe a feasible strategy for the development of novel contraceptives. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by project grants from the MRC (MR/K013343/1 and MR/012492/1), Chief Scientist Office/NHS research Scotland. This work was also supported by NIH R01GM111802, Pew Biomedical Scholars Award 00028642 and Packer Wentz Endowment Will to P.V.L. C.L.R.B is the editor-in-chief of Molecular Human Reproduction, has received lecturing fees from Merck and Ferring, and is on the Scientific Advisory Panel for Ohana BioSciences. C.L.R.B was chair of the World Health Organization Expert Synthesis Group on Diagnosis of Male infertility (2012–2016).

Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens. DOI: http://dx.doi.org/10.7554/eLife.24888.001, eLife digest Seminal plasma, the fluid portion of semen, helps to transport sperm cells to the egg during sexual reproduction. Seminal plasma contains numerous proteins that help the sperm to survive and, in recent years, researchers discovered that it also harbours protein deposits known as amyloid fibrils. Such protein deposits are generally associated with neurodegenerative diseases such as Alzheimer's and Parkinson’s disease, where a build-up of fibrils can damage the nervous system. Semen amyloids, however, are present in the absence of disease, but can boost infection by HIV and other sexually transmitted viruses, by shuttling virus particles to their target cells. Despite these damaging effects, some researchers had suggested that amyloids in semen could be beneficial for humans, though it was unclear what these benefits might be. Roan et al. now set out to assess how semen amyloids affect human sperm activity. The results show that semen amyloids bind to damaged sperm cells and immobilize them, which are then quickly cleared away by immune cells. This could ensure that only the fittest sperm cells reach the egg. These findings suggest that amyloids can potentially serve beneficial roles for reproduction. A next step will be to investigate how semen amyloids trap unwanted sperm and how immune cells know when to remove it. More research is needed to investigate if problems in these processes could lead to infertility in men. DOI: http://dx.doi.org/10.7554/eLife.24888.002

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.

Author(s): Bjorkgren, I; Lishko, PV | Abstract: Since their discovery in late 1970, transient receptor potential (TRP) channels have been implicated in a variety of cellular and physiological functions (Minke, 2010). The superfamily of TRP channels consists of nearly 30 members that are organized into seven major subgroups based on their specic function and sequence similarities (Owsianik et al., 2006; Ramsey et al., 2006). With the exception of TRPN channels that are only found in invertebrates and sh, mammalian genomes contain representatives of all six subfamilies: (1) TRPV (vanilloid); (2) TRPC (canonical); (3) TRPM (melastatin); (4) TRPA (ankyrin); (5) TRPML (mucolipin); and (6) TRPP (polycystin). TRP channels play crucial regulatory roles in many physiological processes, including those associated with reproductive tissues. As calcium-permeable cation channels that respond to a variety of signals (Clapham et al., 2003; Wu et al., 2010), TRP channels exert their role as sensory detectors in both male and female gametes, and play regulatory functions in germ cell development and maturation. Recent evidence obtained from Caenorhabditis elegans studies point to the importance of these proteins during fertilization where certain sperm TRP channels could migrate from a spermatozoon into an egg to ensure successful fertilization and embryo development. In this chapter we discuss how TRP channels can regulate both female and male fertility in different species and their specic roles.

The mir-34/449 family consists of six homologous miRNAs at three genomic loci. Redundancy of miR-34/449 miRNAs and their dominant expression in multiciliated epithelia suggest a functional significance in ciliogenesis. Here we report that mice deficient for all miR-34/449 miRNAs exhibited postnatal mortality, infertility and strong respiratory dysfunction caused by defective mucociliary clearance. In both mouse and Xenopus, miR-34/449-deficient multiciliated cells (MCCs) exhibited a significant decrease in cilia length and number, due to defective basal body maturation and apical docking. The effect of miR-34/449 on ciliogenesis was mediated, at least in part, by post-transcriptional repression of Cp110, a centriolar protein suppressing cilia assembly. Consistent with this, cp110 knockdown in miR-34/449-deficient MCCs restored ciliogenesis by rescuing basal body maturation and docking. Altogether, our findings elucidate conserved cellular and molecular mechanisms through which miR-34/449 regulate motile ciliogenesis.

Key physiological functions of organisms, such as sensory transduction, regulation of heart rate, smooth muscle contraction, bile secretion, endocrine regulation, immune responses, and various pathophysiological conditions, including neuropathic pain, diabetes, kidney failure, and cancer, are

© 2015 American Chemical Society. The basal fungus Allomyces macrogynus (A. Macrogynus) produces motile male gametes displaying well-studied chemotaxis toward their female counterparts. This chemotaxis is driven by sirenin, a sexual pheromone released by the female gametes. The pheromone evokes a large calcium influx in the motile gametes, which could proceed through the cation channel of sperm (CatSper) complex. Herein, we report the total synthesis of sirenin in 10 steps and 8% overall yield and show that the synthetic pheromone activates the CatSper channel complex, indicated by a concentration-dependent increase in intracellular calcium in human sperm. Sirenin activation of the CatSper channel was confirmed using whole-cell patch clamp electrophysiology with human sperm. Based on this proficient synthetic route and confirmed activation of CatSper, analogues of sirenin can be designed as blockers of the CatSper channel that could provide male contraceptive agents.

Ion channels control the sperm ability to fertilize the egg by regulating sperm maturation in the female reproductive tract and by triggering key sperm physiological responses required for successful fertilization such as hyperactivated motility, chemotaxis, and the acrosome reaction. CatSper, a pH-regulated, calcium-selective ion channel, and KSper (Slo3) are core regulators of sperm tail calcium entry and sperm hyperactivated motility. Many other channels had been proposed as regulating sperm activity without direct measurements. With the development of the sperm patch-clamp technique, CatSper and KSper have been confirmed as the primary spermatozoan ion channels. In addition, the voltage-gated proton channel Hv1 has been identified in human sperm tail, and the P2X2 ion channel has been identified in the midpiece of mouse sperm. Mutations and deletions in sperm-specific ion channels affect male fertility in both mice and humans without affecting other physiological functions. The uniqueness of sperm ion channels makes them ideal pharmaceutical targets for contraception. In this review we discuss how ion channels regulate sperm physiology.

Upon ejaculation, mammalian spermatozoa have to undergo a sequence of physiological transformations within the female reproductive tract that will allow them to reach and fertilize the egg. These include initiation of motility, hyperactivation of motility and perhaps chemotaxis toward the egg, and culminate in the acrosome reaction that permits sperm to penetrate the protective vestments of the egg. These physiological responses are triggered through the activation of sperm ion channels that cause elevations of sperm intracellular pH and Ca(2+) in response to certain cues within the female reproductive tract. Despite their key role in sperm physiology and their absolute requirement for the process of fertilization, sperm ion channels remain poorly understood due to the extreme difficulty in application of the patch-clamp technique to spermatozoa. This review covers the topic of sperm ion channels in the following order: first, we discuss how the intracellular Ca(2+) and pH signaling mediated by sperm ion channels controls sperm behavior during the process of fertilization. Then, we briefly cover the history of the methodology to study sperm ion channels, which culminated in the recent development of a reproducible whole-cell patch-clamp technique for mouse and human cells. We further discuss the main approaches used to patch-clamp mature mouse and human spermatozoa. Finally, we focus on the newly discovered sperm ion channels CatSper, KSper (Slo3) and HSper (H(v)1), identified by the sperm patch-clamp technique. We conclude that the patch-clamp technique has markedly improved and shifted our understanding of the sperm ion channels, in addition to revealing significant species-specific differences in these channels. This method is critical for identification of the molecular mechanisms that control sperm behavior within the female reproductive tract and make fertilization possible.

Steroid hormone progesterone released by cumulus cells surrounding the egg is a potent stimulator of human spermatozoa. It attracts spermatozoa towards the egg and helps them penetrate the egg's protective vestments. Progesterone induces Ca(2+) influx into spermatozoa and triggers multiple Ca(2+)-dependent physiological responses essential for successful fertilization, such as sperm hyperactivation, acrosome reaction and chemotaxis towards the egg. As an ovarian hormone, progesterone acts by regulating gene expression through a well-characterized progesterone nuclear receptor. However, the effect of progesterone upon transcriptionally silent spermatozoa remains unexplained and is believed to be mediated by a specialized, non-genomic membrane progesterone receptor. The identity of this non-genomic progesterone receptor and the mechanism by which it causes Ca(2+) entry remain fundamental unresolved questions in human reproduction. Here we elucidate the mechanism of the non-genomic action of progesterone on human spermatozoa by identifying the Ca(2+) channel activated by progesterone. By applying the patch-clamp technique to mature human spermatozoa, we found that nanomolar concentrations of progesterone dramatically potentiate CatSper, a pH-dependent Ca(2+) channel of the sperm flagellum. We demonstrate that human CatSper is synergistically activated by elevation of intracellular pH and extracellular progesterone. Interestingly, human CatSper can be further potentiated by prostaglandins, but apparently through a binding site other than that of progesterone. Because our experimental conditions did not support second messenger signalling, CatSper or a directly associated protein serves as the elusive non-genomic progesterone receptor of sperm. Given that the CatSper-associated progesterone receptor is sperm specific and structurally different from the genomic progesterone receptor, it represents a promising target for the development of a new class of non-hormonal contraceptives.

Elevations of sperm intracellular pH and Ca2+ regulate sperm motility, chemotaxis, capacitation and the acrosome reaction, and play a vital role in the ability of the sperm cell to reach and fertilise the egg. In human spermatozoa, the flagellar voltage-gated proton channel Hv1 is the main H+ extrusion pathway that controls sperm intracellular pH, and the pH-dependent flagellar Ca2+ channel CatSper is the main pathway for Ca2+ entry as measured by the whole-cell patch clamp technique. Hv1 and CatSper channels are co-localized within the principal piece of the sperm flagellum. Hv1 is dedicated to proton extrusion from flagellum and is activated by membrane depolarisation, an alkaline extracellular environment, the endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. The CatSper channel is strongly potentiated by intracellular alkalinisation. Since Hv1 and CatSper channels are located in the same subcellular domain, proton extrusion via Hv1 channels should induce intraflagellar alkalinisation and activate CatSper ion channels. Therefore the combined action of Hv1 and CatSper channels in human spermatozoa can induce elevation of both intracellular pH and Ca2+ required for sperm activation in the female reproductive tract. Here, we discuss how Hv1 and CatSper channels regulate human sperm physiology and the differences in control of sperm intracellular pH and Ca2+ between species.

SummaryHuman spermatozoa are quiescent in the male reproductive system and must undergo activation once introduced into the female reproductive tract. This process is known to require alkalinization of sperm cytoplasm, but the mechanism responsible for transmembrane proton extrusion has remained unknown because of the inability to measure membrane conductance in human sperm. Here, by successfully patch clamping human spermatozoa, we show that proton channel Hv1 is their dominant proton conductance. Hv1 is confined to the principal piece of the sperm flagellum, where it is expressed at unusually high density. Robust flagellar Hv1-dependent proton conductance is activated by membrane depolarization, an alkaline extracellular environment, endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. Hv1 allows only outward transport of protons and is therefore dedicated to inducing intracellular alkalinization and activating spermatozoa. The importance of Hv1 for sperm activation makes it an attractive target for controlling male fertility.PaperFlick

Transient Receptor Potential (TRP) proteins are cation channels composed of a transmembrane domain flanked by large N- and C-terminal cytoplasmic domains. All members of the vanilloid family of TRP channels (TRPV) possess an N-terminal ankyrin repeat domain (ARD). The ARD of mammalian TRPV6, an important regulator of calcium uptake and homeostasis, is essential for channel assembly and regulation. The 1.7 Å crystal structure of the TRPV6-ARD reveals conserved structural elements unique to the ARDs of TRPV proteins. First, a large twist between the fourth and fifth repeats is induced by residues conserved in all TRPV ARDs. Second, the third finger loop is the most variable region in sequence, length and conformation. In TRPV6, a number of putative regulatory phosphorylation sites map to the base of this third finger. Size exclusion chromatography and crystal packing indicate that the TRPV6-ARD does not assemble as a tetramer and is monomeric in solution. Adenosine triphosphate-agarose and calmodulin-agarose pull-down assays show that the TRPV6-ARD does not interact with either ligand, indicating a different functional role for the TRPV6-ARD than in the paralogous thermosensitive TRPV1 channel. Similar biochemical findings are also presented for the highly homologous mammalian TRPV5-ARD. The implications of the structural and biochemical data on the role of the ankyrin repeats in different TRPV channels are discussed.

Calcium ion channels that determine many of the properties of cilia are different in motile cilia as compared to primary cilia and flagella.

During transit through the female reproductive tract, mammalian spermatozoa are exposed to increasing concentrations of progesterone (P4) released by the cumulus oophorus. P4 triggers massive calcium influx into human sperm through activation of the sperm-specific calcium channel CatSper. These properties of human spermatozoa are thought to be unique since CatSper is not progesterone sensitive in rodent sperm. Here, by performing patch clamp recording from spermatozoa from rhesus macaque for the first time, we report that they express P4-sensitive CatSper channel identically to human sperm and react to P4 by inducing responsiveness to zona pellucida, unlike human sperm, which respond directly to P4. We have also determined the physiologic levels of P4 capable of inducing capacitation-associated changes in macaque sperm. Progesterone (1 μM) induced up to a 3-fold increase in the percentage of sperm undergoing the zona pellucida-induced acrosome reaction with the lowest threshold as low as 10 nM of P4. Submicromolar levels of P4 induced a dose-dependent increase in curvilinear velocity and lateral head displacement, while sperm protein tyrosine phosphorylation was not altered. Macaque spermatozoa exposed to 10 μM of P4 developed fully hyperactivated motility. Similar to human sperm, on approaching cumulus mass and binding to zona pellucida, macaque spermatozoa display hyperactivation and undergo an acrosome reaction that coincides with the rise in the sperm intracellular calcium. Taken together, these data indicate that P4 accelerates the completion of capacitation and provides evidence of spermatozoa "priming" as they move into a gradient of progesterone in search for the oocyte.