Background

The noncanonical Wnt receptor and tyrosine kinase Ror2 has been associated with recessive Robinow syndrome (RRS) and dominant brachydactyly type B1. The phenotypes of mouse mutants implicate Ror2 in the development of the heart, lungs, bone, and craniofacial structures, which are affected in RRS. Following a recently identified role of Ror2 in the migration of mouse primordial germ cells, we extensively characterized its expression throughout the fetal internal reproductive system and the postnatal ductal system.

Results

We show that Ror2 gene products are present in the germ cells and somatic cells of the testis and the ovary of both the mouse and human fetus. In reproductive tract structures, we find that Ror2 is expressed in the mesonephros, developing Wolffian and Müllerian ducts, and later in their derivatives, the epididymal epithelium and uterine epithelium.

Conclusions

This study sets the stage to explore function for this tyrosine kinase receptor in novel regions of expression in the developing reproductive system in both mouse and human.

Although much is known about the embryo during implantation, the architecture of the uterine environment in which the early embryo develops is not well understood. We employed confocal imaging in combination with 3D analysis to identify and quantify dynamic changes to the luminal structure of murine uterus in preparation for implantation. When applied to mouse mutants with known implantation defects, this method detected striking peri-implantation abnormalities in uterine morphology that cannot be visualized by histology. We revealed 3D organization of uterine glands and found that they undergo a stereotypical reorientation concurrent with implantation. Furthermore, we extended this technique to generate a 3D rendering of the cycling human endometrium. Analyzing the uterine and embryo structure in 3D for different genetic mutants and pathological conditions will help uncover novel molecular pathways and global structural changes that contribute to successful implantation of an embryo.

Objective

To develop a protocol for cryopreservation and recovery of human endometrial epithelial cells (eECs) retaining molecular and functional characteristics of endometrial epithelium in vivo.

Design

In vitro study using human endometrial cells.

Setting

University research laboratory.

Patient(s)

Endometrial biopsies were obtained from premenopausal women undergoing benign gynecologic procedures.

Intervention(s)

Primary eECs were cryopreserved in 1% fetal bovine serum/10% dimethylsulfoxide in Defined Keratinocyte Serum-Free Medium (KSFM). Recovered cells were observed for endometrial stromal fibroblast (eSF) contamination and subsequently evaluated for morphology, gene expression, and functional characteristics of freshly cultured eECs and in vivo endometrial epithelium.

Main outcome measure(s)

Analysis of eEC morphology and the absence of eSF contamination; evaluation of epithelial-specific gene and protein expression; assessment of epithelial polarity.

Result(s)

Endometrial epithelial cells recovered after cryopreservation (n = 5) displayed epithelial morphology and expressed E-cadherin (CDH1), occludin (OCLN), claudin1 (CLDN1), and keratin18 (KRT18). Compared with eSF, recovered eECs displayed increased (P<.05) expression of epithelial-specific genes AREG, CDH1, DEFB4A, MMP7, and WNT7A, while exhibiting low-to-undetectable (P<.05) stromal-specific genes COL6A3, HOXA11, MMP2, PDGFRB, and WNT5A. Recovered eECs secreted levels of cytokines and growth factors similarly to freshly cultured eECs. Recovered eECs could form a polarized monolayer with high transepithelial electrical resistance (TER) and impermeability to small molecules, and expressed apical/basolateral localization of CDH1 and apical localization of OCLN.

Conclusion(s)

We have developed a protocol for cryopreservation of eECs in which recovered cells after thawing demonstrate morphologic, transcriptomic, and functional characteristics of human endometrial epithelium in vivo.

Meiotic progression requires exquisitely coordinated translation of maternal messenger (m)RNA that has accumulated during oocyte growth. A major regulator of this program is the cytoplasmic polyadenylation element binding protein 1 (CPEB1). However, the temporal pattern of translation at different meiotic stages indicates the function of additional RNA binding proteins (RBPs). Here, we report that deleted in azoospermia-like (DAZL) cooperates with CPEB1 to regulate maternal mRNA translation. Using a strategy that monitors ribosome loading onto endogenous mRNAs and a prototypic translation target, we show that ribosome loading is induced in a DAZL- and CPEB1-dependent manner, as the oocyte reenters meiosis. Depletion of the two RBPs from oocytes and mutagenesis of the 3' untranslated regions (UTRs) demonstrate that both RBPs interact with the Tex19.1 3' UTR and cooperate in translation activation of this mRNA. We observed a synergism between DAZL and cytoplasmic polyadenylation elements (CPEs) in the translation pattern of maternal mRNAs when using a genome-wide analysis. Mechanistically, the number of DAZL proteins loaded onto the mRNA and the characteristics of the CPE might define the degree of cooperation between the two RBPs in activating translation and meiotic progression.

Quantitative analysis of tissues and organs can reveal large-scale patterning as well as the impact of perturbations and aging on biological architecture. Here we develop tools for imaging of single cells in intact organs and computational approaches to assess spatial relationships in 3D. In the mouse ovary, we use nuclear volume of the oocyte to read out quiescence or growth of oocyte-somatic cell units known as follicles. This in-ovary quantification of non-growing follicle dynamics from neonate to adult fits a mathematical function, which corroborates the model of fixed oocyte reserve. Mapping approaches show that radial organization of folliculogenesis established in the newborn ovary is preserved through adulthood. By contrast, inter-follicle clustering increases during aging with different dynamics depending on size. These broadly applicable tools can reveal high dimensional phenotypes and age-related architectural changes in other organs. In the adult mouse pancreas, we find stochastic radial organization of the islets of Langerhans but evidence for localized interactions among the smallest islets.

Mouse ovarian germ cells enter meiosis in a wave that propagates from anterior to posterior, but little is known about contribution of germ cells to initiation or propagation of meiosis. In a Ror2 mutant with diminished germ cell number and migration, we find that overall timing of meiotic initiation is delayed at the population level. We use chemotherapeutic depletion to exclude a profoundly reduced number of germ cells as a cause for meiotic delay. We rule out sex reversal or failure to specify somatic support cells as contributors to the meiotic phenotype. Instead, we find that anomalies in the distribution of germ cells as well as gonad shape in mutants contribute to aberrant initiation of meiosis. Our analysis supports a model of meiotic initiation via diffusible signal(s), excludes a role for germ cells in commencing the meiotic wave and furnishes the first phenotypic demonstration of the wave of meiotic entry. Finally, our studies underscore the importance of considering germ cell migration defects while studying meiosis to discern secondary effects resulting from positioning versus primary meiotic entry phenotypes.