Cyclic Nucleotide-Specific Optogenetics Highlights Compartmentalization of the Sperm Flagellum into cAMP Microdomains.

Inside the female genital tract, mammalian sperm undergo a maturation process called capacitation, which primes the sperm to navigate across the oviduct and fertilize the egg. Sperm capacitation and motility are controlled by 3′,5′-cyclic adenosine monophosphate (cAMP). Here, we show that optogenetics, the control of cellular signaling by genetically encoded light-activated proteins, allows to manipulate cAMP dynamics in sperm flagella and, thereby, sperm capacitation and motility by light. To this end, we used sperm that express the light-activated phosphodiesterase LAPD or the photo-activated adenylate cyclase bPAC. The control of cAMP by LAPD or bPAC combined with pharmacological interventions provides spatiotemporal precision and allows to probe the physiological function of cAMP compartmentalization in mammalian sperm

Re-visiting the Protamine-2 locus: deletion, but not haploinsufficiency, renders male mice infertile

Protamines are arginine-rich DNA-binding proteins that replace histones in elongating spermatids. This leads to hypercondensation of chromatin and ensures physiological sperm morphology, thereby protecting DNA integrity. In mice and humans, two protamines, protamine-1 (Prm1) and protamine-2 (Prm2) are expressed in a species-specific ratio. In humans, alterations of this PRM1/PRM2 ratio is associated with subfertility. By applying CRISPR/Cas9 mediated gene-editing in oocytes, we established Prm2-deficient mice. Surprisingly, heterozygous males remained fertile with sperm displaying normal head morphology and motility. In Prm2-deficient sperm, however, DNA-hypercondensation and acrosome formation was severely impaired. Further, the sperm displayed severe membrane defects resulting in immotility. Thus, lack of Prm2 leads not only to impaired histone to protamine exchange and disturbed DNA-hypercondensation, but also to severe membrane defects resulting in immotility. Interestingly, previous attempts using a regular gene-targeting approach failed to establish Prm2-deficient mice. This was due to the fact that already chimeric animals generated with Prm2+/− ES cells were sterile. However, the Prm2-deficient mouse lines established here clearly demonstrate that mice tolerate loss of one Prm2 allele. As such they present an ideal model for further studies on protamine function and chromatin organization in murine sperm

Molecular mechanism underlying the action of zona-pellucida glycoproteins on mouse sperm

Mammalian oocytes are enveloped by the zona pellucida (ZP), an extracellular matrix of glycoproteins. In sperm, stimulation with ZP proteins evokes a rapid Ca2+ influx via the sperm-specific, pH-sensitive Ca2+ channel CatSper. However, the physiological role and molecular mechanisms underlying ZP-dependent activation of CatSper are unknown. Here, we delineate the sequence of ZP-signaling events in mouse sperm. We show that ZP proteins evoke a rapid intracellular pH i increase that rests predominantly on Na+/H+ exchange by NHA1 and requires cAMP synthesis by the soluble adenylyl cyclase sAC as well as a sufficiently negative membrane potential set by the spem-specific K+ channel Slo3. The alkaline-activated CatSper channel translates the ZP-induced pH i increase into a Ca2+ response. Our findings reveal the molecular components underlying ZP action on mouse sperm, opening up new avenues for understanding the basic principles of sperm function and, thereby, mammalian fertilization

Rotational motion and rheotaxis of human sperm do not require functional CatSper channels and transmembrane Ca2+ signaling.

Navigation of sperm in fluid flow, called rheotaxis, provides long-range guidance in the mammalian oviduct. The rotation of sperm around their longitudinal axis (rolling) promotes rheotaxis. Whether sperm rolling and rheotaxis require calcium (Ca2+ ) influx via the sperm-specific Ca2+ channel CatSper, or rather represent passive biomechanical and hydrodynamic processes, has remained controversial. Here, we study the swimming behavior of sperm from healthy donors and from infertile patients that lack functional CatSper channels, using dark-field microscopy, optical tweezers, and microfluidics. We demonstrate that rolling and rheotaxis persist in CatSper-deficient human sperm. Furthermore, human sperm undergo rolling and rheotaxis even when Ca2+ influx is prevented. Finally, we show that rolling and rheotaxis also persist in mouse sperm deficient in both CatSper and flagellar Ca2+ -signaling domains. Our results strongly support the concept that passive biomechanical and hydrodynamic processes enable sperm rolling and rheotaxis, rather than calcium signaling mediated by CatSper or other mechanisms controlling transmembrane Ca2+ flux

Sperm sensory signaling

Fertilization is exceptionally complex and, depending on the species, happens in entirely different environments. External fertilizers in aquatic habitats, like marine invertebrates or fish, release their gametes into the seawater or freshwater, whereas sperm from most internal fertilizers like mammals cross the female genital tract to make their way to the egg. Various chemical and physical cues guide sperm to the egg. Quite generally, these cues enable signaling pathways that ultimately evoke a cellular Ca2+ response that modulates the waveform of the flagellar beat and, hence, the swimming path. To cope with the panoply of challenges to reach and fertilize the egg, sperm from different species have developed their own unique repertoire of signaling molecules and mechanisms. Here, we review the differences and commonalities for sperm sensory signaling in marine invertebrates (sea urchin), fish (zebrafish), and mammals (mouse, human)

How to control cyclic nucleotide signaling by light

Highlights - Optogenetics controls cyclic nucleotide signaling with spatio-temporal resolution. - Genetic-engineering creates new tools by combining photosensory and effector domains. - Optogenetic control of cyclic nucleotide signaling controls physiological functions. Abstract Optogenetics allows to non-invasively manipulate cellular functions with spatio-temporal precision by combining genetic engineering with the control of protein function by light. Since the discovery of channelrhodopsin has pioneered the field, the optogenetic toolkit has been ever expanding and allows now not only to control neuronal activity by light, but rather a multitude of other cellular functions. One important application that has been established in recent years is the light-dependent control of second messenger signaling. The optogenetic toolkit now allows to control cyclic nucleotide-dependent signaling by light in vitro and in vivo

Elucidating cyclic AMP signaling in subcellular domains with optogenetic tools and fluorescent biosensors

The second messenger 3′,5′-cyclic nucleoside adenosine monophosphate (cAMP) plays a key role in signal transduction across prokaryotes and eukaryotes. Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision. To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited. Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively. In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell. This review provides an overview about optogenetic tools and biosensors to shed light on the subcellular organization of cAMP signaling