http://web.stanford.edu/group/dlab/media/papers/Gradinaru%20cell%202010.pdf

Molecular and Cellular Approaches for Diversifying and Extending Optogenetics

Spatial and temporal control over chemical and biological processes plays a key role in life, where the whole is often much more than the sum of its parts. Quite trivially, the molecules of a cell do not form a living system if they are only arranged in a random fashion. If we want to understand these relationships and especially the problems arising from malfunction, tools are necessary that allow us to design sophisticated experiments that address these questions. Highly valuable in this respect are external triggers that enable us to precisely determine where, when, and to what extent a process is started or stopped. Light is an ideal external trigger: It is highly selective and if applied correctly also harmless. It can be generated and manipulated with well-established techniques, and many ways exist to apply light to living systems--from cells to higher organisms. This Review will focus on developments over the last six years and includes discussions on the underlying technologies as well as their applications.

Light-Controlled Tools

The quest to determine how precise neural activity patterns mediate computation, behavior, and pathology would be greatly aided by a set of tools for reliably activating and inactivating genetically targeted neurons, in a temporally precise and rapidly reversible fashion. Having earlier adapted a light-activated cation channel, channelrhodopsin-2 (ChR2), for allowing neurons to be stimulated by blue light, we searched for a complementary tool that would enable optical neuronal inhibition, driven by light of a second color. Here we report that targeting the codon-optimized form of the light-driven chloride pump halorhodopsin from the archaebacterium Natronomas pharaonis (hereafter abbreviated Halo) to genetically-specified neurons enables them to be silenced reliably, and reversibly, by millisecond-timescale pulses of yellow light. We show that trains of yellow and blue light pulses can drive high-fidelity sequences of hyperpolarizations and depolarizations in neurons simultaneously expressing yellow light-driven Halo and blue light-driven ChR2, allowing for the first time manipulations of neural synchrony without perturbation of other parameters such as spiking rates. The Halo/ChR2 system thus constitutes a powerful toolbox for multichannel photoinhibition and photostimulation of virally or transgenically targeted neural circuits without need for exogenous chemicals, enabling systematic analysis and engineering of the brain, and quantitative bioengineering of excitable cells.

Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike temporal resolution.

In the study of complex mammalian behaviours, technological limitations have prevented spatiotemporally precise control over intracellular signalling processes. Here we report the development of a versatile family of genetically encoded optical tools ('optoXRs') that leverage common structure-function relationships among G-protein-coupled receptors (GPCRs) to recruit and control, with high spatiotemporal precision, receptor-initiated biochemical signalling pathways. In particular, we have developed and characterized two optoXRs that selectively recruit distinct, targeted signalling pathways in response to light. The two optoXRs exerted opposing effects on spike firing in nucleus accumbens in vivo, and precisely timed optoXR photostimulation in nucleus accumbens by itself sufficed to drive conditioned place preference in freely moving mice. The optoXR approach allows testing of hypotheses regarding the causal impact of biochemical signalling in behaving mammals, in a targetable and temporally precise manner.

https://web.stanford.edu/group/dlab/media/papers/Airan%20Nature%202009.pdf

Temporally precise in vivo control of intracellular signalling

Mechanism of action of insecticidal secondary metabolites of plant origin


 
 
 Is a loss of CatSper function in sperm a common cause of unexplained male infertility?
 
 
 
 Loss of CatSper function leads to similar infertility phenotypes in mice and men and represents one of the most common causes of unexplained male infertility.
 
 
 
 Male infertility is often due to low sperm counts, impaired motility, and/or abnormal morphology. However, for a large fraction of infertile men, semen parameters are normal, suggesting that the infertility is rather due to an unexplained dysfunction of the sperm. There has been a growing body of evidence that human sperm dysfunction might involve the sperm-specific Ca2+ channel CatSper (cation channel of sperm) that translates changes in the chemical microenvironment of the female reproductive tract into changes in swimming behavior.
 
 
 
 In an observational study over the course of three years, a prototype of the “CatSper-Activity-Test” (CAT) was used to assess the function of CatSper in sperm from the left-over semen samples of 2286 unselected patients undergoing a semen analysis.
 
 
 
 The CatSper-Activity-Test (CAT) was developed at the Centre of Reproductive Medicine and Andrology, University Hospital Münster and subsequently performed by medical technical assistants in the diagnostic facility of this institution. Requiring only 40 µl of ejaculate, a standard light microscope, and a hands-on-time of a few minutes, the CAT enables a read-out of the activity of CatSper through a motility response: in the CAT buffer, CatSper-intact, but not CatSper-deficient sperm become immotile.
 
 
 
 Using this CatSper-Activity-Test, we identified seven patients suffering from a loss of CatSper function in a cohort of men seeking medical advice for suspected male infertility. Standard semen analysis revealed that the CatSper-deficient patients are (with one exception) normozoospermic and were diagnosed with unexplained infertility. Notably, their sperm not only failed to fertilize the egg naturally but also upon intrauterine insemination (IUI) and in vitro fertilization (IVF), whereas intra-cytoplasmic sperm injection (ICSI) was successful. Two additional CatSper-deficient patients were identified among patients visiting our clinics for other reasons. We show that the loss of CatSper function is predominantly caused by a homozygous deletion of the CATSPER2 gene; one patient featured compound heterozygous variants of the CATSPERE gene. According to the study results, CatSper dysfunction underlies 1.7% of cases of unexplained male infertility, for which a female factor can be excluded.
 
 
 
 Results from this observational study were obtained from a single institute. A multi-center study involving various countries would enable a more precise determination of the prevalence of CatSper-related male infertility.
 
 
 
 The CatSper-Activity-Test reliably identifies CatSper-deficient patients with a hands-on-time of a few minutes and requires neither special equipment nor specific training. We envisage the CatSper-Activity-Test as a novel tool for the early diagnosis of male infertility, thus, contributing to evidence-based treatment decisions and sparing patients unnecessary medical and financial risks.
 
 
 
 not applicable


/pdf/o-017-a-novel-diagnostic-test-identifies-patients-suffering-263x2cav.pdf

O-017 A novel diagnostic test identifies patients suffering from loss of CatSper function

Channel Currents in Rat Intracardiac Neurons Muscarinic Receptor Activation Modulates Calcium

Cylicins are testis specific proteins which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9 mediated gene-editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1-deficiency resulted in male subfertility whereas Cylc2−/-, Cylc1−/y Cylc2+/-, and Cylc1−/y Cylc2−/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human

Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder affecting the function of motile cilia in several organ systems. In PCD, male infertility is caused by defective sperm flagella composition or deficient motile cilia function in the efferent ducts of the male reproductive system. Different PCD-associated genes encoding axonemal components involved in the regulation of ciliary and flagellar beating are also reported to cause infertility due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we performed genetic testing by next generation sequencing techniques, PCD diagnostics including immunofluorescence-, transmission electron-, and high-speed video microscopy on sperm flagella and andrological work up including semen analyses. We identified ten infertile male individuals with pathogenic variants in CCDC39 (one) and CCDC40 (two) encoding ruler proteins, RSPH1 (two) and RSPH9 (one) encoding radial spoke head proteins, and HYDIN (two) and SPEF2 (two) encoding CP-associated proteins, respectively. We demonstrate for the first time that pathogenic variants in RSPH1 and RSPH9 cause male infertility due to sperm cell dysmotility and abnormal flagellar RSPH1 and RSPH9 composition. We also provide novel evidence for MMAF in HYDIN- and RSPH1-mutant individuals. We show absence or severe reduction of CCDC39 and SPEF2 in sperm flagella of CCDC39- and CCDC40-mutant individuals and HYDIN- and SPEF2-mutant individuals, respectively. Thereby, we reveal interactions between CCDC39 and CCDC40 as well as HYDIN and SPEF2 in sperm flagella. Our findings demonstrate that immunofluorescence microscopy in sperm cells is a valuable tool to identify flagellar defects related to the axonemal ruler, radial spoke head and the central pair apparatus, thus aiding the diagnosis of male infertility. This is of particular importance to classify the pathogenicity of genetic defects, especially in cases of missense variants of unknown significance, or to interpret HYDIN variants that are confounded by the presence of the almost identical pseudogene HYDIN2.

/pdf/pathogenic-gene-variants-in-ccdc39-ccdc40-rsph1-rsph9-hydin-2kimi2up.pdf

Timo Strünker

Papers

O-017 A novel diagnostic test identifies patients suffering from loss of CatSper function

Channel Currents in Rat Intracardiac Neurons Muscarinic Receptor Activation Modulates Calcium

Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human

Pathogenic gene variants in CCDC39, CCDC40, RSPH1, RSPH9, HYDIN, and SPEF2 cause defects of sperm flagella composition and male infertility