Tissue clearing and imaging approaches for in toto analysis of the reproductive system†.

New microscopy techniques in combination with tissue clearing protocols and emerging analytical approaches have presented researchers with the tools to understand dynamic biological processes in a three-dimensional context. This paves the road for the exploration of new research questions in reproductive biology, for which previous techniques have provided only approximate resolution. These new methodologies now allow for contextualized analysis of far-larger volumes than was previously possible. Tissue optical clearing and three-dimensional imaging techniques posit the bridging of molecular mechanisms, macroscopic morphogenic development, and maintenance of reproductive function into one cohesive and comprehensive understanding of the biology of the reproductive system. In this review, we present a survey of the various tissue clearing techniques and imaging systems, as they have been applied to the developing and adult reproductive system. We provide an overview of tools available for analysis of experimental data, giving particular attention to the emergence of artificial intelligence-assisted methods and their applicability to image analysis. We conclude with an evaluation of how novel image analysis approaches that have been applied to other organ systems could be incorporated into future experimental evaluation of reproductive biology.

Fetal cannabidiol (CBD) exposure alters thermal pain sensitivity, problem-solving, and prefrontal cortex excitability.

Thousands of people suffer from nausea with pregnancy each year. Nausea can be alleviated with cannabidiol (CBD), a primary component of cannabis that is widely available. However, it is unknown how fetal CBD exposure affects embryonic development and postnatal outcomes. CBD binds and activates receptors that are expressed in the fetal brain and are important for brain development, including serotonin receptors (5HT1A), voltage-gated potassium (Kv)7 receptors, and the transient potential vanilloid 1 receptor (TRPV1). Excessive activation of each of these receptors can disrupt neurodevelopment. Here, we test the hypothesis that fetal CBD exposure in mice alters offspring neurodevelopment and postnatal behavior. We administered 50 mg/kg CBD in sunflower oil or sunflower oil alone to pregnant mice from embryonic day 5 through birth. We show that fetal CBD exposure sensitizes adult male offspring to thermal pain through TRPV1. We show that fetal CBD exposure decreases problem-solving behaviors in female CBD-exposed offspring. We demonstrate that fetal CBD exposure increases the minimum current required to elicit action potentials and decreases the number of action potentials in female offspring layer 2/3 prefrontal cortex (PFC) pyramidal neurons. Fetal CBD exposure reduces the amplitude of glutamate uncaging-evoked excitatory post-synaptic currents, consistent with CBD-exposed female problem-solving behavior deficits. Combined, these data show that fetal CBD exposure disrupts neurodevelopment and postnatal behavior in a sex specific manner.

OoCount: A Machine-Learning Based Approach to Mouse Ovarian Follicle Counting and Classification.

The number and distribution of ovarian follicles in each growth stage provides a reliable readout of ovarian health and function. Leveraging techniques for three-dimensional (3D) imaging of ovaries in toto has the potential to uncover total, accurate ovarian follicle counts. However, because of the size and holistic nature of these images, counting oocytes is time consuming and difficult. The advent of deep-learning algorithms has allowed for the rapid development of ultra-fast, automated methods to analyze microscopy images. In recent years, these pipelines have become more user-friendly and accessible to non-specialists. We used these tools to create OoCount, a high-throughput, open-source method for automatic oocyte segmentation and classification from fluorescent 3D microscopy images of whole mouse ovaries using a deep-learning convolutional neural network (CNN) based approach. We developed a fast tissue-clearing and spinning disk confocal-based imaging protocol to obtain 3D images of whole mount perinatal and adult mouse ovaries. Fluorescently labeled oocytes from 3D images of ovaries were manually annotated in Napari to develop a machine learning training dataset. This dataset was used to retrain StarDist using a CNN within DL4MicEverywhere to automatically label all oocytes in the ovary. In a second phase, we utilize Accelerated Pixel and Object Classification, a Napari plugin, to classify labeled oocytes and sort them into growth stages. Here, we provide an end-to-end protocol for producing high-quality 3D images of the perinatal and adult mouse ovary, obtaining follicle counts and staging. We also demonstrate how to customize OoCount to fit images produced in any lab. Using OoCount, we can obtain accurate counts of oocytes in each growth stage in the perinatal and adult ovary, improving our ability to study ovarian function and fertility. Summary sentence: This protocol introduces OoCount, a high-throughput, open-source method for automatic oocyte segmentation and classification from fluorescent 3D microscopy images of whole mouse ovaries using a machine learning-based approach.

Chemogenetic activation of CRF neurons as a model of chronic stress produces sex-specific physiological and behavioral effects.

Trauma and chronic stress exposure are the strongest predictors of lifetime neuropsychiatric disease presentation. These disorders often have significant sex biases, with females having higher incidences of affective disorders such as major depression, anxiety, and PTSD. Understanding the mechanisms by which stress exposure heightens disease vulnerability is essential for developing novel interventions. Current rodent stress models consist of a battery of sensory, homeostatic, and psychological stressors that are ultimately integrated by corticotropin-releasing factor (CRF) neurons to trigger corticosteroid release. These stress paradigms, however, often differ between research groups in the type, timing, and duration of stressors utilized. These inconsistencies, along with the variability of individual animals' perception and response to each stressor, present challenges for reproducibility and translational relevance. Here, we hypothesized that a more direct approach using chemogenetic activation of CRF neurons would recapitulate the effects of traditional stress paradigms and provide a high-throughput method for examining stress-relevant phenotypes. Using a transgenic approach to express the Gq-coupled Designer Receptor Exclusively Activated by Designer Drugs (DREADD) receptor hM3Dq in CRF-neurons, we found that the DREADD ligand clozapine-N-oxide (CNO) produced an acute and robust activation of the hypothalamic-pituitary-adrenal (HPA) axis, as predicted. Interestingly, chronic treatment with this method of direct CRF activation uncovered a novel sex-specific dissociation of glucocorticoid levels with stress-related outcomes. Despite hM3Dq-expressing females producing greater corticosterone levels in response to CNO than males, hM3Dq-expressing males showed significant typical physiological stress sensitivity with reductions in body and thymus weights. hM3Dq-expressing females while resistant to the physiological effects of chronic CRF activation, showed significant increases in baseline and fear-conditioned freezing behaviors. These data establish a novel mouse model for interrogating stress-relevant phenotypes and highlight sex-specific stress circuitry distinct for physiological and limbic control that may underlie disease risk.

Extracellular vesicles are dynamic regulators of maternal glucose homeostasis during pregnancy.

Homeostatic regulation of the maternal milieu during pregnancy is critical for maternal and fetal health. The placenta facilitates critical communication between maternal and fetal compartments, in part, through the production of extracellular vesicles (EVs). EVs enable tissue synchrony via cell-cell and long-distance communication and are at their highest circulating concentration during pregnancy. While much work has been done investigating how physiological challenges in pregnancy affect the fetus, the role of placental communication in maternal health has not been well examined. We previously identified placental O-glycosyl transferase (OGT), a glucose-sensing enzyme, as a target of maternal stress where OGT levels and activity affected the O-glycosylation of proteins critical for EV cargo loading and secretion. Here, we hypothesized that placental OGT plays an essential role in maternal homeostatic regulation during pregnancy via its regulation of maternal circulating EV concentrations. Our studies found that changes to key metabolic factors over the circadian cycle, including glucocorticoids, insulin, and glucose, were significantly associated with changes in circulating EV concentration. Targeting placental OGT in mice, we found a novel significant positive relationship between placental OGT and maternal circulating EV concentration that was associated with improving maternal glucose tolerance during pregnancy. Finally, an intravenous elevation in EVs, matching the concentration of EVs during pregnancy, shifted non-pregnant female glucose sensitivity, blunted glucose variance, and improved synchrony of glucose uptake. These data suggest an important and novel role for circulating EVs as homeostatic regulators important in maternal health during pregnancy.

Developmental transcriptomic patterns can be altered by transgenic overexpression of Uty.

The genetic material encoded on X and Y chromosomes provides the foundation by which biological sex differences are established. Epigenetic regulators expressed on these sex chromosomes, including Kdm6a (Utx), Kdm5c, and Ddx3x have far-reaching impacts on transcriptional control of phenotypic sex differences. Although the functionality of UTY (Kdm6c, the Y-linked homologue of UTX), has been supported by more recent studies, its role in developmental sex differences is not understood. Here we test the hypothesis that UTY is an important transcriptional regulator during development that could contribute to sex-specific phenotypes and disease risks across the lifespan. We generated a random insertion Uty transgenic mouse (Uty-Tg) to overexpress Uty. By comparing transcriptomic profiles in developmental tissues, placenta and hypothalamus, we assessed potential UTY functional activity, comparing Uty-expressing female mice (XX + Uty) with wild-type male (XY) and female (XX) mice. To determine if Uty expression altered physiological or behavioral outcomes, adult mice were phenotypically examined. Uty expression masculinized female gene expression patterns in both the placenta and hypothalamus. Gene ontology (GO) and gene set enrichment analysis (GSEA) consistently identified pathways including immune and synaptic signaling as biological processes associated with UTY. Interestingly, adult females expressing Uty gained less weight and had a greater glucose tolerance compared to wild-type male and female mice when provided a high-fat diet. Utilizing a Uty-overexpressing transgenic mouse, our results provide novel evidence as to a functional transcriptional role for UTY in developing tissues, and a foundation to build on its prospective capacity to influence sex-specific developmental and health outcomes.

The composition of human vaginal microbiota transferred at birth affects offspring health in a mouse model.

Newborns are colonized by maternal microbiota that is essential for offspring health and development. The composition of these pioneer communities exhibits individual differences, but the importance of this early-life heterogeneity to health outcomes is not understood. Here we validate a human microbiota-associated model in which fetal mice are cesarean delivered and gavaged with defined human vaginal microbial communities. This model replicates the inoculation that occurs during vaginal birth and reveals lasting effects on offspring metabolism, immunity, and the brain in a community-specific manner. This microbial effect is amplified by prior gestation in a maternal obesogenic or vaginal dysbiotic environment where placental and fetal ileum development are altered, and an augmented immune response increases rates of offspring mortality. Collectively, we describe a translationally relevant model to examine the defined role of specific human microbial communities on offspring health outcomes, and demonstrate that the prenatal environment dramatically shapes the postnatal response to inoculation.