Abstract
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.
Introduction
The oviduct is an essential organ for natural conception and pregnancy. It serves as the site for gamete transport and maturation, hosts fertilization, and supports the transport and development of the embryo prior to implantation (Li & Winuthayanon 2017, Coy et al. 2012). Studies of oviductal function date back nearly a century (Westman 1926). Over the years, technological advancements ranging from molecular genetics to imaging have enabled novel insights into the role of the oviduct and its interactions with gametes and embryos (Yanagimachi 2022), such as sperm attachment to the epithelium and subsequent hyperactivation (Suarez & Pacey 2006); the estrogen- and progesterone-regulated ciliary and muscular activities that drive the fluid flow (Barton et al. 2020); and the secretions promoting cleavage and development (Ghersevich et al. 2015). Such knowledge provides critical guidance for reproductive health care, but the process within the oviduct – for example, fertilization and gamete/embryo transport – remains far from elucidation in mammalian models. Historically, observations on any part of this process were largely performed under in vitro or ex vivo conditions. However, these conditions are insufficient to uncover how the reproductive process takes place in the native oviductal environment, which is structurally complex, constantly moving, and modulated by hormones and other stimuli (Maillo et al. 2016, Barton et al. 2020, Kölle et al. 2020). A lack of in vivo imaging has long posed a major hurdle for investigating the hidden dynamics inside the oviduct, complicating the study of oviductal function.
The movements and activities of gametes and embryos are excellent indicators of oviductal function. With the emergence of in vivo imaging approaches in the mouse model, such dynamics have recently started to be understood in the native oviductal environment. These observations serve multiple roles: generating new insights, validating prior knowledge, explaining findings from discrete time points, connecting gamete/embryo behaviors with oviductal activities, and stimulating novel hypotheses and studies. The methods themselves open the possibility of understanding the mammalian oviduct and its associated reproductive process in vivo. We believe this recent advancement is a result of the cross-disciplinary integration of engineering and biology, and we hope to convey the excitement of working with this interface to explore the wonders of a new life’s beginning. Here, we first consider the significance of in vivo studies in the context of reproductive health and solutions. Then, we describe in vivo imaging methods and the insights that were revealed by those methods in the past decade. Finally, we present our perspectives on some pressing applications to focus on and the technical innovations to come.
The need for in vivo imaging
It is extremely difficult to study a complex and dynamic reproductive process without seeing it; given the varying environment inside the oviduct that has yet to be fully understood, in vivo imaging is critical. This need exists for both unraveling the mechanism of reproductive disorders and improving assisted reproductive technologies (ARTs).
There are multiple unknown aspects of the oviductal transport in relation to reproductive disorders, such as oviduct-related infertility and ectopic pregnancy. Consider tubal ectopic pregnancy (tEP) as an example: failing to deliver the preimplantation embryo to the uterus creates embryo retentions in the oviduct, acting as a prerequisite in tEP (Shaw et al. 2010); however, it remains largely unclear how the oviduct transports oocytes and embryos in a timely fashion (Shao 2010). Without in vivo imaging, dissecting the roles of oviductal components in oocyte/embryo transport after manipulating the oviduct is limited to evaluating the final outcome, such as examining whether the embryos are in the oviduct or the uterus at a single time point (Wang et al. 2004, Ning et al. 2014). This answers the question about the requirement, but how the manipulation leads to the outcome cannot be assessed. It is inspiring to see that in vivo imaging has recently begun to address this hurdle in the study of disordered embryo transport in the mouse model (Bianchi et al. 2021). In particular, an ink/dye-based in vivo imaging method (Hino & Yanagimachi 2019) was utilized to directly show how the oviductal fluid flow was altered, leading to the embryo retention at the isthmic–ampullary junction (IAJ) in Adgrd1-deficient mice (Bianchi et al. 2021). As shown in this recent work, dynamic phenotyping inside the oviduct in vivo is imperative to uncovering the relation between molecular genetic factors and disrupted reproductive outcomes. With a range of mouse models created toward investigation of potential causes of embryo retention in the oviduct (Shao 2010, Qian et al. 2018, Herrera et al. 2020, Yuan et al. 2021), in vivo imaging is an emerging key component of the phenotyping tool kit for an improved understanding of tEP.
The success of ARTs, such as in vitro fertilization (IVF), has brought hope to over a million couples suffering from reduced fertility. While the cumulative birth rate from multiple ART cycles approaches that of natural conception (Luke et al. 2012), the pregnancy rate is significantly lower (Mansour et al. 2014). Embryos and children conceived through ARTs have a higher risk of obstetric and perinatal complications (Pandey et al. 2012) and a higher chance of imprinting disorders (Lazaraviciute et al. 2014), respectively. In IVF, fertilization and preimplantation development takes place outside the oviduct in a laboratory setting until embryos are ready for implantation into the uterus. Studies suggest that epigenetic factors associated with a change in the gamete/embryo environment likely contribute to the adverse outcomes of ARTs (Niemitz & Feinberg 2004, Lucas 2013). Therefore, understanding the in vivo oviductal environment and the in vivo gamete/embryo–oviduct interactions is crucial to guiding further optimization of IVF (Ménézo et al. 2015, Li & Winuthayanon 2017, Pérez-Cerezales et al. 2018). This perspective is neatly illustrated by Avilés et al. (2015): ‘It can be assumed that the efficacy of ART will improve as fast as our knowledge of the in vivo process increases’. Biochemical factors within the oviduct have been extensively studied and proven to improve the efficiency of IVF (Avilés et al. 2010, Ghersevich et al. 2015), while biomechanical factors remain largely unexplored and require in vivo tools for investigation. For example, recent in vivo imaging of the mouse oviduct revealed that, after fertilization, large groups of embryos appeared relatively stationary within the lower ampulla despite active oviductal contractions producing flows (Wang & Larina 2021), suggesting that embryos may experience flow-induced shear forces during their initial development. Also, mechanical interactions between the embryo and the oviductal epithelium are believed to initiate embryo–maternal cross-talk that could facilitate preimplantation development and transport of the embryos (Kölle et al. 2020). These cumulate toward the need for in vivo imaging to first understand, then simulate in vitro, the biomechanical dynamics of the reproductive process in the oviduct with the goal of improving ARTs.
While we focus on dynamics and in vivo imaging in this article, it is important to note that in vitro imaging sets the structural and morphological foundation for studying dynamic aspects of the mouse oviduct (Agduhr 1927). In particular, cellular heterogeneity and tubular morphology were recently revealed in unprecedented detail from images of fixed mouse oviduct samples (Harwalkar et al. 2021), which uncovered consistent coiling patterns, revealed how the mucosal folds transit along the oviduct, showed the distribution patterns of multiciliated cells and their spatial variations, and demonstrated the locations of oocytes/embryos in the oviduct at different days post coitum. These created a valuable basis for investigating the oviductal function. Only by building on such imaging-based studies of the anatomical and cellular characteristics can the in vivo dynamics be properly interpreted. In our discussions, we highlight that in vivo insights need to be placed in the context of in vitro and also ex vivo works to maximize their value.
In vivo optical access
When imaging gametes and preimplantation embryos, their small sizes mandate optical modalities. Although using light for imaging provides subcellular resolution, light attenuation in biological tissues significantly limits the penetration depth to a few millimeters where micro-level resolution can be maintained. As a result, directly probing inside the oviduct through the skin and muscle layers by using light is challenging. Gaining optical access to the oviduct has thus been an important aspect of in vivo imaging, with approaches including direct surgical exposure and intravital window implantation (Fig. 1).
In vivo optical access to the mouse oviduct. (A) Exposed reproductive organs placed in a saline-filled dish for imaging. Adapted from Muro et al. (2016), Creative Commons license CC BY-NC 4.0. (B) A perfusion collar setup for temperature-controlled bathing of the exposed reproductive organs. Adapted from Hino & Yanagimachi (2019) with permission. (C) The original design of intravital window for imaging of ovary. Adapted from Bochner et al. (2015), Creative Commons license CC BY 4.0. (D) Further optimized intravital window setup for imaging of the oviduct (top). Bright-field images showing the in vivo mouse oviduct through the intravital window (bottom). Adapted from Wang & Larina (2021) with permission.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
In vivo optical access to the mouse oviduct. (A) Exposed reproductive organs placed in a saline-filled dish for imaging. Adapted from Muro et al. (2016), Creative Commons license CC BY-NC 4.0. (B) A perfusion collar setup for temperature-controlled bathing of the exposed reproductive organs. Adapted from Hino & Yanagimachi (2019) with permission. (C) The original design of intravital window for imaging of ovary. Adapted from Bochner et al. (2015), Creative Commons license CC BY 4.0. (D) Further optimized intravital window setup for imaging of the oviduct (top). Bright-field images showing the in vivo mouse oviduct through the intravital window (bottom). Adapted from Wang & Larina (2021) with permission.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
In vivo optical access to the mouse oviduct. (A) Exposed reproductive organs placed in a saline-filled dish for imaging. Adapted from Muro et al. (2016), Creative Commons license CC BY-NC 4.0. (B) A perfusion collar setup for temperature-controlled bathing of the exposed reproductive organs. Adapted from Hino & Yanagimachi (2019) with permission. (C) The original design of intravital window for imaging of ovary. Adapted from Bochner et al. (2015), Creative Commons license CC BY 4.0. (D) Further optimized intravital window setup for imaging of the oviduct (top). Bright-field images showing the in vivo mouse oviduct through the intravital window (bottom). Adapted from Wang & Larina (2021) with permission.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
Surgical exposure of the oviduct through an incision of the dorsal or abdominal body wall has been a widely used strategy for small animal models (Boling & Blandau 1971a, Halbert et al. 1976a,b, Battalia & Yanagimachi 1979, Burton et al. 2015, Wang et al. 2015, Ishikawa et al. 2016, Muro et al. 2016, Wang & Larina 2018b) and also the sheep model (Druart et al. 2009). Multiple setups have been reported, with two representative ones shown in Figs. 1A and B. Similar to the standard procedure for zygote injection in transgenic mouse production (Ittner & Götz 2007), a surgical clamp can be used to hold the fat pad associated with the ovary and stabilize the oviduct for short-duration imaging, where different regions of the mouse oviduct can be easily accessed from the top (Wang & Larina 2018a). To prevent tissue dehydration and enable long-duration imaging, the exposed reproductive organs can be placed in a petri dish filled with 37ºC phosphate-buffered saline (Fig. 1A), and imaging can be performed from both top (Ishikawa et al. 2016) and bottom (Muro et al. 2016). Specifically, bottom access utilizes a glass-bottom dish with a 4 mm notch in the side to accommodate the uterus, and the ovarian fat pad is secured to the dish bottom with adhesive (Muro et al. 2016). With this petri dish configuration, chemical treatment of the oviduct is convenient, since target solutions can be directly applied to the medium bathing the oviduct. To study the role of muscular contractions in sperm transport, prifinium bromide, an anticholinergic drug, was used with this setup to suppress contractions (Ishikawa et al. 2016). To maintain the oviduct as close to the physiological state as possible, a perfusion collar setup was developed for hamsters (Battalia & Yanagimachi 1979) and recently adopted for mice (Hino & Yanagimachi 2019). Within the collar, the medium completely submerges the oviduct, and through active circulation, the medium temperature can be kept at the desired level for a prolonged period (Fig. 1B). These factors help to preserve the peristaltic motion of the oviduct that was shown to contribute to sperm reaching the fertilization site (Hino & Yanagimachi 2019).
Longitudinal imaging is of particular interest when studying reproductive process, and the ability to repeatedly locate the same oviductal region is conductive to understanding the dynamics at multiple time scales. The intravital window is the primary approach for longitudinal, high-resolution optical imaging of internal organs in the mouse model (Alieva et al. 2014, Huang et al. 2021), and recent advancements in window design and implementation have led to superior imaging capabilities and discoveries that are otherwise hard to achieve (Kitamura et al. 2017, Huang et al. 2020). The development of intravital windows for the female reproductive organs was largely initiated by the need for longitudinal microscopy of ovarian cancer in the mouse model (Bochner et al. 2015). The excellent window design (Fig. 1C) enabled visualization of the physiological response of the ovary to hormonal changes, the process of tumor infiltration into the ovary, and tumor cell movements inside the oviduct (Bochner et al. 2015). The window features small eyelets on the rim for implantation, tissue-supporting petals for gluing the fat pad to secure the ovary, and frame extensions for holding the window stable during imaging (Fig. 1C).
Focusing on the oviduct, the window design and experimental procedures were further optimized (Fig. 1D) to allow for imaging of the oviductal morphology and function (Wang et al. 2018), as well as the structure (Moore et al. 2019) and dynamics (Wang & Larina 2021) of oocytes/embryos moving inside the oviduct. This window is 3D printed, making it low cost and disposable after a single use (Wang & Larina 2020). Mice with the window implanted can mate with successful embryo implantation on the window side, showing no major effects of the window implantation procedure on oviductal function in transporting gametes and embryos (Moore et al. 2019). Using this approach, the beat of the oviductal motile cilia, as well as the oviductal contraction and its wave propagation, were detected, visualized, and quantified in vivo (Wang et al. 2018). Demonstrations of both prolonged and longitudinal imaging through the intravital window (Wang et al. 2018, Wang & Larina 2021) revealed the promise of a detailed dynamic investigation of the reproductive process inside the mouse oviduct.
Box 1 Fluorescence imaging – convenient contrast for in vivo visualization
Fluorescence imaging represents the use of fluorescent contrast to achieve molecular or structural visualizations that are highly specific to the targeted specimen. Widely used in biological research, fluorescent contrast is based on the photon absorption and emission property of fluorescent proteins or dyes, and a separation of the emission light from the excitation light enables the user to observe the location and dynamics of targets labeled by fluorescent substances. Generally, this light separation can be obtained by a modular fluorescence filter set for specific excitation–emission wavelengths, where a dichromatic beamsplitter is used to reflect shorter wavelengths (containing excitation wavelengths) and to pass longer wavelengths (containing emission wavelengths), and excitation and emission filters are used to further select the excitation and emission bands. Due to its simple implementation, fluorescent contrast has been integrated with different optical imaging modalities to form a variety of microscopic techniques, such as widefield, confocal, and light-sheet fluorescence microscopy. Thus, multiple imaging scales (resolutions and field of view) and different imaging speeds are available to accommodate a range of applications. For example, widefield imaging with a low-magnification objective can generate a simultaneous, large visualization area for screening, laser scanning confocal imaging could provide a sub-micron spatial resolution, and a light-sheet illumination allows for capturing dynamics at over 300 volumes per second. Additionally, various detection methods of fluorescence contrast have led to extended imaging capabilities, such as localization of single molecules with super-resolution methods and probing the molecular environment with lifetime imaging. Except when relying on autofluorescence of the specimen, fluorescence imaging requires labeling. This can be achieved by either genetically engineering the organism to produce site-specific fluorescent proteins or externally introducing fluorescence to the target. With the rapid advancements in genetic engineering and molecular techniques, fluorescence labeling is becoming more feasible, convenient, and efficient for highly specific molecular and structural imaging.
Box 2 Optical coherence tomography – label-free, 3D in vivo imaging
In contrast with fluorescence microscopy, optical coherence tomography (OCT) is a label-free imaging modality that largely relies on the elastic scattering of light from tissue. Often referred to as an optical analog to ultrasound imaging, OCT relies on broadband light containing a range of wavelengths, measures backscattered light from the sample, and uses an interferometric approach to distinguish the light coming from different depths within the tissue. In today’s Fourier domain OCT, the in-depth profile (Z-direction) of the backscattered light, termed an A-scan, is obtained as a single exposure of wavelength-resolved light followed by a fast Fourier transform. Scanning the A-scan in one transverse direction (X-direction) forms a two-dimensional (2D) image, termed a B-scan, and scanning the B-scan over the other transverse direction (Y-direction) generates a three-dimensional (3D) volume. By directly mapping the backscattered light intensity over 3D space, OCT provides the structural image representing the distribution of optical scattering property within the sample. Compared with ultrasound imaging, OCT has a reduced imaging depth that is largely limited by significant light–tissue interactions but a better resolution due to the relatively short wavelength of light. This puts OCT in a unique position in terms of imaging scale. With a 1–2 mm depth in optically scattering tissues and a micro-level spatial resolvability, OCT fills the imaging scale gap between high-frequency ultrasound and confocal microscopy. OCT is considered a fast imaging modality, with A-scan rates up to the order of MHz, which can produce volume rates up to tens of Hertz. In addition to structural imaging that reveals the morphology and architecture of tissues, OCT has been developed for functional imaging that enabled various important applications across a range of biomedical fields. The major functional imaging capabilities of OCT include speckle- and phase-variance OCT for angiography, Doppler OCT to quantitatively assess the blood flow, spectroscopic OCT to probe blood oxygen saturation, polarization-sensitive OCT to map the birefringence properties of tissue, and OCT elastography for characterization of tissue elasticity. Such functional imaging contrasts in addition to the high-resolution 3D structural image suggest great potential for the investigation of dynamic, structurally, metabolically, and mechanically complex processes, such as those inside the oviduct.
Spermatozoa in vivo in the oviduct
Using a single-lens microscope of his own design, Leeuwenhoek discovered sperm and first described their morphology in the 1670s (Leeuwenhoek 1679). Over hundreds of years, sperm motility, their behaviors, and their fascinating abilities to respond to chemicals (chemotaxis), temperature gradients (thermotaxis), and flow fields (rheotaxis) have been largely studied ex vivo. Investigations of sperm activities within the oviduct, the native fertilization environment, have just started recently, pointing to an exciting frontier in the study of mammalian fertilization in vivo.
For studies of sperm outside of the oviduct, sperm cells are easily distinguished based on their unique shape and movement. However, delineating sperm morphology through the tissues of the oviduct wall has been challenging, and thus, identification of sperm in vivo inside the oviduct requires an appropriate combination of imaging contrast and scale.
Fluorescence imaging (Box 1)
Without fluorescent labeling, bright-field microscopy can locate sperm through the oviduct wall at high magnification (Suarez 1987), preferably by capturing the tail movement to improve the sensitivity and specificity of the detection. However, this approach is difficult and significantly limits the capability of studying sperm activities over a relatively large region inside the oviduct. Widefield fluorescence microscopy is a powerful solution for quickly and conveniently locating the sperm in situ in different segments of the mouse oviduct and investigating their behavior (La Spina et al. 2016, Ded et al. 2020). Double-labeling mouse sperm with fluorescent markers has been the strategy used for in vivo studies (Ishikawa et al. 2016, Muro et al. 2016). For studies involving artificial insemination, the cauda epididymal sperm can be incubated with Hoechst 33342 and MitoTracker Green FM to label the nucleus and mitochondria, respectively (Ishikawa et al. 2016). Alternatively, transgenic mice (B6D2F1-Tg (CAG/su9-DsRed2, Acr3-EGFP) RBGS002Osb) have been engineered for investigating ejaculated sperm from natural mating (Hasuwa et al. 2010), where the sperm acrosome and mitochondria, respectively, express green fluorescent proteins and red fluorescent proteins (Fig. 2A). Not only can such sperm be efficiently located in vivo through the oviduct wall (Fig. 2B), but the dual fluorescence also allows for the identification of sperm after acrosome reaction and monitoring the acrosome status (Muro et al. 2016), which is of significant value in understanding sperm behavior. Notably, the fluorescent sperm can be located after they have penetrated the zona pellucida of oocytes within the ampulla of the oviduct (Muro et al. 2016), providing the possibility of imaging fertilization in vivo.
Imaging contrasts for sperm identification in vivo. (A) Dual-fluorescently labeled sperm in the mouse oviduct. (B) A snapshot of time-lapse imaging of the migration of fluorescent sperm in the oviduct isthmus of a live mouse. ‘a’ labels the extramural segment of the utero-tubal junction (UTJ); ‘b-d’ labels the isthmus. (A) and (B) adapted from Muro et al. (2016), Creative Commons license CC BY-NC 4.0. (C) A tortuous trajectory of sperm in a petri dish compared with a microparticle. The scale bars are 100 µm. Green lines represent the tracking of sperm locations with a time interval of 0.9 s. (D) 3D trajectories of non-sperm and sperm cells in the oviduct in vivo. The scale bars are 50 µm. The tracks have a 0.9-s time interval. Adapted from Wang & Larina (2018b) with permission.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
Imaging contrasts for sperm identification in vivo. (A) Dual-fluorescently labeled sperm in the mouse oviduct. (B) A snapshot of time-lapse imaging of the migration of fluorescent sperm in the oviduct isthmus of a live mouse. ‘a’ labels the extramural segment of the utero-tubal junction (UTJ); ‘b-d’ labels the isthmus. (A) and (B) adapted from Muro et al. (2016), Creative Commons license CC BY-NC 4.0. (C) A tortuous trajectory of sperm in a petri dish compared with a microparticle. The scale bars are 100 µm. Green lines represent the tracking of sperm locations with a time interval of 0.9 s. (D) 3D trajectories of non-sperm and sperm cells in the oviduct in vivo. The scale bars are 50 µm. The tracks have a 0.9-s time interval. Adapted from Wang & Larina (2018b) with permission.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
Imaging contrasts for sperm identification in vivo. (A) Dual-fluorescently labeled sperm in the mouse oviduct. (B) A snapshot of time-lapse imaging of the migration of fluorescent sperm in the oviduct isthmus of a live mouse. ‘a’ labels the extramural segment of the utero-tubal junction (UTJ); ‘b-d’ labels the isthmus. (A) and (B) adapted from Muro et al. (2016), Creative Commons license CC BY-NC 4.0. (C) A tortuous trajectory of sperm in a petri dish compared with a microparticle. The scale bars are 100 µm. Green lines represent the tracking of sperm locations with a time interval of 0.9 s. (D) 3D trajectories of non-sperm and sperm cells in the oviduct in vivo. The scale bars are 50 µm. The tracks have a 0.9-s time interval. Adapted from Wang & Larina (2018b) with permission.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
Optical coherence tomography (Box 2)
Label-free imaging with OCT provides the advantage of millimeter-field-of-view and 3D visualization over the entire depth of the oviductal lumen, but sperm cells appear as bright spots in the OCT image, the same as other cells and particles. For sperm identification in the OCT volume, a functional method was developed based on unique characteristics of the trajectory of sperm movement (Wang & Larina 2018b). Specifically, when probed at an interval of 0.9 s, the trajectory of motile sperm features frequent changes of directions, distinct from those induced by bulk movement (Fig. 2C). A parameter characterizing this feature, the standard deviation of direction variation (SDofDV), proved to be effective in distinguishing motile sperm ex vivo within the environment of the oviduct ampulla (Wang & Larina 2018b). With the threshold of SDofDV set based on non-sperm cells in the ampulla in vivo, motile sperm can be identified in the ampulla in vivo, showing a more tortuous 3D path of movement in comparison to the non-sperm cell (Fig. 2D). This functional motile sperm identification method with OCT allows imaging of sperm trajectories in 3D at the native site of fertilization, revealing sperm activities volumetrically within the dynamic oviductal environment. The ability to measure sperm velocity and locate the sperm relative to the ampulla epithelium set the stage for the quantitative study of sperm behavior in vivo in the mouse model.
In vivo insights
In vivo studies so far have largely focused on observation and analysis of sperm movement and transport within the mouse oviduct, including both the isthmus and the ampulla. Sperm were seen attached to the epithelium in the isthmus, and along with peristaltic oviductal contractions, free-swimming sperm were observed moving back and forth in the lumen (Muro et al. 2016). Over 20 min, the region where the sperm heads attach shifted from the lower isthmus to the upper isthmus (Muro et al. 2016). When further studying the role of oviductal contraction in sperm transport (Ishikawa et al. 2016), tightly packed sperm assemblages were found in the isthmus, and their movements followed the contraction-induced shuttling flows of oviductal fluids (Fig. 3A, R1-R2). The assemblages became loose as moving toward the ampulla (Fig. 3A, R3-R4), and no assemblages were seen close to the ampulla (Fig. 3A, R5-R6). From the isthmus to the ampulla, the sperm density reduced, as did the amplitude and frequency of the shuttling flows that were not present in regions close to the ampulla (Fig. 3A). When suppressing muscular activity with Padrin, no sperm assemblage or shuttling movements were observed, and sperm swam randomly with only a small number found in the region leading to the ampulla (Ishikawa et al. 2016). These findings suggest that oviductal contractions play a role in transporting sperm assemblages through the isthmus but not in the region close to the ampulla. Considering the strong shuttling flows that move the sperm assemblages, these observations imply that the sperm taxes (rheotaxis, chemotaxis, and thermotaxis) which have been largely studied ex vivo (Li & Winuthayanon 2017) are unlikely to be the primary cues guiding sperm transport through the isthmus. This was further confirmed by in vivo assessment, with the oviduct kept within an actively perfused physiological environment and with the use of Indian ink to seed the oviductal fluid (Hino & Yanagimachi 2019). Specifically, the fluid flow within the oviduct was found to be predominantly and overall adovarian (toward the ovary) at all times throughout the estrous cycle and early pregnancy, and interestingly, arresting the oviductal contraction did not stop the adovarian fluid flow which could also be produced by a large volume of secretion from the entire length of the isthmus (Hino & Yanagimachi 2019). Based on the observation of rapid adovarian flow in the mouse oviduct, Hino and Yanagimachi proposed that there could be more of both live and dead sperm delivered to the fertilization site than expected, and a sperm-trapping action by the cumulus cell clouds could lead to fertilization (Hino & Yanagimachi 2019).
Sperm dynamics observed in the mouse oviduct in vivo. (A) Illustration of sperm transport in the oviduct isthmus in the context of oviduct contractions. Adapted from Ishikawa et al. (2016), Creative Commons license CC BY-NC 4.0. (B) Illustration of sperm movements in the oviduct ampulla relative to the oviductal wall and mucosa folds.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
Sperm dynamics observed in the mouse oviduct in vivo. (A) Illustration of sperm transport in the oviduct isthmus in the context of oviduct contractions. Adapted from Ishikawa et al. (2016), Creative Commons license CC BY-NC 4.0. (B) Illustration of sperm movements in the oviduct ampulla relative to the oviductal wall and mucosa folds.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
Sperm dynamics observed in the mouse oviduct in vivo. (A) Illustration of sperm transport in the oviduct isthmus in the context of oviduct contractions. Adapted from Ishikawa et al. (2016), Creative Commons license CC BY-NC 4.0. (B) Illustration of sperm movements in the oviduct ampulla relative to the oviductal wall and mucosa folds.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
In the oviduct ampulla, the site of fertilization, diverse sperm movements and activities were observed in vivo in the mouse model (Wang and Larina 2018b), as summarized in Fig. 3B. Sperm cells entering the ampulla tended to move toward the epithelium (termed a sperm collection process), which agrees with the in vitro observations (El-Sherry et al. 2014, Kantsler et al. 2014). After entering the near-epithelium region, the speed of the sperm appeared to be dependent on their distance from the epithelium, featuring a higher speed when closer to the epithelium, which is covered by motile cilia. In vitro studies showed that the beat of cilia induces stronger flow closer to the ciliated surface (Jonas et al. 2011) and that the speed of the sperm changes in response to the fluid flow speed through the rheotaxis (El-Sherry et al. 2014, Kantsler et al. 2014). Together, these observations suggest that the near-epithelium movement of sperm could be a rheotaxis response to the flow field induced by the ciliary beat. Grouping of sperm and separation of sperm aggregate were observed in vivo, producing parallel movements and revealing dynamic interactions among sperm in the ampulla. Also, different patterns of sperm movements were visualized between adjacent mucosa folds, where complex physical and chemical factors could exist and contribute to regulating sperm activities. These in vivo observations suggested new regulatory relations between the sperm and the ampulla environment for fertilization, which could potentially be explored through quantitative, time-resolved 3D analyses.
Oocytes and embryos in vivo in the oviduct
After ovulation, oocytes surrounded by cumulus cells are delivered into the oviduct by ciliary activities at the oviduct infundibulum (Talbot et al. 1999). This marks the start of oviductal transport of oocytes and later preimplantation embryos to the uterus for implantation. Since the early work (Black & Asdell 1958, Greenwald 1961, Boling & Blandau 1971b, Halbert et al. 1976a,b), experiments studying how the oviduct drives and controls this process have been primarily performed in live, small animal models because the function of the oviduct is difficult to maintain once excised. However, detailed dynamics of the oocyte/embryo movement were largely unavailable when assessing transport in response to manipulations of the oviduct.
Wide-field microscopy
Given the relatively large size of oocytes and preimplantation embryos, they can be directly located through the oviduct wall in the mouse model by widefield microscopy with just white-light illumination and detection. This is particularly convenient and effective for observing the cumulus–oocyte complex in the ampulla (Fig. 4A), where a large number of cell complexes significantly expand the lumen (especially in the superovulation case), resulting in an even thinner and more transparent oviduct wall. In contrast, imaging oocytes or embryos in the isthmus through the same bright-field imaging approach could be challenging, due to the increased wall thickness in the isthmus and the faster oocyte/embryo movement.
Imaging in vivo dynamics of oocytes/embryos in the mouse oviduct. (A) Widefield microscopy of cumulus–oocyte complexes inside the oviduct ampulla of superovulated mice with white-light illumination and detection. Adapted from La Spina et al. (2016) with permission. (B) In vivo 3D dynamic OCT imaging of cumulus–oocyte complexes in the lower ampulla (top). A sample trajectory of an oocyte and mapping of movement speed (bottom). The scale bars are 200 µm (top) and 30 µm (bottom). (C) Illustration of the distinct oocyte/embryo dynamics during transport through the oviduct. (B) and (C) adapted from Wang & Larina (2021) with permission.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
Imaging in vivo dynamics of oocytes/embryos in the mouse oviduct. (A) Widefield microscopy of cumulus–oocyte complexes inside the oviduct ampulla of superovulated mice with white-light illumination and detection. Adapted from La Spina et al. (2016) with permission. (B) In vivo 3D dynamic OCT imaging of cumulus–oocyte complexes in the lower ampulla (top). A sample trajectory of an oocyte and mapping of movement speed (bottom). The scale bars are 200 µm (top) and 30 µm (bottom). (C) Illustration of the distinct oocyte/embryo dynamics during transport through the oviduct. (B) and (C) adapted from Wang & Larina (2021) with permission.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
Imaging in vivo dynamics of oocytes/embryos in the mouse oviduct. (A) Widefield microscopy of cumulus–oocyte complexes inside the oviduct ampulla of superovulated mice with white-light illumination and detection. Adapted from La Spina et al. (2016) with permission. (B) In vivo 3D dynamic OCT imaging of cumulus–oocyte complexes in the lower ampulla (top). A sample trajectory of an oocyte and mapping of movement speed (bottom). The scale bars are 200 µm (top) and 30 µm (bottom). (C) Illustration of the distinct oocyte/embryo dynamics during transport through the oviduct. (B) and (C) adapted from Wang & Larina (2021) with permission.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
Optical coherence tomography (Box 2)
The large size and distinct shape of oocytes and embryos make OCT ideal for imaging them through the oviductal wall in vivo in the mouse model (Wang & Larina 2021). Specifically, the microscale resolution not only enables distinction of the oocyte and embryo from other cells and particles but also resolves cellular details for staging embryos (Moore et al. 2019). The large 3D field of view provides volumetric visualizations of oocytes/embryos together with the oviductal structure and morphology (Fig. 4B). The fast imaging speed also permits following the oocyte/embryo movement throughout different sections of the oviduct. These advantages have made it possible to track and measure the movement of oocytes and embryos in 3D as they are transported inside the oviduct (Fig. 4B), and their detailed dynamics can be analyzed within the context of the oviductal activities, such as during muscle contraction (Wang & Larina 2021).
In vivo insights
In vivo imaging in the mouse revealed more complex dynamics of oocytes/embryos than previously thought, and under normal conditions (natural ovulation and mating), new patterns of oocyte/embryo movements in relation to the oviductal contraction were observed (Wang & Larina 2021). Figure 4C summarizes such findings. In the upper ampulla, the group of cumulus–oocyte complexes formed a circular movement at an average speed of 7–18.4 µm/s, which was hypothesized to be driven by the beat of the motile cilia lining the epithelium. Interestingly, as a fast circulating cloud, the group of cumulus–oocyte complexes slowly moved toward the lower ampulla (the site of fertilization). A luminal constriction along the way formed a ‘gate’ that controlled the net advancement of the entire cloud by the gradual opening of the lumen. The ‘gate’ formation and opening, likely produced by tonic muscle contraction and relaxation, respectively, suggested a role for muscular activities, not in driving the oocytes to the fertilization site but potentially in regulating the timing of transport. This agrees with earlier findings that, under blocked oviductal contraction, oocytes can still be delivered to the lower ampulla (Halbert et al. 1976a,b, 1989). This previous observation that the ciliary beat itself can successfully transport oocytes does not necessarily mean that contractions of the oviduct do not contribute to this process. The detailed observation of the ‘gate’ dynamics in vivo suggested a new mechanism for how muscular activities contribute to oviductal transport. In the lower ampulla, the movement pattern changed from circling to oscillating, which coincided with muscle contraction and relaxation in the upper isthmus. After fertilization, embryo groups were observed in the lower ampulla that can remain relatively stable and intact, despite the surrounding dynamics in the environment. Separations of embryos from the group were also seen. Although not much related to human reproduction, embryo grouping at the initial developmental stage might be of interest for further study in vivo. Embryos entering the upper and mid-isthmus exhibited bidirectional long-distance movements accompanied by peristaltic oviductal contractions. After reaching the lower isthmus close to the utero-tubal junction (UTJ), the embryos were accumulated within a small region for further transport to the uterus. As the oviductal transport direction of oocytes/embryos is opposite to that of sperm, the underlying mechanism regulating the entire transport process is likely dependent on the estrous timing and fertilization state and is region specific inside the oviduct. The described dynamics under the normal reproductive state could serve as a basis for further investigation into the function of the oviduct in both healthy and disordered conditions.
Emerging directions and outlooks
Emerging applications and novel biological hypotheses drive technological development, while technical advancements enable new applications stimulating new biological hypotheses. The relationship between reproductive biology and in vivo imaging is no exception. The need to understand the reproductive process inside the mammalian oviduct has pushed forward the development of in vivo imaging approaches, and it is exciting to see that, as a result, several areas of reproductive studies are evolving rapidly, particularly the study of oocyte/embryo retention and the fertilization process.
Oocyte/embryo retention in the oviduct
Understanding the cause of tEP is critical for improving the clinical management of this life-threatening disorder. Although some risk factors have been identified, the molecular, cellular, and functional mechanisms remain largely unknown (Shaw et al. 2010). Laboratory mice generally do not experience tEP (Corpa 2006); however, oocyte/embryo retention, which is a prerequisite of tEP, has been reported in an increasing number of mouse models, offering valuable opportunities to investigate and elucidate its underlying mechanisms. Such models include CB1-deficient mice (Wang et al. 2004), Dicer conditional mutants (Hong et al. 2008, Gonzalez & Behringer 2009), mice with either silenced or enhanced H2S signaling (Ning et al. 2014), a moderate (5 mg/kg) or high dose (30 mg/kg) of caffeine exposure (Qian et al. 2018), Esr1 conditional mutants (Herrera et al. 2020), and Adgrd1-deficient mice (Bianchi et al. 2021). In vivo imaging and characterization of oocyte/embryo retention in these mouse models could link the molecular genetic aspects to the oviductal functions, thus elucidating the entire path leading to transport failure. Excitingly, the recent study on Adgrd1-deficient mice started incorporating in vivo imaging to reveal the direct functional cause of oocyte/embryo retention in the ampulla (Bianchi et al. 2021). By assessing the oviductal flow in vivo (Fig. 5A), the authors found that the oviduct lacking Adgrd1 on the epithelium cannot attenuate the adovarian fluid flow postovulation, thus maintaining a balance between the abovarian (away from the ovary) and adovarian flows at the IAJ, which retained the oocytes/embryos inside the ampulla (Bianchi et al. 2021). This highlighted the value of in vivo dynamic phenotyping for investigating disordered processes of oocyte/embryo transport in the oviduct.
In vivo dynamics at the oviduct isthmic–ampullary junction (IAJ). (A) Kymographs of the injected dye in the oviduct of the Adgrd1 mutant and control at 1.5 days postconception in vivo showed that oviductal flow in the control segregated the dye into boluses and moved them gradually toward the ampulla, while the dye in the mutant oviduct instantaneously filled the entire oviduct. Adapted from Bianchi et al. (2021), Creative Commons license CC BY 4.0. (B) A time-lapse 3D visualization of one individual embryo separating from the embryo group and having a bidirectional movement at the IAJ in vivo. Arrows point at the same embryo over time. The scale bars are 200 µm. Adapted from Wang & Larina (2021) with permission.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
In vivo dynamics at the oviduct isthmic–ampullary junction (IAJ). (A) Kymographs of the injected dye in the oviduct of the Adgrd1 mutant and control at 1.5 days postconception in vivo showed that oviductal flow in the control segregated the dye into boluses and moved them gradually toward the ampulla, while the dye in the mutant oviduct instantaneously filled the entire oviduct. Adapted from Bianchi et al. (2021), Creative Commons license CC BY 4.0. (B) A time-lapse 3D visualization of one individual embryo separating from the embryo group and having a bidirectional movement at the IAJ in vivo. Arrows point at the same embryo over time. The scale bars are 200 µm. Adapted from Wang & Larina (2021) with permission.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
In vivo dynamics at the oviduct isthmic–ampullary junction (IAJ). (A) Kymographs of the injected dye in the oviduct of the Adgrd1 mutant and control at 1.5 days postconception in vivo showed that oviductal flow in the control segregated the dye into boluses and moved them gradually toward the ampulla, while the dye in the mutant oviduct instantaneously filled the entire oviduct. Adapted from Bianchi et al. (2021), Creative Commons license CC BY 4.0. (B) A time-lapse 3D visualization of one individual embryo separating from the embryo group and having a bidirectional movement at the IAJ in vivo. Arrows point at the same embryo over time. The scale bars are 200 µm. Adapted from Wang & Larina (2021) with permission.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
Based on the locations inside the oviduct where retained embryos were found, it is believed that the anatomical boundaries, including the IAJ and UTJ, could be critical in generating retentions for embryos. Thus, understanding how oocytes/embryos are normally transported through these two regions is of high significance. Recent in vivo imaging focused on the dynamics at the IAJ (Wang & Larina 2021), which suggested that transport through this location is performed as a queue of individual embryos with repeated bidirectional movements at the IAJ (Fig. 5B). Since embryos form groups in the lower ampulla, which do not pass through the IAJ as a whole, the IAJ possibly serves the role of breaking embryo groups into the individuals that were observed in the upper and mid-isthmus (Wang & Larina 2021). With specific and targeted disruption of the activities of oviductal smooth muscle, cilia, and secretory cells, further insights that precisely identify the functional control of oocyte/embryo transport at the IAJ and UTJ are expected, which will be a major step toward understanding tEP.
Fertilization in the oviduct
From discovering the sperm capacitation (Austin 1951, Chang 1951) to uncovering the role of CatSper in sperm motility and male fertility (Ren et al. 2001, Ren & Xia 2010), reproductive biologists have gained much understanding of sperm and fertilization (Okabe 2018); however, studying the process of mammalian fertilization in vivo remains a challenge. Now, we are closer than ever before to making it a reality. The identification and localization of sperm cells in the mouse oviduct in vivo provided a promising route to enable imaging of the fertilization process within its native environment. In fact, with fluorescence-labeled sperm, the process of sperm interaction with cumulus–oocyte complexes has been imaged inside the ampulla of the oviduct isolated from the female mouse (Hino et al. 2016). The interesting observation showed that an acrosome-reacted sperm cell moved in and out of the cumuli oophori of two fertilized oocytes before heading toward a third oocyte that was unfertilized (Fig. 6A). Sperm passing through the oocyte zona pellucida was not captured in the oviduct, but the observed process revealed how a spermatozoon entered the cumulus oophorus. The surprising efficiency of the sperm at identifying and moving away from fertilized oocytes suggested a mechanism for the sperm to quickly recognize the differences in zonae pellucidae between fertilized and unfertilized oocytes (Hino et al. 2016). Further in vivo imaging studies within the dynamic oviductal environment are expected to bring new findings about the regulation of fertilization (Yanagimachi 2022).
Toward in vivo imaging of fertilization. (A) Visualization of a fluorescence-labeled mouse spermatozoon moving in and out of the cumuli oophori of two fertilized oocytes and entering the cumulus oophorus of an unfertilized oocyte in the oviduct ampulla. In the left panel, the yellow arrows point at fertilized oocytes, and the white arrow points at an unfertilized oocyte. In the right panel, the arrow points at the sperm. Adapted from Hino et al. (2016), Creative Commons license CC BY-NC 4.0. (B) Label-free cross-sectional 3D OCT imaging of cumulus-oocyte complexes in the mouse oviduct ampulla. The scale bar is 100 µm.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
Toward in vivo imaging of fertilization. (A) Visualization of a fluorescence-labeled mouse spermatozoon moving in and out of the cumuli oophori of two fertilized oocytes and entering the cumulus oophorus of an unfertilized oocyte in the oviduct ampulla. In the left panel, the yellow arrows point at fertilized oocytes, and the white arrow points at an unfertilized oocyte. In the right panel, the arrow points at the sperm. Adapted from Hino et al. (2016), Creative Commons license CC BY-NC 4.0. (B) Label-free cross-sectional 3D OCT imaging of cumulus-oocyte complexes in the mouse oviduct ampulla. The scale bar is 100 µm.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
Toward in vivo imaging of fertilization. (A) Visualization of a fluorescence-labeled mouse spermatozoon moving in and out of the cumuli oophori of two fertilized oocytes and entering the cumulus oophorus of an unfertilized oocyte in the oviduct ampulla. In the left panel, the yellow arrows point at fertilized oocytes, and the white arrow points at an unfertilized oocyte. In the right panel, the arrow points at the sperm. Adapted from Hino et al. (2016), Creative Commons license CC BY-NC 4.0. (B) Label-free cross-sectional 3D OCT imaging of cumulus-oocyte complexes in the mouse oviduct ampulla. The scale bar is 100 µm.
Citation: Reproduction 165, 2; 10.1530/REP-22-0250
With widefield fluorescence imaging being largely limited to 2D visualizations, the 3D imaging capability of OCT is attractive for pursuing quantitative dynamics of the fertilization process. However, the existing sperm identification method (Wang & Larina 2018b) cannot be used after the sperm enter the cumulus oophorus, as direct tracking becomes challenging through densely packed cumulus cells. Novel contrasts for in vivo imaging of the motile sperm within the cumulus–oocyte complex are needed and are under development (Tian et al. 2022). In addition to its distinct moving trajectory, a motile sperm cell also present unique movements of its flagellum (Gaffney et al. 2011). Although the resolution of OCT cannot resolve the sperm flagella, their dynamics could produce changes in the OCT signals over time, like the imaging of ciliary beat with OCT (Oldenburg et al. 2012, Wang et al. 2015, Jing et al. 2017). Analyzing those changes could potentially distinguish motile sperm from other cells or particles in the oviduct. This type of contrast mechanism will significantly improve the efficiency of sperm identification in vivo, while challenges exist in optimizing the imaging parameters, such as the speed, and in processing the OCT signals. With the rapid advancement in OCT technologies (Kim et al. 2015, de Boer et al. 2017) and also an increasing involvement of OCT in studying developmental biology (Men et al. 2016, Wang et al. 2020), we are optimistic about seeing new OCT-based contrasts soon for in vivo sperm imaging in the mouse oviduct. Together with the powerful imaging scale of OCT that can resolve the cumulus–oocyte complex in the oviduct ampulla (Fig. 6B), such technical innovations will bring an enhanced approach for imaging fertilization and other reproductive events in vivo.
Conclusions
In vivo imaging of gametes and embryos in the oviduct is creating a new frontier in studying the processes leading to fertilization and pregnancy, particularly in the mouse model. The continuously refined optical access to the mouse oviduct and advancements in imaging strategies and methods have uncovered a range of dynamics for understanding the detailed role of the oviduct in mammalian reproduction. These insights and further technical innovations promise to give rise to more in vivo studies elucidating the reproductive process in its native state.
Declaration of interest
The authors declare no conflict of interest.
Funding
This work was supported by the National Institutes of Health (grant R21EB028409 to S.W. and grant R01EB027099 and R01HD096335 to I.V.L.).
Author contribution statement
SW and IVL designed this review. SW drafted the manuscript. IVL revised the manuscript.
Acknowledgements
The authors would like to thank Andre C. Faubert (Stevens Institute of Technology) and Maria Larina (The University of Texas at Austin) for their critical reading of the manuscript.
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