Author response: Integration of mouse ovary morphogenesis with developmental dynamics of the oviduct, ovarian ligaments, and rete ovarii (2022)

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Morphogenetic events during the development of the fetal ovary are crucial to the establishment of female fertility. However, the effects of structural rearrangements of the ovary and surrounding reproductive tissues on ovary morphogenesis remain largely uncharacterized. Using tissue clearing and lightsheet microscopy, we found that ovary folding correlated with regionalization into cortex and medulla. Relocation of the oviduct to the ventral aspect of the ovary led to ovary encapsulation, and mutual attachment of the ovary and oviduct to the cranial suspensory ligament likely triggered ovary folding. During this process, the rete ovarii (RO) elaborated into a convoluted tubular structure extending from the ovary into the ovarian capsule. Using genetic mouse models in which the oviduct and RO are perturbed, we found the oviduct is required for ovary encapsulation. This study reveals novel relationships among the ovary and surrounding tissues and paves the way for functional investigation of the relationship between architecture and differentiation of the mammalian ovary. Editor's evaluation This work presents a very detailed study on the morphological processes governing mouse ovary morphogenesis from E14.5 to birth using recently developed methods. As a reference to ovary morphogenesis and the establishment of the female genital tract, the manuscript is of interest to all developmental biologists as it will serve as a reference to whole embryo morphogenesis, in particular vertebrate ovary morphogenetics processes. https://doi.org/10.7554/eLife.81088.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest In humans and other mammals, the female reproductive organs, or ovaries, develop early in life, while the young are still in their mother’s womb. Ovaries contain several different compartments, including the ovarian follicles. These are small groups of cells that produce reproductive hormones, and each follicle also has the potential to produce one egg for fertilisation. The ovaries are further surrounded by different tissues that develop alongside them. These include the oviducts, which carry fertilised eggs from the ovaries into the womb, and ligaments, which anchor the ovaries to the wall of the body cavity. During the development of ovaries, ovarian follicles are sorted into two distinct groups. The first, called medullary follicles, are lost before puberty. The second group, or cortical follicles, remain in a state of ‘suspended animation’ until puberty. After that, they act as a ‘reserve’ of eggs for the rest of the reproductive lifespan. Once each cortical follicle has produced an egg, it is not replenished. This means that proper follicle sorting is crucial for establishing female fertility, and therefore the ability to conceive. The mechanisms behind follicle sorting, however, are still poorly understood. McKey et al. set out to determine how the ovary’s structure changed during its development. In the experiments, high-resolution microscopy techniques were used to reconstruct ovaries of mice in 3D across different stages of development. This revealed that the ends of each ovary started folding towards each other just before birth, and that the folding also happened at the same time as follicle sorting. Simultaneous changes in the shape and orientation of the ligaments suggested that these tissues might direct the folding, for example by pushing or pulling on the rest of the ovary. These results suggest that the changes in ovary structure in early life are critically linked to the establishment of the ovary’s egg reserves. McKey et al. hope that this study will pave the way to a better understanding of infertility and, ultimately, better treatments. Introduction During mouse fetal development, the gonad first arises on the ventromedial aspect of the mesonephros (Brambell and Hill, 1927; Harikae et al., 2013; Karl and Capel, 1998; Windley and Wilhelm, 2015). In XX embryos, the gonads develop into ovaries, where supporting cells become granulosa cells, interstitial cells become theca cells and other stromal cell types, and germ cells become oocytes (Nef et al., 2019; Rastetter et al., 2014; Stévant et al., 2019; Wilhelm et al., 2013). As these cells undergo differentiation between E11.5 and birth, they progressively assemble into individual ovarian follicles, characterized by the presence of one oocyte surrounded by two or three pre-granulosa cells (Lei and Spradling, 2016; Lei and Spradling, 2013; Liu et al., 2010; Mork et al., 2012; Pepling and Lei, 2018; Pepling, 2012; Zheng et al., 2014).The advent of single-cell transcriptomics has provided a wealth of information on the cellular profiles and gene expression trajectories within the developing ovary, presenting an unprecedented view of the molecular and cellular pathways at play during mammalian ovary differentiation and follicle formation (Li et al., 2017; Liu et al., 2010; Mayère et al., 2022; Niu and Spradling, 2020; Stévant et al., 2019). However, transcriptomics alone do not address the spatial and structural components that underlie ovary regionalization and patterning. In fact, while granulosa cells and oocytes are locally assembling into follicles, the ovary as a whole undergoes dramatic changes in shape and cell composition, including the integration of extrinsic components such as lymphatics, mesenchymal theca cells, and innervation (Liu et al., 2015; McKey et al., 2019; Svingen et al., 2012). The events that occur during ovary morphogenesis are poorly defined and lack context, owing in part to the scarcity of molecular and imaging tools to study the ovary in its native position. Similarly, tissues closely associated with the ovary, such as the Müllerian duct (MD), ovarian ligaments, and the rete ovarii (RO), that also undergo dramatic transitions during development Adham et al., 2002; Ford et al., 2021; Lee et al., 2011; Wenzel and Odend’hal, 1985 have been studied individually, but not in the context of ovary morphogenesis. Consequently, their impact on ovary morphogenesis has been overlooked. With recent advances in tissue clearing and 3D imaging technology, it is now possible to visualize structural changes at the level of the whole gonad or even the whole embryo (Bunce et al., 2021; Fiorentino et al., 2021; Soygur et al., 2021a; Soygur and Laird, 2021b). We recently developed a tissue-clearing method that combines CUBIC and iDISCO (McKey et al., 2020; Renier et al., 2014; Susaki et al., 2015), and optimized 3D imaging methods using lightsheet microscopy to visualize the fetal and perinatal ovary in situ, in the context of its surrounding tissues (McKey et al., 2019). Using our 3D-imaging methods and high-resolution confocal microscopy, we provide a detailed and contextual description of mouse ovary morphogenesis from E14.5 to birth. We define three discrete hallmarks of ovary morphogenesis: (1) ovary folding, (2) specification of medullary and cortical compartments, and (3) ovary encapsulation. We integrated this morphometric approach with a detailed study of three tissues closely associated with the ovary: the ovarian ligaments, the MD and the RO, and found compelling correlations between the developmental dynamics of these tissues and ovary morphogenesis. Finally, we found that different combinations of Pax2 and Pax8 deletion alleles perturb the oviduct and/or regions of the RO differentially, and lead to disruptions in ovary morphogenesis. Results 3D models of developing ovaries provide a new perspective on ovary morphology The gonad first arises on the coelomic surface of the mesonephros. Early gonads are generally visualized and studied in complex with the mesonephros, which hosts the developing reproductive ducts. The mesonephric duct, which gives rise to the Wolffian duct (WD), is present from the onset of gonadogenesis (Brambell, 1927; Kobayashi and Behringer, 2003; Zhao and Yao, 2019). However, the MD develops later by invagination of the mesonephric epithelium between E11.5 and E13.5 in the mouse (Huang et al., 2014; Mullen and Behringer, 2014; Orvis and Behringer, 2007; Roly et al., 2018). While both WD and MD initially develop in all embryos, only one is maintained depending on the sex of the embryo. In female embryos, the WD fully regresses by E15.5 (Zhao et al., 2021), while the MD is maintained and gives rise to the oviduct (most proximate to the ovary), the uterus, the cervix, and the anterior aspect of the vagina (Kobayashi and Behringer, 2003). We chose E14.5 as the starting stage for our study, as this precedes the major changes in shape and conformation that are specific to the female gonad. To determine the optimal angles to assess ovary morphology, we first performed immunostaining of whole XX embryos at E14.5 with antibodies against FOXL2, which is expressed in pre-granulosa cells and serves as a marker of the gonadal domain, and antibodies against PAX8, which is expressed in the epithelium of the reproductive ducts and cells of the developing kidney. These samples were cleared using iDISCO and imaged with lightsheet microscopy (Bunce et al., 2021; Renier et al., 2014). We generated 3D models of the labeled structures using isosurface segmentation to provide a morphological view of the urogenital complex in situ (Figure 1A, Figure 1—video 1). We found that at E14.5, the bilateral gonad/mesonephros complexes resided in the posterior third of the embryo, in a plane orthogonal to the midline axis of the embryo (Figure 1A) located on the ventro-lateral aspect of the developing kidneys (Figure 1B). A recent study combining contextual 3D images of the developing gonads in the whole embryo with quantitative morphometric analysis showed that the entire genital ridge rotates inward from E10.5 to E12.5 (Bunce et al., 2021), thus by E12.5, the gonads appear on the medial aspect of the mesonephros. Consistent with these observations, at E14.5, the reproductive ducts were located lateral to the gonads, which were oriented toward each other in the medial axis (Figure 1B). We used the 3D objects representing the ovaries and ducts in this image to define the six views that are presented throughout this study (Figure 1C). The caudal and cranial views best illustrated the position and shifts of the ducts relative to the ovary (Figure 1B), while the dorsal, medial, lateral, and ventral views provided information on the shape and regionalization of the ovary itself (Figure 1C). Dorsal and ventral views also provided context on the position and development of the individual ducts and mesonephric tubules inside the mesonephros (Figure 1C, top panels). Lateral and medial views were optimal for observing the dorsal/ventral regionalization of the ovary and the interface between the gonad and the mesonephros (Figure 1C, bottom panels). Figure 1 with 1 supplement see all Download asset Open asset 3D models of developing ovaries provide a new perspective of ovary morphology. (A) 3D model generated by isosurface segmentation of lightsheet images taken of a whole XX embryo at E14.5 immunostained for PAX8 (kidney, reproductive ducts, green, red) and FOXL2 (gonad, cyan). (B) Caudal and cranial views of the surfaces representing the urogenital system, with and without the kidney surfaces, isolated from the whole embryo images in (A). (C) Close-ups of dorsal, ventral, medial, and lateral views of the surfaces representing the ovary and reproductive ducts, isolated from the images in (A). MD, Müllerian duct; MT, mesonephric tubule; WD, Wolffian duct. Yellow asterisks indicate the location of the infundibulum of the presumptive oviduct for reference. Compasses on the bottom left of each panel indicate the orientation of the ovary: A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral. Scale bars, 100 μm. The ovary transitions from elongated to crescent-shaped during late gestation Having defined the contextual components of ovary development at E14.5 and the optimal ways of visualizing morphogenetic changes, we next studied the shape of the ovary from E14.5 to birth. For this, we used immunostaining with antibodies against FOXL2 and first imaged whole-mount gonad/mesonephros complexes using confocal microscopy (Figure 2A). At E14.5, the ovary was still shaped as an elongated tissue developing on the medial face of the mesonephros. By E17.5, the ovary adopted a rounder conformation. The dorsal side eventually resembled a crescent shape in E18.5 embryos (Figure 2A). This change in conformation was accompanied by a hollowing of the FOXL2 domain in the center of the dorsal face of the ovary (Figure 2A). We next investigated the dynamics of the FOXL2+ domain in whole ovaries cleared with iDISCO+CUBIC and imaged with lightsheet microscopy (Figure 2—figure supplement 1A; Figure 2—video 1). The advantage of this method is that the sample was imaged in its native conformation, in a cylinder of agarose, and thus did not undergo artificial deformation resulting from mounting on a slide (Figure 2—figure supplement 1A,B). With these 3D models, we better visualized the conformation of the ovary in space and observed a similar shift from an elongated to a crescent shape between E14.5 and birth (postnatal day 0, P0) (Figure 2B). This was most visible on the dorsal and ventral sides of the ovary, where anterior and posterior regions appeared to rise toward the medial axis and come closer to each other. This was associated with an increase in the inflexion toward the center of the ovary, and with an ingression of the medial face of the ovary, leading to an increase in the curvature of the lateral face (Figure 2B). Our data thus suggest that the change in conformation of the ovary is representative of a folding process resulting in the center inflexion of the ovary and relocation of anterior and posterior poles. Figure 2 with 2 supplements see all Download asset Open asset The ovary transitions from elongated to crescent-shaped during late gestation. (A) Optical sections from confocal Z-stacks of whole immunostaining of ovary/mesonephros complexes at E14.5, E17.5, and E18.5 immunostained for FOXL2 (cyan) and counterstained with Hoechst nuclear dye (grayscale). (B) 3D model generated by isosurface segmentation of lightsheet images taken of whole ovaries at E14.5, E17.5, and P0 immunostained for FOXL2 (cyan). Top large panels represent the dorsal view, while small bottom panels illustrate ventral, medial, and lateral views of the same ovary. Dashed arrows illustrate the change in conformation of the ovary as it develops: red arrows, relocation of the anterior and posterior poles; green arrows, medial inflexion; orange arrows, increase in lateral concavity. (C) Cranial and caudal views of the surface of the ovaries presented in (B). White arrows point to the dorsal protrusion of the ovarian domain. A, anterior pole; MD, Müllerian duct; Mn, mesonephros; Ov, ovary; P, posterior pole. Compasses on the bottom left of each panel indicate the orientation of the ovary: A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral. Scale bars, 100 μm. Upon closer observation, we found that the anterior portion of the ovary appeared to thicken and shift up above the middle of the ovary, suggesting that the anterior region is subject to more deformation than the posterior region (Figure 2B). Caudal and cranial views of the process showed that while the ventral surface of the ovary remained smooth during ovary folding, a lateral protrusion extended from the mid-region of the dorsal face between E14.5 and E17.5 (Figure 2C, white arrows). Ovary folding results in specification of the medullary and cortical compartments of the ovary Previous studies have shown that the location of individual follicles inside the ovary carries functional significance (Cordeiro et al., 2015; Mork et al., 2012; Suzuki et al., 2015; Zheng et al., 2014). Although follicles assembled during late gestation in the medullary compartment of the ovary initiate growth shortly after birth, they become atretic during the juvenile period and are all depleted by the onset of puberty. In parallel, a new wave of de novo follicle formation occurs around birth in the cortical compartment (Mork et al., 2012; Niu and Spradling, 2020; Zheng et al., 2014). These constitute the finite pool of ovarian follicles that remain quiescent throughout the juvenile period and begin to sequentially activate and initiate growth cyclically at the onset of puberty and until menopause, thus supporting the entire female reproductive lifespan (Duncan and Gerton, 2018; Mork et al., 2012; Zheng et al., 2014). While this functional regionalization points to the influence of ovarian architecture on the organization and outcome of follicles within the ovary, both the origin of the cortical and medullary compartments and the nature of the signals and the mechanisms that drive this differential patterning remain unclear. The process of ovary folding as well as the differences in the conformation of the ventral and dorsal surfaces of the ovary led us to investigate whether ovary folding is responsible for the regionalization of the ovary into cortical and medullary compartments. The ovarian cortex is covered with ovarian surface epithelium (OSE), which is the female-specific derivative of the gonadal coelomic epithelium. We used the dynamics of the OSE to serve as a proxy for the establishment of the ovarian cortex during morphogenesis using the Lgr5-GFP-IRES-CreER (Lgr5-Gfp) transgenic reporter, in which cells of the OSE are labeled with GFP (red in these images) (Figure 3; Ng et al., 2014). At E14.5, the OSE was only visible in the most medial aspect of the ovary from the dorsal side but covered the ventral portion of the ovarian surface, as demonstrated by the overlap of FOXL2 staining and GFP expression (Figure 3A and B). The OSE continued to cover the ventral and lateral faces of the ovary throughout the time-course (Figure 3C–F). From E15.5 to P0, the OSE became more visible from the dorsal side as the anterior and posterior domains folded toward the center. As this occurred, the FOXL2+/LGR5− domain became gradually restricted to the dorsal extrusion of the ovarian domain, and to the middle region of the ovary from the dorsal face (Figure 3C–F). The appearance of the LGR5+ domain on the dorsal side coincided with the inflexion of the middle region of the ovary, which presumably resulted in an internalization of the cells originally present on the dorsal surface of the ovary to form the ovarian medulla, and to establish the hilum, which serves as a conduit for ovary vasculature and innervation (Figure 3D, E and F). Figure 3 Download asset Open asset Ovary folding is concomitant with specification of the medullary and cortical compartments of the ovary. (A, C, E) Optical sections from confocal Z-stacks of whole ovary/mesonephros complexes from mice at E14.5, and P0 immunostained for GFP and FOXL2 and counterstained with Hoechst nuclear dye (grayscale). in the top were taken from the dorsal side and the bottom from the ventral or lateral Yellow asterisks indicate the location of the infundibulum of the presumptive oviduct for reference. D, 3D models generated by isosurface segmentation of lightsheet images taken of whole ovaries from mice at E14.5, E17.5, and P0 immunostained for FOXL2 (cyan) and GFP Top panels represent the dorsal view, middle panels represent the medial view, and small bottom panels illustrate ventral and lateral views of the same ovary. Yellow arrows in indicate the of the ovarian surface epithelium from the ventral to the dorsal side of the ovarian domain. White arrows point to the MD, Müllerian duct; Mn, mesonephros. Compasses on the bottom left of each panel indicate the orientation of the ovary for the entire A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral. Scale bars, 100 μm. The and relocation of the MD the ovary fully Having characterized the structural changes of the ovarian domain, we next investigated the developmental dynamics of tissues closely associated with the ovary, starting with the The invagination and of the MD occurs from E11.5 to E13.5 in the mouse and is by a of the duct and functional regionalization into uterus, and vagina (Kobayashi and Behringer, 2003; Roly et al., 2018). The presumptive oviduct develops to the ovary and undergoes during late gestation et al., 2021; et al., We investigated the correlations between the dramatic changes in oviduct conformation and ovary morphogenesis. For this, we first used 3D of the ovary and MD on isosurface segmentation of lightsheet images of FOXL2 and PAX8 The shift in position of the MD with to the ovary was best visualized from caudal and cranial views (Figure At E14.5, the MD was fully and to the entire axis of the ovary, on its aspect (Figure The infundibulum is the cranial of the presumptive which in is responsible for The infundibulum was initially located the anterior of the ovary (Figure At E17.5, while the infundibulum only to the region of the ovary and the presumptive remained to the axis of the ovary, the presumptive oviduct and to the ovary (Figure This shift was more in the anterior region of the oviduct (Figure By P0, the presumptive oviduct and into its and this was associated with a shift to the ventral side of the ovary (Figure Figure Download asset Open asset The and relocation of the Müllerian duct the ovary fully (A) 3D model generated by isosurface segmentation of lightsheet images taken of whole ovaries at E14.5, E17.5, and P0 immunostained for FOXL2 (cyan) and PAX8 A, anterior pole; P, posterior pole. (B) from confocal Z-stacks of whole ovary/mesonephros complexes at E14.5 E17.5 and P0 immunostained for PAX8 and and counterstained with Hoechst nuclear dye (grayscale). in the top were taken from the dorsal side and the bottom from the lateral Yellow asterisks indicate the location of the infundibulum of the presumptive oviduct for reference. (C) 3D models generated by isosurface segmentation of lightsheet images taken of whole ovaries at E14.5 E17.5 and P0 immunostained for FOXL2 (cyan) and PAX8 and counterstained with Hoechst nuclear dye (grayscale). The generated with the for of the entire including the ovarian capsule. White point to the surface of the ovary, which the and not with the ovarian capsule. Top panels represent the ventral view, middle panels represent the lateral view, and small bottom panels illustrate dorsal and medial views of the same ovary. arrows indicate the change in position of the infundibulum relative to the plane of the ovary. asterisks indicate the center of the ovary. White arrows indicate the of mesonephric tissue around the ovary in the anterior and posterior regions arrows). Yellow asterisks indicate the location of the infundibulum of the presumptive oviduct for reference. MD, Müllerian duct; Mn, mesonephros; ovarian ovarian Ov, ovary; Compasses on the bottom left of each panel indicate the orientation of the ovary for the entire A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral. Scale bars, 100 μm. We next investigated how the relocation of the MD correlated with ovary encapsulation. We immunostained whole-mount gonads with antibodies against PAX8 to the epithelium of the MD and to the vasculature et al., as a way to visualize the ovarian capsule. These samples were imaged with confocal microscopy (Figure In parallel, we used 3D of the ovary, MD, and ovarian on isosurface segmentation of lightsheet images of FOXL2, PAX8, and Hoechst Consistent with results shown in Figure at E14.5, the MD was located the ovary, with the infundibulum on the lateral the anterior (Figure Figure At this the ovary was in direct with the and was not the mesonephros and ovary were (Figure We used isosurface segmentation of the Hoechst in our lightsheet images to surfaces representing the entire ovary/mesonephros complex (Figure and to the ovarian domain visible (Figure cyan). that the FOXL2 domain is surrounded by the OSE labeled in these which for the space between the isosurface and the FOXL2 domain at all stages (Figure white and not with the ovarian which is at At E17.5, the infundibulum from the lateral to the medial portion of the ovary and from the anterior to the center of the ovary (Figure Figure This was associated with a lateral to medial of cranial mesonephric the presumptive ovarian that the cranial region of the ovary (Figure arrows). At this the middle portion of the MD around the side of the ovary and was visible from the dorsal perspective (Figure the process was best visualized from the lateral view, where the cranial region of the ovary was from the but was covered by mesonephric on the medial by the MD (Figure Figure By P0, the infundibulum continued its cranial to caudal and was now located at the middle of the medial side of the ovary, the of the (Figure Figure This change was associated with a continued of cranial mesonephric which now covered the cranial of the ovary. At this we also observed a caudal of and the was now with the infundibulum on the medial side of the ovary (Figure From the ventral the P0 ovary appeared fully with the of the oviduct the ventral of the ovarian (Figure By the of this process, the infundibulum of the oviduct was in the where it later will responsible for These data that the of the ovary occurs during late gestation and is associated with the and regionalization of the MD to form the This us to investigate potential components that structural change in both the ovary and the oviduct The ligament and cranial suspensory ligament the ovary to the MD and body wall and act as of ovary folding and encapsulation. The position of the ovary within the body is maintained by two the and the The and are that the uterus, and ovary, the position of the reproductive inside the body and a conduit for and et al., While the is a different are defined by the they the the uterus, the the ovary, and the the The caudal of the is to the dorsal body while its cranial region transitions into the which the ovary to the dorsal body wall the et al., and these structures are of the ovary, we that developmental dynamics of the and provide into the mechanisms of ovary morphogenesis. We first used immunostaining against the marker as a proxy for visualizing the in whole urogenital complexes from E15.5 to E18.5 (Figure We found that the and a structure on the aspect of the reproductive and ovary at E15.5 and E14.5 (Figure Figure labeled also with smooth (Figure supplement 1A, At the was visible as a of tissue on the