Combined iDISCO and CUBIC tissue clearing and lightsheet microscopy for in toto analysis of the adult mouse ovary†.

At any given time, the ovary contains a number of follicles in distinct growth stages, each with a set of identifying characteristics. Although follicle counting and staging using histological stains on paraffin-embedded ovary sections has been the gold standard in assessing ovarian health in fertility studies, the final counts rely on extrapolation factors that diverge greatly among studies. These methods also limit our ability to investigate spatial aspects of ovary organization. Recent advances in optical tissue clearing and lightsheet microscopy have permitted comprehensive analysis of intact tissues. In this study, we set out to determine the best clearing and imaging methods to generate 3D images of the complete adult mouse ovary that could be used for accurate assessments of ovarian follicles. We found that a combination of iDISCO and CUBIC was the best method to clear the immunostained ovary. Using lightsheet microscopy, we generated 3D images of the intact ovary and performed qualitative assessments of follicles at all stages of development. This study is an important step toward developing quantitative computational models that allow rapid and accurate assessments of growing and quiescent primordial follicles, and to investigate the integrity of extrinsic ovarian components including vascular and neuronal networks.

Neural crest-derived neurons invade the ovary but not the testis during mouse gonad development.

Testes and ovaries undergo sex-specific morphogenetic changes and adopt strikingly different morphologies, despite the fact that both arise from a common precursor, the bipotential gonad. Previous studies showed that recruitment of vasculature is critical for testis patterning. However, vasculature is not recruited into the early ovary. Peripheral innervation is involved in patterning development of many organs but has been given little attention in gonad development. In this study, we show that while innervation in the male reproductive complex is restricted to the epididymis and vas deferens and never invades the interior of the testis, neural crest-derived innervation invades the interior of the ovary around E16.5. Individual neural crest cells colonize the ovary, differentiate into neurons and glia, and form a dense neural network within the ovarian medulla. Using a sex-reversing mutant mouse line, we show that innervation is specific to ovary development, is not dependent on the genetic sex of gonadal or neural crest cells, and may be blocked by repressive guidance signals elevated in the male pathway. This study reveals another aspect of sexually dimorphic gonad development, establishes a precise timeline and structure of ovarian innervation, and raises many questions for future research.

A grafted ovarian fragment rescues host fertility after chemotherapy.

STUDY QUESTION: Can host fertility be rescued by grafting of a fragment of a healthy ovary soon after chemotherapy? SUMMARY ANSWER: We found that grafting a green fluorescent protein (GFP)-positive fragment from a healthy isogenic ovary to the left ovary of a chemo-treated host rescued function and fertility of the grafted host ovary, and resulted in the production of host-derived offspring as late as the sixth litter after chemotherapy (CTx) treatment, whereas none of the ungrafted controls produced a second litter. WHAT IS KNOWN ALREADY: In women and girls undergoing chemotherapy, infertility and premature ovarian failure are frequent outcomes. There are accumulating reports of improved endocrine function after autotransplantation of an ovarian fragment, raising the possibility that the transplant is beneficial to the endogenous ovary. STUDY DESIGN, SIZE, DURATION: We first established a CTx treatment regimen that resulted in the permanent loss of fertility in 100% of female mice of the FVB inbred strain. We grafted an isogenic ovary fragment from a healthy female homozygous for a GFP transgene to the left ovary of 100 CTx-treated hosts, and compared fertility to 39 ungrafted controls in 6 months of continuous matings, using GFP to distinguish offspring derived from the graft, and those derived from the host. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunofluoresece and western blot analysis of 39 treated ovaries during and 15 days after CTx treatment revealed elevated apoptosis, rapid loss of granulosa cells and an increased recruitment of growing follicles. Using immunofluorescence and confocal imaging, we tracked the outcome of the grafted tissue over 4 months and its effect on the adjacent and contralateral ovary of the host. MAIN RESULTS AND THE ROLE OF CHANCE: Fifty-three percent of grafted females produced a second litter whereas none of the ungrafted females produced a second litter. The likelihood that this could occur by chance is very low (P < 0.0001). LIMITATIONS, REASONS FOR CAUTION: These results are shown only in mice, and whether or how they might apply to chemotherapy patients subjected to different CTx regimens is not yet clear. WIDER IMPLICATIONS OF THE FINDINGS: Our experiments prove that rescue of a chemo-treated ovary is possible, and establish a system to investigate the mechanism of rescue and to identify the factors responsible with the long-term goal of developing therapies for preservation of ovarian endocrine function and fertility in women undergoing chemotherapy. LARGE SCALE DATA: No large datasets were produced. STUDY FUNDING/COMPETING INTERESTS: Duke University Medical Center Chancellor's Discovery Grant to BC; ESJ was supported by an NRSA 5F31CA165545; SK was supported by NIH RO1 GM08033; RWT was supported by the Duke University School of Medicine Ovarian Cancer Research Fellowship; XBM was supported by CONICYT. The authors have no conflicts of interest to declare.

Mouse germ cell clusters form by aggregation as well as clonal divisions.

After their arrival in the fetal gonad, mammalian germ cells express E-cadherin and are found in large clusters, similar to germ cell cysts in Drosophila. In Drosophila, germ cells in cysts are connected by ring canals. Several molecular components of intercellular bridges in mammalian cells have been identified, including TEX14, a protein required for the stabilization of intercellular bridges, and several associated proteins that are components of the cytokinesis complex. This has led to the hypothesis that germ cell clusters in the mammalian gonad arise through incomplete cell divisions. We tested this hypothesis by generating chimeras between GFP-positive and GFP-negative mice. We show that germ cell clusters in the fetal gonad arise through aggregation as well as cell division. Intercellular bridges, however, are likely restricted to cells of the same genotype.

BAX-mediated cell death affects early germ cell loss and incidence of testicular teratomas in Dnd1(Ter/Ter) mice.

A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1(Ter/Ter)) results in a significant early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129Sv/J background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost or what factors directly control the strain-specific phenotype variation. To determine the mechanism underlying early PGC loss we crossed Dnd1(Ter/Ter) embryos to a Bax-null background and found that germ cells were partially rescued. Surprisingly, on a mixed genetic background, rescued male germ cells also generated fully developed teratomas at a high rate. Double-mutant females on a mixed background did not develop teratomas, but were fertile and produced viable off-spring. However, when Dnd1(Ter/Ter) XX germ cells developed in a testicular environment they gave rise to the same neoplastic clusters as mutant XY germ cells in a testis. We conclude that BAX-mediated apoptosis plays a role in early germ cell loss and protects from testicular teratoma formation on a mixed genetic background.