Purinergic Signaling in Spermatogenesis.

Adenosine triphosphate (ATP) serves as the essential source of cellular energy. Over the last two decades, however, ATP has also attracted increasing interest as an extracellular signal that activates purinergic plasma membrane receptors of the P2 family. P2 receptors are divided into two types: ATP-gated nonselective cation channels (P2X) and G protein-coupled receptors (P2Y), the latter being activated by a broad range of purine and pyrimidine nucleotides (ATP, ADP, UTP, and UDP, among others). Purinergic signaling mechanisms are involved in numerous physiological events and pathophysiological conditions. Here, we address the growing body of evidence implicating purinergic signaling in male reproductive system functions. The life-long generation of fertile male germ cells is a highly complex, yet mechanistically poorly understood process. Given the relatively sparse innervation of the testis, spermatogenesis relies on both endocrine control and multi-directional paracrine communication. Therefore, a detailed understanding of such paracrine messengers, including ATP, is crucial to gain mechanistic insight into male reproduction.⁠.

Integrity of and damage to wings, feather vanes and serrations in barn owls.

The silent flight of owls is well known. It has served as role model for the designs of new airplane wings and ventilators. One of the structural features that underlies silent flight is the serrated leading edge of the wing that is mainly formed by the tenth primary flight feather (P10). We examined here how much the wings, the P10 feather and the serrations in different populations of barn owls reflect the intact situation. First, when the P10 feather molts, no or fewer serrations are present. Furthermore, damage to feathers and serrations may occur. Damage may be due to several factors like broken feather tips, lost rami, barbules, or broken tips of serrations. The latter may cause a narrowing of the outer vane of the P10 feather. We quantitatively assessed damage by counting the number of wings with missing or broken primary feathers, the number of wings with a narrowed outer vane of the P10 feather, and the number of serrations with reduced length. Considerable damage occurred on wings and feathers on both the macroscopic and microscopic levels. The observed damage most likely influences flight performance. More damage occurred in Galapagos barn owls than in North American and European barn owls. The Galapagos population may be more vulnerable than the other populations because it may at least temporarily be in a bad nutritional state and, thus, postpone molt.

Testicular adenosine acts as a pro-inflammatory molecule: role of testicular peritubular cells.

Extracellular ATP has been described to be involved in inflammatory cytokine production by human testicular peritubular cells (HTPCs). The ectonucleotidases ENTPD1 and NT5E degrade ATP and have been reported in rodent testicular peritubular cells. We hypothesized that if a similar situation exists in human testis, ATP metabolites may contribute to cytokine production. Indeed, ENTPD1 and NT5E were found in situ and in vitro in HTPCs. Malachite green assays confirmed enzyme activities in HTPCs. Pharmacological inhibition of ENTPD1 (by POM-1) significantly reduced pro-inflammatory cytokines evoked by ATP treatment, suggesting that metabolites of ATP, including adenosine, are likely involved. We focused on adenosine and detected three of the four known adenosine receptors in HTPCs. One, A2B, was also found in situ in peritubular cells of human testicular sections. The A2B agonist BAY60-6583 significantly elevated levels of IL6 and CXCL8, a result also obtained with adenosine and its analogue NECA. Results of siRNA-mediated A2B down-regulation support a role of this receptor. In mouse peritubular cells, in contrast to HTPCs, all four of the known adenosine receptors were detected; when challenged with adenosine, cytokine expression levels significantly increased. Organotypic short-term testis cultures yielded comparable results and indicate an overall pro-inflammatory action of adenosine in the mouse testis. If transferable to the in vivo situation, our results may implicate that interference with the generation of ATP metabolites or interference with adenosine receptors could reduce inflammatory events in the testis. These novel insights may provide new avenues for treatment of sterile inflammation in male subfertility and infertility.

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells.

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.

ATP activation of peritubular cells drives testicular sperm transport.

Spermatogenesis, the complex process of male germ cell proliferation, differentiation, and maturation, is the basis of male fertility. In the seminiferous tubules of the testes, spermatozoa are constantly generated from spermatogonial stem cells through a stereotyped sequence of mitotic and meiotic divisions. The basic physiological principles, however, that control both maturation and luminal transport of the still immotile spermatozoa within the seminiferous tubules remain poorly, if at all, defined. Here, we show that coordinated contractions of smooth muscle-like testicular peritubular cells provide the propulsive force for luminal sperm transport toward the rete testis. Using a mouse model for in vivo imaging, we describe and quantify spontaneous tubular contractions and show a causal relationship between peritubular Ca2+ waves and peristaltic transport. Moreover, we identify P2 receptor-dependent purinergic signaling pathways as physiological triggers of tubular contractions both in vitro and in vivo. When challenged with extracellular ATP, transport of luminal content inside the seminiferous tubules displays stage-dependent directionality. We thus suggest that paracrine purinergic signaling coordinates peristaltic recurrent contractions of the mouse seminiferous tubules to propel immotile spermatozoa to the rete testis.

As sperm develop in the testis, the immature cells must make their way through a maze of small tubes known as seminiferous tubules. However, at this stage, the cells do not yet move the long tails that normally allow them to 'swim'; it is therefore unclear how they are able to move through the tubules. Now, Fleck, Kenzler et al. have showed that, in mice, muscle-like cells within the walls of seminiferous tubules can create waves of contractions that push sperm along. Further experiments were then conducted on cells grown in the laboratory. This revealed that a signaling molecule called ATP orchestrates the moving process by activating a cascade of molecular events that result in contractions. Fleck, Kenzler et al. then harnessed an advanced microscopy technique to demonstrate that this mechanism occurs in living mice. Together, these results provide a better understanding of how sperm mature, which could potentially be relevant for both male infertility and birth control.

TRPV4 is the temperature-sensitive ion channel of human sperm.

Ion channels control the ability of human sperm to fertilize the egg by triggering hyperactivated motility, which is regulated by membrane potential, intracellular pH, and cytosolic calcium. Previous studies unraveled three essential ion channels that regulate these parameters: (1) the Ca2+ channel CatSper, (2) the K+ channel KSper, and (3) the H+ channel Hv1. However, the molecular identity of the sperm Na+ conductance that mediates initial membrane depolarization and, thus, triggers downstream signaling events is yet to be defined. Here, we functionally characterize DSper, the Depolarizing Channel of Sperm, as the temperature-activated channel TRPV4. It is functionally expressed at both mRNA and protein levels, while other temperature-sensitive TRPV channels are not functional in human sperm. DSper currents are activated by warm temperatures and mediate cation conductance, that shares a pharmacological profile reminiscent of TRPV4. Together, these results suggest that TRPV4 activation triggers initial membrane depolarization, facilitating both CatSper and Hv1 gating and, consequently, sperm hyperactivation.

Distinct purinergic signaling pathways in prepubescent mouse spermatogonia.

Spermatogenesis ranks among the most complex, yet least understood, developmental processes. The physiological principles that control male germ cell development in mammals are notoriously difficult to unravel, given the intricate anatomy and complex endo- and paracrinology of the testis. Accordingly, we lack a conceptual understanding of the basic signaling mechanisms within the testis, which control the seminiferous epithelial cycle and thus govern spermatogenesis. Here, we address paracrine signal transduction in undifferentiated male germ cells from an electrophysiological perspective. We identify distinct purinergic signaling pathways in prepubescent mouse spermatogonia, both in vitro and in situ. ATP-a dynamic, widespread, and evolutionary conserved mediator of cell to cell communication in various developmental contexts-activates at least two different spermatogonial purinoceptor isoforms. Both receptors operate within nonoverlapping stimulus concentration ranges, display distinct response kinetics and, in the juvenile seminiferous cord, are uniquely expressed in spermatogonia. We further find that spermatogonia express Ca(2+)-activated large-conductance K(+) channels that appear to function as a safeguard against prolonged ATP-dependent depolarization. Quantitative purine measurements additionally suggest testicular ATP-induced ATP release, a mechanism that could increase the paracrine radius of initially localized signaling events. Moreover, we establish a novel seminiferous tubule slice preparation that allows targeted electrophysiological recordings from identified testicular cell types in an intact epithelial environment. This unique approach not only confirms our in vitro findings, but also supports the notion of purinergic signaling during the early stages of spermatogenesis.