Mouse Oviductal Epithelium Research Articles

Abstract AP16: MODELING ENDOMETRIOID AND HIGH GRADE SEROUS CARCINOMAS IN THE MOUSE USING CRISPR/CAS9-MEDIATED SOMATIC GENE EDITING IN FALLOPIAN TUBE EPITHELIUM

Abstract Genetically engineered mouse models (GEMMs) have contributed significantly to our understanding of the role of specific gene defects in human cancers. GEMMs that recapitulate many of the molecular and biological characteristics of various histologic subtypes of ovarian carcinomas have already been developed. However, conventional methods of GEMM development based on Cre-lox technology require the time-consuming and labor-intensive processes of transgenic line production and cross-breeding, a problem that is exacerbated when developing cancer GEMMs based on multiple genetic defects. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system potentially offers a more rapid and versatile alternative platform for tumor modeling in the mouse, as it can be used to target multiple genomic loci simultaneously by specifying a 20-nt targeting sequence for each gene of interest. The development of gynecological cancer GEMMs using CRISPR/Cas9-mediated conditional gene editing has not yet been reported. We have tested this approach to generate endometrioid carcinoma (EC) and high-grade serous carcinoma (HGSC) in the mouse oviductal epithelium. We first selected potentially optimal 20-nt sequences (guide RNAs) targeting the murine orthologs of the Apc and Pten tumor suppressor genes for the EC model, and the Brca1, Trp53, Rb1, and Nf1 genes for the HGSC model. Efficient cutting mediated by each guide RNA was validated in vitro by the Surveyor nuclease assay in NIH3T3 mouse fibroblasts. Next, we generated two transgenic mouse lines: one carrying a transgene with guide RNAs targeting Apc and Pten in tandem as a single guide RNA (sgAP) and a second carrying a transgene with a single guide RNA targeting Brca1, Trp53, Rb1, and Nf1 (sgBPRN). These transgenic lines were then crossed with Ovgp1-iCre-ERT2 and Rosa26LSL-Cas9-EGFP (Jackson laboratory) mice to allow for conditional (Tamoxifen-regulated) inactivation of Apc-Pten or Brca1-Trp53-Rb1-Nf1 specifically in the FTE. Endometrioid carcinomas were identified in Ovgp1-iCreERT2;Rosa26LSL-Cas9-EGFP;sgAP mice by 20 weeks post tamoxifen and early HGSC was present in an Ovgp1-iCreERT2; Rosa26LSL-Cas9-EGFP;sgBPRN mouse 34 weeks post tamoxifen treatment. The tumors derived from Cre-CRISPR/Cas9-sgRNA technology showed similar morphology and immunophenotypic characteristics to tumors arising in the models using Cre-lox technology based on the same genetic defects. Insertions/deletions (Indels) in tumor DNA were found in all of the targeted genes near the expected cut-sites, and confirmed by Sanger sequencing of cloned PCR products. All of the Indels resulted in predicted premature protein truncation and loss of function. Our results show that CRISPR/Cas9-sgRNA system genome editing can be used successfully to model gynecological cancers in mice. Citation Format: Rong Wu, Carmine Stolfi, Yali Zhai, Eric R. Fearon, and Kathleen R. Cho. MODELING ENDOMETRIOID AND HIGH GRADE SEROUS CARCINOMAS IN THE MOUSE USING CRISPR/CAS9-MEDIATED SOMATIC GENE EDITING IN FALLOPIAN TUBE EPITHELIUM [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP16.

17\u03b2-Estradiol sensitizes ovarian surface epithelium to transformation by suppressing Disabled-2 expression

Estrogen replacement therapy increases the risk of human ovarian cancer and exogenous estradiol accelerates the onset of ovarian cancer in mouse models. This study uses primary cultures of mouse ovarian surface epithelium (OSE) to demonstrate that one possible mechanism by which estrogen accelerates the initiation of ovarian cancer is by up-regulation of microRNA-378 via the ESR1 pathway to result in the down-regulation of a tumour suppressor called Disabled-2 (Dab2). Estrogen suppression of Dab2 was reproducible in vivo and across many cell types including mouse oviductal epithelium and primary cultures of human ovarian cancer cells. Suppression of Dab2 resulted in increased proliferation, loss of contact inhibition, morphological dysplasia, and resistance to oncogene-induced senescence – all factors that can sensitize OSE to transformation. Given that DAB2 is highly expressed in healthy human OSE and is absent in the majority of ovarian tumours, this study has taken the first steps to provide a mechanistic explanation for how estrogen therapy may play a role in the initiation of ovarian cancer.

PAX2 maintains the differentiation of mouse oviductal epithelium and inhibits the transition to a stem cell-like state.

Recent studies have provided evidence that the secretory cells of the fallopian tube (oviduct) are a probable origin for high-grade serous ovarian carcinoma. In addition to secretory cells, the fallopian tube epithelium consists of ciliated cells and CD44+ undifferentiated stem-like cells. Loss of PAX2 expression is recognized as an early event in epithelial transformation, but the specific role of PAX2 in this transition is unknown. The aim of this study was to define the role of PAX2 in oviductal epithelial (OVE) cells and its response to transforming growth factor β1 (TGFβ), characterizing specifically its potential involvement in regulating stem cell-like behaviors that may contribute to formation of cancer-initiating cells. Treatment of primary cultures of mouse OVE cells with TGFβ induced an epithelial-mesenchymal transition (EMT) associated with decreased expression of PAX2 and an increase in the fraction of cells expressing CD44. PAX2 knockdown in OVE cells and overexpression in ovarian epithelial cells confirmed that PAX2 inhibits stem cell characteristics and regulates the degree of epithelial differentiation of OVE cells. These results suggest that loss of PAX2, as occurs in serous tubal intraepithelial carcinomas, may shift secretory cells to a more mesenchymal phenotype associated with stem-like features.

Progesterone Prevents High-Grade Serous Ovarian Cancer by Inducing Necroptosis of p53-Defective Fallopian Tube Epithelial Cells

High-grade serous ovarian carcinoma (HGSOC) originates mainly from the fallopian tube (FT) epithelium and always carries early TP53 mutations. We previously reported that tumors initiate in the FT fimbria epithelium because of apoptotic failure and the expansion of cells with DNA double-strand breaks (DSB) caused by bathing of the FT epithelial cells in reactive oxygen species (ROSs) and hemoglobin-rich follicular fluid (FF) after ovulation. Because ovulation is frequent and HGSOC is rare, we hypothesized that luteal-phase progesterone (P4) could eliminate p53-defective FT cells. Here we show that P4, via P4 receptors (PRs), induces necroptosis in Trp53-/- mouse oviduct epithelium and in immortalized human p53-defective fimbrial epithelium through the TNF-α/RIPK1/RIPK3/MLKL pathway. Necroptosis occurs specifically at diestrus, recovers at the proestrus phase of the estrus cycle, and can be augmented with P4 supplementation. These results reveal the mechanism of the well-known ability of progesterone to prevent ovarian cancer.

GPR39 is region-specifically expressed in mouse oviduct correlating with the Zn2+ distribution

G-protein-coupled receptor 39 (GPR39) plays a role in cellular and physiological processes, including insulin secretion, cell death inhibition, wound healing, and obesity. Increasing evidence suggests that GPR39 is potently stimulated by zinc ions (Zn2+) and is therefore considered a putative Zn2+ receptor. Given the importance of Zn2+ in the reproductive system, we proposed that GPR39 might have a functional role in the reproductive system. However, the localization of GPR39 in the reproductive system remains unknown. Here, we used mice expressing a Gpr39 promoter-driven LacZ reporter system to detect Gpr39 expression in the reproductive system at different phases of the estrous cycle and found an interesting region-specific distribution of Gpr39 in the mouse oviduct epithelium, with strong expression at the ampulla and weak expression at the isthmus, which was consistent with the results using reverse transcription polymerase chain reaction and immunofluorescence. Moreover, using ZnSeAMG staining, we found that Zn2+, the putative ligand of GPR39, also found a distribution similar to GPR39 expression, suggesting that their potential interaction mediates fertilization and embryo transportation.

Abstract 2684: Modeling a prototypical type I ovarian carcinoma in the mouse based on oviductal versus ovarian epithelial transformation: which is superior

Abstract Type I ovarian carcinomas are relatively indolent tumors that are nonetheless deadly if detected too late. Endometrioid carcinoma (EC) is a prototypical Type I ovarian cancer subtype. Existing mouse models of the disease based on transformation of the ovarian surface epithelium (OSE) take advantage of known EC genetic signatures but fail to capture fully the clinical disease progression and tumor phenotype. We previously described a mouse ovarian EC model in which the Apc and Pten tumor suppressor genes are conditionally deleted in the OSE via bursal injection of adenovirus expressing Cre recombinase (AdCre). We now report a new mouse model of EC in which the Ovgp1 (oviductal glycoprotein) promoter regulates expression of tamoxifen-inducible Cre recombinase specifically in the mouse oviductal epithelium (equivalent to human fallopian tube epithelium [FTE]), and not in the OSE or endometrium. Ovgp1-iCreERT2;Apcfl/fl;Ptenfl/fl mice treated with tamoxifen (TAM) develop oviductal tumors over months rather than weeks. The oviductal EC morphology closely resembles that of human ovarian ECs, with overt gland formation throughout, whereas ovarian bursal injection of AdCre in Ovgp1-iCreERT2;Apcfl/fl;Ptenfl/fl mice results in OSE-derived ECs that are poorly differentiated and include a prominent spindle cell component, consistent with our prior work. OSE-derived tumors progress rapidly, with none of the mice surviving beyond 14 weeks post-AdCre injection. Littermates with FTE-derived tumors survive much longer (>30 weeks post TAM). The slow progression and late (typically >16 weeks post TAM) metastasis to ovary and omentum associated with the FTE-derived tumors are more akin to the relatively indolent behavior characteristic of Type I ovarian carcinoma in humans. To further evaluate which of the two types of mouse tumors more closely resemble human ovarian tumors, we profiled gene expression in 6 FTE- and 5 OSE-derived Apc/Pten-deficient tumors, and compared the groups to each other and to data from 99 human ovarian carcinomas we analyzed previously (including 37 endometrioid, 41 serous, and 13 clear cell, and 8 mucinous carcinomas). Based on gene expression, the FTE-derived tumors are more correlated to every class of human ovarian tumors (p < 2 X10−6 in all 4 comparisons), with highest correlation to human ECs (particularly those with mutant CTNNB1) and mucinous carcinomas. This new model of EC, based on transformation of Mullerian epithelium rather than the OSE, will likely prove more suitable for translational applications, including pre-clinical testing of novel therapeutics for ovarian EC. Citation Format: Rong Wu, Yali Zhai, Rork Kuick, Paloma Garcia, Anum Naseem, Eric R. Fearon, kathleen R. Cho. Modeling a prototypical type I ovarian carcinoma in the mouse based on oviductal versus ovarian epithelial transformation: which is superior. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2684.

Dynamics of planar cell polarity protein Vangl2 in the mouse oviduct epithelium

The planar cell polarity (PCP) pathway regulates morphogenesis in various organs. The polarized localization is a key feature of core PCP factors for orchestrating cell polarity in an epithelial sheet. Several studies using Drosophila melanogaster have investigated the mechanism of the polarized localization. However, to what extent these mechanisms are conserved and how the polarization of core PCP factors is maintained in mature vertebrates are still open questions. Here, we addressed these questions by analyzing the dynamics of Vangl2, a member of core PCP factors, in the mouse oviduct epithelium. Multiple core PCP factors including Vangl2 were expressed in the mouse oviduct in postnatal stages. Vangl1, Vangl2 and Frizzled6 had polarized localization in the oviduct epithelium. Exogenously introduced expression of green fluorescent protein (GFP)-tagged core PCP factors by electroporation revealed that Vangl1, Vangl2 and Prickle2 are localized on the ovarian side of the cell periphery in the oviduct. To visualize the Vangl2 dynamics, we generated the R26-Vangl2-EGFP transgenic mice. In these mice, Vangl2-EGFP was ubiquitously expressed and showed polarized localization in multiple organs including the oviduct, the trachea, the lateral ventricle and the uterus. Fluorescence recovery after photobleaching (FRAP) analysis in the mature oviduct revealed that Vangl2 in the enriched subdomain of cell periphery (cellular edge) was more stable than Vangl2 in the less-enriched cellular edge. Furthermore, when a subregion of a Vangl2-enriched cellular edge was bleached, the Vangl2-enriched subregion neighboring the bleached region in the same cellular edge tended to decrease more intensities than the neighboring sub-region in the next Vangl2-enriched cellular edge. Finally, the polarization of Vangl2 was observed in nocodazole treated mouse viduct, suggesting the maintenance of Vangl2 asymmetry is independent of microtubule formation. Taken together, we revealed the characteristics of Vangl2 dynamics in the oviduct epithelium, and found that Vangl2 forms stable complex at the enriched cellular edge and forms compartments. Our data collectively suggest that the mechanism for maintenance of Vangl2 asymmetry in mature mouse oviduct is different from the microtubule dependent polarized transport model, which has been proposed for the reinforcement of the asymmetry of two core PCP proteins, Flamingo and Dishevelled, in the developing fly.

Celsr1 is required for the generation of polarity at multiple levels of the mouse oviduct.

The oviduct is an important organ in reproduction where fertilization occurs, and through which the fertilized eggs are carried to the uterus in mammals. This organ is highly polarized, where the epithelium forms longitudinal folds along the ovary-uterus axis, and the epithelial multicilia beat towards the uterus to transport the ovulated ova. Here, we analyzed the postnatal development of mouse oviduct and report that multilevel polarities of the oviduct are regulated by a planar cell polarity (PCP) gene, Celsr1. In the epithelium, Celsr1 is concentrated in the specific cellular boundaries perpendicular to the ovary-uterus axis from postnatal day 2. We found a new feature of cellular polarity in the oviduct - the apical surface of epithelial cells is elongated along the ovary-uterus axis. In Celsr1-deficient mice, the ciliary motion is not orchestrated along the ovary-uterus axis and the transport ability of beating cilia is impaired. Epithelial cells show less elongation and randomized orientation, and epithelial folds show randomized directionality and ectopic branches in the mutant. Our mosaic analysis suggests that the geometry of epithelial cells is primarily regulated by Celsr1 and as a consequence the epithelial folds are aligned. Taken together, we reveal the characteristics of the multilevel polarity formation processes in the mouse oviduct epithelium and suggest a novel function of the PCP pathway for proper tissue morphogenesis.

Abstract 3875: NUAK2, a gene with a putative driver mutation in ovarian cancer, is regulated through the murine estrus cycle and loss is associated with worse outcome in ovarian cancer

Abstract Molecular events leading to ovarian cancer are poorly understood but ovulatory hormones and a high number of lifetime ovulations increase risk. The most common histotype, serous ovarian cancer, is thought to arise from ovarian surface epithelium or inclusion cysts although recent evidence has implicated fallopian tube epithelium and other Müllerian tissues as potential sites of origin. The aim of this study was to identify genes that may play a critical role in the pathogenesis of ovarian cancer from candidates that we have previously identified as regulated during the murine estrus cycle in microdissected ovarian surface epithelium and matched oviduct. We examined estrus regulated gene profiles in normal mouse ovarian and oviduct epithelium for genes (i) differentially expressed in ovarian cancer compared to normal controls; (ii) with copy number aberration in ovarian cancer; and (iii) with reported mutation in solid tumors. We identified over 350 genes that are regulated in the normal mouse ovarian epithelium during the estrus cycle and dysregulated in cancer including four genes found to be mutated in various tumour types (Catalogue Of Somatic Mutations In Cancer) and around 50 genes that have copy number aberration in ovarian cancer (The Cancer Genome Atlas). A similar number of genes were regulated in the normal mouse oviduct epithelium during the estrus cycle and dysregulated in ovarian cancer including six that are mutated in various tumor types and around 35 with copy number aberration in ovarian cancer. Interestingly, there was very little similarity between the ovarian and oviduct epithelium gene lists with only 19 genes in common, however, pathway analysis revealed over-representation of genes involved in cell cycle, particularly the spindle assembly checkpoint, and extracellular matrix/cell adhesion in both lists. We identified NUAK family, SNF1-like kinase, 2 (NUAK2) as upregulated in ovarian epithelium during the proestrus phase of the murine estrus cycle, upregulated in ovarian cancer and with a candidate driver mutation for breast and ovarian cancer. Expression of NUAK2 was examined by immunohistochemistry in human ovary (n=10), fallopian tube (n=9), and ovarian cancer (n=119). This showed that NUAK2 expression was highest in fallopian tube, intermediate in OSE and lost in ∼20% of ovarian cancer cases. Kaplan-Meier curves with log-rank test revealed reduced expression of NUAK2 was significantly associated with reduced overall survival in ovarian cancer (p<0.04). Future work involves functional analysis of key genes, including NUAK2, in human cell lines. Defining genes that are activated in normal ovarian and oviduct epithelial cells in the course of ovulation that are also dysregulated in cancer has identified a number of molecular pathways and novel candidate genes that may contribute to the development of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3875. doi:10.1158/1538-7445.AM2011-3875

Reconstruction of Oviduct and Demonstration of Epithelial Fate Determination in Mice1

The mouse oviductal epithelium is a simple monolayer until Postnatal Day 7 and subsequently consists of differentiated secretory cells and ciliated cells. In adult oviduct, the two types of epithelial cells are unevenly distributed; ciliated cells are dominant in the ampulla and secretory cells are dominant in the isthmus. Recombinants of enzymatically separated epithelial and mesenchymal tissues of oviducts were grafted under kidney capsule for 4 wk. The recombinants developed structures with a lumen covered with a monolayer of ciliated cells and secretory cells, demonstrating that the recombinant tissues reconstructed oviductal structure. Geographically (ampulla versus isthmus) heterotypic recombinants were prepared from neonatal oviducts at Day 3. The epithelia in reconstructed oviducts took the patterns of cell distribution depending on the origin of the mesenchymal tissues. The results indicate that the mesenchyme geographically has distinct abilities to determine undifferentiated epithelial cells to ciliated cells or secretory cells in the mouse oviduct.

In vivo gene transfer to the mouse oviduct epithelium

Objective: To transfect the mouse oviduct in vivo. Design: Prospective study. Setting: Research laboratory. Animal(s): Sixteen female Swiss albino mice. Intervention(s): The left oviduct of 10 female mice was instilled with a liposome DNA solution. In addition, 2 control mice received liposome solution, 2 received phosphate-buffered saline solution, and 2 received no injection. Main Outcome Measure(s): The expression of the gene transfected (β-galactosidase) was detected in the oviduct epithelium with the use of a routine histochemical analysis. Result(s): The 90% of the female mice that were transfected with liposome/β-Gal complexes expressed the gene in the oviduct mucosa. The controls did not show β-Gal expression. Conclusion(s): Cationic liposome/DNA complexes can be used for in vivo transfection of the mouse fallopian tubes. The foreign gene expression occurs in clusters of cells located along the mucosa of the isthmus and juncture regions.

Immunocytochemistry of Ion Transport Mediators in the Genital Tract of Female Rodents

With immunocytochemistry, we have determined distribution of sodium, potassium-adenosine triphosphatase (Na+, K+-ATPase) and of three isoenzymes of carbonic anhydrase (CA) and have shown absence of the chloride channel, Band 3 protein, in the genital tract of female rodents. Staining for Na+,K+-ATPase was strongest in the ampullary oviduct and uterine glands in the mouse. In the mouse and rat ovary, immunostaining evidenced CA I, II, and III in theca interna cells where the enzyme could affect the pH of follicular fluid. The zona pellucida of the ovary and cytoplasmic foci in follicular granulosa cells stained for content of only CA I in mouse ovary, suggesting synthesis of a zona pellucida component by granulosa cells. CA II in mouse oviductal epithelium increased from the negative infundibulum to the variably positive ampulla and isthmus to the uniformly positive interstitial segment. The content of CA III varied inversely to that of CA II. The prevalence of CA II-positive cells apparently corresponded with that of nonciliated cells, whereas abundance of CA III-positive cells concurred with that of ciliated cells in regions of the mouse oviduct. The rat oviduct lacked CA II but, like that of the mouse, showed CA III in the proximal region. The staining for CA II in surface epithelium exceeded the reactivity in glandular epithelium in the mouse uterus, except during estrus. In contrast, rat uterus evidenced CA II in glandular but not surface epithelium. These results testify to possible significance of various ion transport mechanisms for biologic activities of diverse cells in the female genital tract.

Progressive proliferative changes in the oviduct of mice following developmental exposure to diethylstilbestrol.

Structural malformation of the oviduct has been reported in experimental animal models and women following prenatal exposure to diethylstilbestrol (DES). To study histological changes in the oviduct in the absence of gross structural malformation, neonatal CD-1 mice were treated with DES (2 micrograms/pup/day) on days 1-5 of age. Focal epithelial hyperplasia was present at 1 month of age in 16 out of 18 (89%) of the DES-treated mice. At 4 months of age, general epithelial hyperplasia with multiple gland-like structures into and through the muscle wall of the oviduct was observed in 90% of the treated mice; by 12 months of age, epithelial hyperplasia and pseudogland formation were seen in 100% of the DES-exposed animals. Epithelial hyperplasia and gland formation were not observed in control mice. The alteration induced by DES in the differentiation and proliferation of mouse oviductal epithelium suggests that the oviduct is a target for DES toxic effects. In addition, there was a progression of the epithelial changes with age. The histological changes described in this study may be partially responsible for the decreased fertility previously reported in this mouse model. Similar changes in the oviduct of DES-exposed women remain to be determined.

Freeze-fracture images of the zonula occludens in the mouse oviduct epithelium.

Freeze-fracture images of zonulae occludentes of the oviduct epithelium of adult mice were observed in the electron microscope. The structure of zonulae occludentes varies depending on the types of oviduct epithelial cells. Those between two secretory cells consist of 5--14 (9.2 +/- 2.0) strands interposed between the luminal and the lateral plasma membrane. The strands run irregularly making well developed anastomoses with one another. The tight junction between two ciliated cells consists of 4--14 (8.5 +/- 2.3) strands. Among them the upper 3--9 strands are unique in shape. The strands run regularly, compactly and parallel to the luminal surface and to each other with a few anastomoses. The distance between the adjacent strands is about 40 nm. Under this characteristic part several strands are very loosely distributed and run irregularly. The pattern of the strands in the zonula occludens between a secretory cell and a ciliated one resembles that between adjacent ciliated cells. The junction consists of 4--13 (8.3 +/- 2.0) strands. The characteristically dense and parallel arrangement of the strands demonstrated in this study is presumed to be related to the ciliary movement. All the zonulae occludentes in the mouse oviduct epithelium are "very tight" in type. No large gap junctions are seen in the mouse oviduct epithelium.

Transfer and Localisation of Maternal Serum Antigens by Mouse Preimplantation Embryos and Oviductal Epithelium

Two hours after systemic injection of bovine plasma albumin (BPA) into pregnant mice, albumin-like antigen was detected by indirect immunohistological methods within the cytoplasm of oviductal and preimplantation uterine embryos whether ovulation was spontaneous or induced by hormone injection. Although fluorescence, localising antigen similar to or identical with the systemically injected foreign protein, was present in embryos in all oviductal regions and at all cleavage stages, the intraembryonic location of the transferred serum molecules differed from embryo stage. Most ootids and two-celled blastomeres contained large intracytoplasmic areas of intense fluorescence randomly associated with non-fluorescent or dimly fluorescent areas in the same cell. By four- and eight-celled stages, albumin-like antigen was localised at the periphery of blastomeres; less was found deep within embryos. By morula and blastocyst stages, blastomeres differed from each other in fluorescence intensity although intracellular fluorescence was homogeneous. Transferred BPA antigen, present in both pronuclei, probably was absent from blastomere nuclei. Ootid zonae pellucidae contained BPA antigen but none was detected in zonae surrounding cleaving embryos. Little foreign albumin was detected in oviductal epithelium. It is concluded that morphological, as well as biochemical, differentiation occurs during mammalian cleavage and it is suggested that maternal macromolecular contributions to mammalian preimplantation embryos may be necessary for normal development in vivo.