Structural analysis of the female reptile reproductive system by micro-computed tomography and optical coherence tomography†.

Volumetric data provide unprecedented structural insight to the reproductive tract and add vital anatomical context to the relationships between organs. The morphology of the female reproductive tract in non-avian reptiles varies between species, corresponding to a broad range of reproductive modes and providing valuable insight to comparative investigations of reproductive anatomy. However, reproductive studies in reptilian models, such as the brown anole studied here, have historically relied on histological methods to understand the anatomy. While these methods are highly effective for characterizing the cell types present in each organ, histological methods lose the 3D relationships between images and leave the architecture of the organ system poorly understood. We present the first comprehensive volumetric analyses of the female brown anole reproductive tract using two non-invasive, non-destructive imaging modalities: micro-computed tomography (microCT) and optical coherence tomography (OCT). Both are specialized imaging technologies that facilitate high-throughput imaging and preserve three-dimensional information. This study represents the first time that microCT has been used to study all reproductive organs in this species and the very first time that OCT has been applied to this species. We show how the non-destructive volumetric imaging provided by each modality reveals anatomical context including orientation and relationships between reproductive organs of the anole lizard. In addition to broad patterns of morphology, both imaging modalities provide the high resolution necessary to capture details and key anatomical features of each organ. We demonstrate that classic histological features can be appreciated within whole-organ architecture in volumetric imaging using microCT and OCT, providing the complementary information necessary to understand the relationships between tissues and organs in the reproductive system. This side-by-side imaging analysis using microCT and OCT allows us to evaluate the specific advantages and limitations of these two methods for the female reptile reproductive system.

In vivo dynamic volumetric imaging of mouse testis and epididymis with optical coherence tomography†.

The implementation of live imaging in reproductive research is crucial for studying the physiological dynamics. Sperm transport is a highly dynamic process regulated by tubular contractions and luminal flows within the male reproductive tract. However, due to the lack of imaging techniques to capture these dynamics in vivo, there is little information on the physiological and biomechanical regulation of sperm transport through the male reproductive tract. Here, we present a functional in vivo imaging approach using optical coherence tomography, enabling live, label-free, depth-resolved, three-dimensional, high-resolution visualization of the mouse testis and epididymis. With this approach, we spatiotemporally captured tubular contractility in mouse testis and epididymis, as well as microstructures of these reproductive organs. Our findings demonstrated that the contraction frequency varies significantly depending on the epididymal regions, suggesting the spatial regulation of epididymal contractility. Furthermore, we implemented quantitative measurements of the contraction wave and luminal transport through the epididymal duct, revealing the physiological dynamics within the male reproductive tract. The results show that the contraction wave propagates along the epididymal duct and the wave propagation velocity was estimated in vivo. In conclusion, this is the first study to develop in vivo dynamic volumetric imaging of the male reproductive tract, which allows for quantitative analysis of the dynamics associated with sperm transport. This study sets a platform for various studies investigating normal and abnormal male reproductive physiology as well as the pharmacological and environmental effects on reproductive functions in mouse models, ultimately contributing to a comprehensive understanding of male reproductive disorders.

Open-source, highly efficient, post-acquisition synchronization for 4D dual-contrast imaging of the mouse embryonic heart over development with optical coherence tomography.

Dynamic imaging of the beating embryonic heart in 3D is critical for understanding cardiac development and defects. Optical coherence tomography (OCT) plays an important role in embryonic heart imaging with its unique imaging scale and label-free contrasts. In particular, 4D (3D + time) OCT imaging enabled biomechanical analysis of the developing heart in various animal models. While ultrafast OCT systems allow for direct volumetric imaging of the beating heart, the imaging speed remains limited, leading to an image quality inferior to that produced by post-acquisition synchronization. As OCT systems become increasingly available to a wide range of biomedical researchers, a more accessible 4D reconstruction method is required to enable the broader application of OCT in the dynamic, volumetric assessment of embryonic heartbeat. Here, we report an open-source, highly efficient, post-acquisition synchronization method for 4D cardiodynamic and hemodynamic imaging of the mouse embryonic heart. Relying on the difference between images to characterize heart wall movements, the method provides good sensitivity to the cardiac activity when aligning heartbeat phases, even at early stages when the heart wall occupies only a small number of pixels. The method works with a densely sampled single 3D data acquisition, which, unlike the B-M scans required by other methods, is readily available in most commercial OCT systems. Compared with an existing approach for the mouse embryonic heart, this method shows superior reconstruction quality. We present the robustness of the method through results from different embryos with distinct heart rates, ranging from 1.24 Hz to 2.13 Hz. Since the alignment process operates on a 1D signal, the method has a high efficiency, featuring sub-second alignment time while utilizing ∼100% of the original image files. This allows us to achieve repeated, dual-contrast imaging of mouse embryonic heart development. This new, open-source method could facilitate research using OCT to study early cardiogenesis.

Optical coherence tomography for dynamic investigation of mammalian reproductive processes.

The biological events associated with mammalian reproductive processes are highly dynamic and tightly regulated by molecular, genetic, and biomechanical factors. Implementation of live imaging in reproductive research is vital for the advancement of our understanding of normal reproductive physiology and for improving the management of reproductive disorders. Optical coherence tomography (OCT) is emerging as a promising tool for dynamic volumetric imaging of various reproductive processes in mice and other animal models. In this review, we summarize recent studies employing OCT-based approaches toward the investigation of reproductive processes in both, males and females. We describe how OCT can be applied to study structural features of the male reproductive system and sperm transport through the male reproductive tract. We review OCT applications for in vitro and dynamic in vivo imaging of the female reproductive system, staging and tracking of oocytes and embryos, and investigations of the oocyte/embryo transport through the oviduct. We describe how the functional OCT approach can be applied to the analysis of cilia dynamics within the male and female reproductive systems. We also discuss the areas of research, where OCT could find potential applications to progress our understanding of normal reproductive physiology and reproductive disorders.

Dynamics of gametes and embryos in the oviduct: what can in vivo imaging reveal?

In brief: In vivo imaging of gametes and embryos in the oviduct enables new studies of the native processes that lead to fertilization and pregnancy. This review article discusses recent advancements in the in vivo imaging methods and insights which contribute to understanding the oviductal function. Abstract: Understanding the physiological dynamics of gametes and embryos in the fallopian tube (oviduct) has significant implications for managing reproductive disorders and improving assisted reproductive technologies. Recent advancements in imaging of the mouse oviduct in vivo uncovered fascinating dynamics of gametes and embryos in their native states. These new imaging approaches and observations are bringing exciting momentum to uncover the otherwise-hidden processes orchestrating fertilization and pregnancy. For mechanistic investigations, in vivo imaging in genetic mouse models enables dynamic phenotyping of gene functions in the reproductive process. Here, we review these imaging methods, discuss insights recently revealed by in vivo imaging, and comment on emerging directions, aiming to stimulate new in vivo studies of reproductive dynamics.

In vivo volumetric depth-resolved imaging of cilia metachronal waves using dynamic optical coherence tomography.

Motile cilia are dynamic hair-like structures covering epithelial surfaces in multiple organs. The periodic coordinated beating of cilia creates waves propagating along the surface, known as the metachronal waves, which transport fluids and mucus along the epithelium. Motile ciliopathies result from disrupted coordinated cilia beating and are associated with serious clinical complications, including reproductive disorders. Despite the recognized clinical significance, research of cilia dynamics is extremely limited. Here, we present quantitative imaging of cilia metachronal waves volumetrically through tissue layers using dynamic optical coherence tomography (OCT). Our method relies on spatiotemporal mapping of the phase of intensity fluctuations in OCT images caused by the ciliary beating. We validated our new method ex vivo and implemented it in vivo to visualize cilia metachronal wave propagation within the mouse fallopian tube. This method can be extended to the assessment of physiological cilia function and ciliary dyskinesias in various organ systems, contributing to better management of pathologies associated with motile ciliopathies.

2021 JOSA A Emerging Researcher Best Paper Prize: editorial.

JOSA A Editor-in-Chief, Olga Korotkova, Feature Editor, Johannes Courtial, and members of the 2021 Emerging Researcher Best Paper Prize Committee announce the recipient of the 2021 prize for the best paper published by an emerging researcher in the Journal.

Mouse embryo phenotyping with optical coherence tomography.

With the explosion of gene editing tools in recent years, there has been a much greater demand for mouse embryo phenotyping, and traditional methods such as histology and histochemistry experienced a methodological renaissance as they became the principal tools for phenotyping. However, it is important to explore alternative phenotyping options to maximize time and resources and implement volumetric structural analysis for enhanced investigation of phenotypes. Cardiovascular phenotyping, in particular, is important to perform in vivo due to the dramatic structural and functional changes that occur in heart development over relatively short periods of time. Optical coherence tomography (OCT) is one of the most exciting advanced imaging techniques emerging within the field of developmental biology, and this review provides a summary of how it is currently being implemented in mouse embryo investigations and phenotyping. This review aims to provide an understanding of the approaches used in optical coherence tomography and how they can be applied in embryology and developmental biology, with the overall aim of bridging the gap between biology and technology.

Myometrial progesterone receptor determines a transcription program for uterine remodeling and contractions during pregnancy.

The uterine myometrium expands and maintains contractile quiescence before parturition. While the steroid hormone progesterone blocks labor, the role of progesterone signaling in myometrial expansion remains elusive. This study investigated the myometrial functions of the progesterone receptor, PGR. Pgr ablation in mouse smooth muscle leads to subfertility, oviductal embryo retention, and impaired myometrial adaptation to pregnancy. While gross morphology between mutant and control uteri are comparable, mutant uteri manifest a decrease of 76.6% oxytocin-stimulated contractility in a pseudopregnant context with a reduced expression of intracellular calcium homeostasis genes including Pde5a and Plcb4. At mid-pregnancy, the mutant myometrium exhibits discontinuous myofibers and disarrayed extracellular matrix at the conceptus site. Transcriptome of the mutant mid-pregnant uterine wall manifests altered muscle and extracellular matrix profiles and resembles that of late-pregnancy control tissues. A survey of PGR occupancy, H3K27ac histone marks, and chromatin looping annotates cis-acting elements that may direct gene expression of mid-pregnancy uteri for uterine remodeling. Further analyses suggest that major muscle and matrix regulators Myocd and Ccn2 and smooth muscle building block genes are PGR direct downstream targets. Cataloging enhancers that are topologically associated with progesterone downstream genes reveals distinctive patterns of transcription factor binding motifs in groups of enhancers and identifies potential regulatory partners of PGR outside its occupying sites. Finally, conserved correlations are found between estimated PGR activities and RNA abundance of downstream muscle and matrix genes in human myometrial tissues. In summary, PGR is pivotal to direct the molecular program for the uterus to remodel and support pregnancy.

Following the Beat: Imaging the Valveless Pumping Function in the Early Embryonic Heart.

In vertebrates, the coordinated beat of the early heart tube drives cardiogenesis and supports embryonic growth. How the heart pumps at this valveless stage marks a fascinating problem that is of vital significance for understanding cardiac development and defects. The developing heart achieves its function at the same time as continuous and dramatic morphological changes, which in turn modify its pumping dynamics. The beauty of this muti-time-scale process also highlights its complexity that requires interdisciplinary approaches to study. High-resolution optical imaging, particularly fast, four-dimensional (4D) imaging, plays a critical role in revealing the process of pumping, instructing numerical modeling, and enabling biomechanical analyses. In this review, we aim to connect the investigation of valveless pumping mechanisms with the recent advancements in embryonic cardiodynamic imaging, facilitating interactions between these two areas of study, in hopes of encouraging and motivating innovative work to further understand the early heartbeat.

Dynamic volumetric imaging and cilia beat mapping in the mouse male reproductive tract with optical coherence tomography.

Spermatozoa transport within the male reproductive tract is a highly dynamic and biologically important reproductive event. However, due to the lack of live volumetric imaging technologies and quantitative measurements, there is little information on the dynamic aspect and regulation of this process. Here, we presented ex vivo dynamic volumetric imaging of the mouse testis, efferent duct, epididymis, and vas deferens at a micro-scale spatial resolution with optical coherence tomography (OCT). Micro computed tomography imaging is presented as a reference for the proposed OCT imaging. Application of functional OCT analysis allowed for 3D mapping of the cilia beat frequency in the efferent duct, which volumetrically visualized the spatial distribution of the ciliated cells and corresponding ciliary activities. Potentially these analyses could be expanded to in vivo settings through intravital approach. In summary, this study demonstrated that OCT has a great potential to investigate the microstructure and dynamics, such as cilia beating, muscle contractions, and sperm transport, within the male reproductive tract.

The testis-specific E3 ubiquitin ligase RNF133 is required for fecundity in mice.

BACKGROUND: Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin-proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated E3 ubiquitin-protein ligases that function during spermatogenesis remain unknown. RESULTS: We identified four evolutionarily conserved testis-specific E3 ubiquitin-protein ligases [RING finger protein 133 (Rnf133); RING finger protein 148 (Rnf148); RING finger protein 151 (Rnf151); and Zinc finger SWIM-type containing 2 (Zswim2)]. Using the CRISPR/Cas9 system, we generated and analyzed the fertility of mutant mice with null alleles for each of these E3-encoding genes, as well as double and triple knockout (KO) mice. Male fertility, male reproductive organ, and sperm-associated parameters were analyzed in detail. Fecundity remained largely unaffected in Rnf148, Rnf151, and Zswim2 KO males; however, Rnf133 KO males displayed severe subfertility. Additionally, Rnf133 KO sperm exhibited abnormal morphology and reduced motility. Ultrastructural analysis demonstrated that cytoplasmic droplets were retained in Rnf133 KO spermatozoa. Although Rnf133 and Rnf148 encode paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases based on their protein structures, there was limited functional redundancy of these proteins. In addition, we identified UBE2J1 as an E2 ubiquitin-conjugating protein that interacts with RNF133. CONCLUSIONS: Our studies reveal that RNF133 is a testis-expressed E3 ubiquitin-protein ligase that plays a critical role for sperm function during spermiogenesis. Based on the presence of a transmembrane domain in RNF133 and its interaction with the ER containing E2 protein UBE2J1, we hypothesize that these ubiquitin-regulatory proteins function together in ER quality control during spermatogenesis.

Ultra-fast dynamic line-field optical coherence elastography.

In this work, we present an ultra-fast line-field optical coherence elastography system (LF-OCE) with an 11.5 MHz equivalent A-line rate. The system was composed of a line-field spectral domain optical coherence tomography system based on a supercontinuum light source, Michelson-type interferometer, and a high-speed 2D spectrometer. The system performed ultra-fast imaging of elastic waves in tissue-mimicking phantoms of various elasticities. The results corroborated well with mechanical testing. Following validation, LF-OCE measurements were made in in situ and in in vivo rabbit corneas under various conditions. The results show the capability of the system to rapidly image elastic waves in tissues.

In vivo dynamic 3D imaging of oocytes and embryos in the mouse oviduct.

Developmental biologists have always relied on imaging to shed light on dynamic cellular events. However, processes such as mammalian fertilization and embryogenesis are generally inaccessible for direct imaging. In consequence, how the oviduct (fallopian tube) facilitates the transport of gametes and preimplantation embryos continues to be unanswered. Here we present a combination of intravital window and optical coherence tomography for dynamic, volumetric, in vivo imaging of oocytes and embryos as they are transported through the mouse oviduct. We observed location-dependent circling, oscillating, and long-distance bi-directional movements of oocytes and embryos that suggest regulatory mechanisms driving transport and question established views in the field. This in vivo imaging approach can be combined with a variety of genetic and pharmacological manipulations for live functional analysis, bringing the potential to investigate reproductive physiology in its native state.

2020 JOSA A Emerging Researcher Best Paper Prize: editorial.

JOSA A Editor-in-Chief P. Scott Carney, Feature Editor Johannes Courtial, and members of the 2020 Emerging Researcher Best Paper Prize Committee announce the recipient of the 2020 prize for the best paper published by an emerging researcher in the Journal.

Dynamic Imaging of Mouse Embryos and Cardiac Development in Static Culture.

Dynamic imaging is a powerful approach to assess the function of a developing organ system. The heart is a dynamic organ that undergoes quick morphological and mechanical changes through early embryonic development. Defining the embyonic mouse heart's normal function is important for our own understanding of human heart development and will inform us on treatments and prevention of congenital heart defects (CHD). Traditional methods such as ultrasound or fluorescence-based microscopy are suitable for live dynamic imaging, are excellent to visualize structure and connect gene expression to phenotypes, but can be of low quality in resolving fine features and lack imaging depth and scale to fully appreciate organ morphogenesis. Additionally, previous methods can be limited in accommodating a live imaging apparatus capable of sustaining whole embryo development for extended periods time. Optical coherence tomography (OCT) is unique in this circumstance because acquisition of three-dimensional images without contrast reagents, at single cell resolution make it a suitable modality to visualize fine structures in the developing embryo. OCT setups are highly customizable for live imaging because of the tethered imaging arm, due to its setup as a fiber-based interferometer. OCT allows for 4D (3D + time) functional imaging of living mouse embryos and can provide functional and mechanical information to ascertain how the heart's pump function changes through development. In this chapter, we will focus on how we use OCT to visualize live heart dynamics at different stages of development and provide mechanical information to reveal functional properties of the developing heart.

Embryonic Mouse Cardiodynamic OCT Imaging.

The embryonic heart is an active and developing organ. Genetic studies in mouse models have generated great insight into normal heart development and congenital heart defects, and suggest mechanical forces such as heart contraction and blood flow to be implicated in cardiogenesis and disease. To explore this relationship and investigate the interplay between biomechanical forces and cardiac development, live dynamic cardiac imaging is essential. Cardiodynamic imaging with optical coherence tomography (OCT) is proving to be a unique approach to functional analysis of the embryonic mouse heart. Its compatibility with live culture systems, reagent-free contrast, cellular level resolution, and millimeter scale imaging depth make it capable of imaging the heart volumetrically and providing spatially resolved information on heart wall dynamics and blood flow. Here, we review the progress made in mouse embryonic cardiodynamic imaging with OCT, highlighting leaps in technology to overcome limitations in resolution and acquisition speed. We describe state-of-the-art functional OCT methods such as Doppler OCT and OCT angiography for blood flow imaging and quantification in the beating heart. As OCT is a continuously developing technology, we provide insight into the future developments of this area, toward the investigation of normal cardiogenesis and congenital heart defects.

Optogenetic cardiac pacing in cultured mouse embryos under imaging guidance.

The mouse embryo is an established model for investigation of regulatory mechanisms controlling cardiac development and congenital heart defects in humans. Since cultured mouse embryos are very sensitive to any manipulations and environmental fluctuations, controlled alterations in mouse embryonic cardiac function are extremely challenging, which is a major hurdle in mammalian cardiac biomechanics research. This manuscript presents first optogenetic manipulation of cardiodynamics and hemodynamics in cultured mouse embryos. Optogenetic pacing was combined with 4D (3D + time) optical coherence tomography structural and Doppler imaging, demonstrating that embryonic hearts under optogenetic pacing can function efficiently and produce strong blood flows. This study demonstrates that the presented method is a powerful tool giving quick, consistent, reversible control over heart dynamics and blood flow under real time visualization, enabling various live cardiac biomechanics studies toward better understanding of normal cardiogenesis and congenital heart defects in humans.

Label-free optical imaging in developmental biology [Invited].

Application of optical imaging in developmental biology marks an exciting frontier in biomedical optics. Optical resolution and imaging depth allow for investigation of growing embryos at subcellular, cellular, and whole organism levels, while the complexity and variety of embryonic processes set multiple challenges stimulating the development of various live dynamic embryonic imaging approaches. Among other optical methods, label-free optical techniques attract an increasing interest as they allow investigation of developmental mechanisms without application of exogenous markers or fluorescent reporters. There has been a boost in development of label-free optical imaging techniques for studying embryonic development in animal models over the last decade, which revealed new information about early development and created new areas for investigation. Here, we review the recent progress in label-free optical embryonic imaging, discuss specific applications, and comment on future developments at the interface of photonics, engineering, and developmental biology.