Molecular contribution to embryonic aneuploidy and karyotypic complexity in initial cleavage divisions of mammalian development.

Embryonic aneuploidy is highly complex, often leading to developmental arrest, implantation failure or spontaneous miscarriage in both natural and assisted reproduction. Despite our knowledge of mitotic mis-segregation in somatic cells, the molecular pathways regulating chromosome fidelity during the error-prone cleavage-stage of mammalian embryogenesis remain largely undefined. Using bovine embryos and live-cell fluorescent imaging, we observed frequent micro-/multi-nucleation of mis-segregated chromosomes in initial mitotic divisions that underwent unilateral inheritance, re-fused with the primary nucleus or formed a chromatin bridge with neighboring cells. A correlation between a lack of syngamy, multipolar divisions and asymmetric genome partitioning was also revealed, and single-cell DNA-seq showed propagation of primarily non-reciprocal mitotic errors. Depletion of the mitotic checkpoint protein BUB1B (also known as BUBR1) resulted in similarly abnormal nuclear structures and cell divisions, as well as chaotic aneuploidy and dysregulation of the kinase-substrate network that mediates mitotic progression, all before zygotic genome activation. This demonstrates that embryonic micronuclei sustain multiple fates, provides an explanation for blastomeres with uniparental origins, and substantiates defective checkpoints and likely other maternally derived factors as major contributors to the karyotypic complexity afflicting mammalian preimplantation development.

Metabolomics analysis of follicular fluid coupled with oocyte aspiration reveals importance of glucocorticoids in primate periovulatory follicle competency.

Gonadotropin administration during infertility treatment stimulates the growth and development of multiple ovarian follicles, yielding heterogeneous oocytes with variable capacity for fertilization, cleavage, and blastocyst formation. To determine how the intrafollicular environment affects oocyte competency, 74 individual rhesus macaque follicles were aspirated and the corresponding oocytes classified as failed to cleave, cleaved but arrested prior to blastulation, or those that formed blastocysts following in vitro fertilization. Metabolomics analysis of the follicular fluid (FF) identified 60 unique metabolites that were significantly different between embryo classifications, of which a notable increase in the intrafollicular ratio of cortisol to cortisone was observed in the blastocyst group. Immunolocalization of the glucocorticoid receptor (GR, NR3C1) revealed translocation from the cytoplasm to nucleus with oocyte maturation in vitro and, correlation to intrafollicular expression of the 11-hydroxy steroid dehydrogenases that interconvert these glucocorticoids was detected upon an ovulatory stimulus in vivo. While NR3C1 knockdown in oocytes had no effect on their maturation or fertilization, expansion of the associated cumulus granulosa cells was inhibited. Our findings indicate an important role for NR3C1 in the regulation of follicular processes via paracrine signaling. Further studies are required to define the means through which the FF cortisol:cortisone ratio determines oocyte competency.

Ablation of conceptus PTGS2 expression does not alter early conceptus development and establishment of pregnancy in the pig†.

Pig conceptuses secrete estrogens (E2), interleukin 1 beta 2 (IL1B2), and prostaglandins (PGs) during the period of rapid trophoblast elongation and establishment of pregnancy. Previous studies established that IL1B2 is essential for rapid conceptus elongation, whereas E2 is not essential for conceptus elongation or early maintenance of the corpora lutea. The objective of the present study was to determine if conceptus expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and release of PG are important for early development and establishment of pregnancy. To understand the role of PTGS2 in conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing PTGS2 using CRISPR/Cas9 technology. Wild-type (PTGS2+/+) and null (PTGS2-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. Immunolocalization of PTGS2 and PG production was absent in cultured PTGS2-/- blastocysts on day 7. PTGS2+/+ and PTGS2-/- blastocysts were transferred into surrogate gilts, and the reproductive tracts were collected on either days 14, 17, or 35 of pregnancy. After flushing the uterus on days 14 and 17, filamentous conceptuses were cultured for 3 h to determine PG production. Conceptus release of total PG, prostaglandin F2⍺ (PGF2α), and PGE in culture media was lower with PTGS2-/- conceptuses compared to PTGS2+/+ conceptuses. However, the total PG, PGF2α, and PGE content in the uterine flushings was not different. PTGS2-/- conceptus surrogates allowed to continue pregnancy were maintained beyond 30 days of gestation. These results indicate that pig conceptus PTGS2 is not essential for early development and establishment of pregnancy in the pig.

Assessing equine embryo developmental competency by time-lapse image analysis.

The timing of early mitotic events during preimplantation embryo development is important for subsequent embryogenesis in many mammalian species, including mouse and human, but, to date, no study has closely examined mitotic timing in equine embryos from oocytes obtained by ovum pick-up. Here, cumulus-oocyte complexes were collected by transvaginal follicular aspiration, matured invitro and fertilised via intracytoplasmic sperm injection. Each fertilised oocyte was cultured up to the blastocyst stage and monitored by time-lapse imaging for the measurement of cell cycle intervals and identification of morphological criteria indicative of developmental potential. Of the 56 fertilised oocytes, 35 initiated mitosis and 11 progressed to the blastocyst stage. Analysis of the first three mitotic divisions in embryos that formed blastocysts determined that typical blastocyst timing (median±IQR) is 30.0±17.5min, 8.8±1.7h and 0.6±1.4h respectively. Frequent cellular fragmentation, multipolar divisions and blastomere exclusion suggested that equine embryos likely contend with a high incidence of chromosomal missegregation. Indeed, chromosome-containing micronuclei and multinuclei with extensive DNA damage were observed throughout preimplantation embryogenesis. This indicates that time-lapse image analysis may be used as a non-invasive method to assess equine embryo quality in future studies.

New perspective on conceptus estrogens in maternal recognition and pregnancy establishment in the pig†.

The proposed signal for maternal recognition of pregnancy in pigs is estrogen (E2), produced by the elongating conceptuses between days 11 to 12 of pregnancy with a more sustained increase during conceptus attachment and placental development on days 15 to 30. To understand the role of E2 in porcine conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing aromatase (CYP19A1) using CRISPR/Cas9 technology. Wild-type (CYP19A1+/+) and (CYP19A1-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer, which were transferred into recipient gilts. Elongated and attaching conceptuses were recovered from gilts containing CYP19A1+/+ or CYP19A1-/- embryos on day 14 and 17 of pregnancy. Total E2 in the uterine flushings of gilts with CYP19A1-/- embryos was lower than recipients containing CYP19A1+/+ embryos with no difference in testosterone, PGF2α, or PGE2 on either day 14 or 17. Despite the loss of conceptus E2 production, CYP19A1-/- conceptuses were capable of maintaining the corpora lutea. However, gilts gestating CYP19A1-/- embryos aborted between days 27 and 31 of gestation. Attempts to rescue the pregnancy of CYP19A1-/- gestating gilts with exogenous E2 failed to maintain pregnancy. However, CYP19A1-/- embryos could be rescued when co-transferred with embryos derived by in vitro fertilization. Endometrial transcriptome analysis revealed that ablation of conceptus E2 resulted in disruption of a number biological pathways. Results demonstrate that intrinsic E2 conceptus production is not essential for pre-implantation development, conceptus elongation, and early CL maintenance, but is essential for maintenance of pregnancy beyond 30 days .

Generation of Mouse for Conditional Expression of Forkhead Box A2.

Forkhead box A2 (FOXA2) is a pioneer transcription factor involved in organ development, function, and cancer. In the uterus, FOXA2 is essential for pregnancy and expressed specifically in the glands of the endometrium. Loss of FOXA2 function occurs during development of endometrial cancer in humans. The current study describes the development of a mouse model for conditional expression of mouse FOXA2. Using a system consisting of a minigene located at the Rosa26 locus, we generated a CAG-S-mFOXA2 allele in embryonic stem cells and subsequently in mice; before activation, the minigene is silent because of a floxed stop cassette inserted between the promoter and the transgene. To validate functionality, mice with the CAG-S-mFOXA2 allele were crossed with progesterone receptor (Pgr)-Cre mice and lactotransferrin (Ltf)-iCre mice that express Cre in the immature and adult uterus, respectively. In immature Pgr-Cre-CAG-S-mFoxa2 mice, FOXA2 protein was expressed in the luminal epithelium (LE), glandular epithelium (GE), stroma, and inner layer of the myometrium. Interestingly, FOXA2 protein was not observed in most of the LE of uteri from adult Pgr-Cre-CAG-S-mFoxa2 mice, although FOXA2 was maintained in the stroma, GE, and myometrium. The adult Pgr-Cre-CAG-S-mFoxa2 females were completely infertile. In contrast, Ltf-iCre-CAG-S-mFoxa2 mice were fertile with no detectable histological differences in the uterus. The adult uterus of Pgr-Cre-CAG-S-mFoxa2 mice was smaller, contained few endometrial glands, and displayed areas of partially stratified LE and GE. This transgenic mouse line is a valuable resource to elucidating and exploring FOXA2 function.

Progesterone effects on extracellular vesicles in the sheep uterus.

Progesterone (P4) acts via the endometrium to promote conceptus growth and implantation for pregnancy establishment. Many cells release extracellular vesicles (EVs) that are membrane-bound vesicles of endosomal and plasma membrane origin. In sheep, endometrial-derived EVs were found to traffic to the conceptus trophectoderm. Thus, EVs are hypothesized to be an important mode of intercellular communication by transferring select RNAs, proteins, and lipids between the endometrium and conceptus. Electron microscopy analysis found that the endometrial luminal and glandular epithelia were the primary source of EVs in the uterus of cyclic sheep. Size exclusion chromatography and nanoparticle tracking analysis (NTA) found that total EV number in the uterine lumen increased from day 10 to 14 in cyclic sheep. Next, ewes were ovariectomized and hormone replaced to determine effects of P4 on the endometrium and EVs in the uterine lumen. Transcriptome analyses found that P4 regulated 1611 genes and nine miRNAs in the endometrium. Total EV number in the uterine lumen was increased by P4 treatment. Small RNA sequencing of EVs detected expression of 768 miRNAs and determined that P4 regulated seven of those miRNAs. These studies provide fundamental new information on how P4 influences endometrial function to regulate conceptus growth for pregnancy establishment in sheep.

GATA2/3-TFAP2A/C transcription factor network couples human pluripotent stem cell differentiation to trophectoderm with repression of pluripotency.

To elucidate the molecular basis of BMP4-induced differentiation of human pluripotent stem cells (PSCs) toward progeny with trophectoderm characteristics, we produced transcriptome, epigenome H3K4me3, H3K27me3, and CpG methylation maps of trophoblast progenitors, purified using the surface marker APA. We combined them with the temporally resolved transcriptome of the preprogenitor phase and of single APA+ cells. This revealed a circuit of bivalent TFAP2A, TFAP2C, GATA2, and GATA3 transcription factors, coined collectively the "trophectoderm four" (TEtra), which are also present in human trophectoderm in vivo. At the onset of differentiation, the TEtra factors occupy multiple sites in epigenetically inactive placental genes and in OCT4 Functional manipulation of GATA3 and TFAP2A indicated that they directly couple trophoblast-specific gene induction with suppression of pluripotency. In accordance, knocking down GATA3 in primate embryos resulted in a failure to form trophectoderm. The discovery of the TEtra circuit indicates how trophectoderm commitment is regulated in human embryogenesis.

Extracellular Vesicles Originate from the Conceptus and Uterus During Early Pregnancy in Sheep.

Cells release diverse types of membrane-bound vesicles of endosomal and plasma membrane origin, termed exosomes and microvesicles, respectively. Extracellular vesicles (EVs) represent an important mode of intercellular communication by transferring select RNAs, proteins, and lipids between cells. The present studies tested the hypothesis that the elongating ovine conceptus and uterus produces EVs that mediate conceptus-maternal interactions during early pregnancy. In Study 1, EVs were purified from uterine luminal fluid of Day 14 cyclic sheep. The EVs were fluorescently labeled with PKH67 dye and infused into the uterine lumen of pregnant sheep for 6 days using an osmotic pump. On Day 14, labeled EVs were observed in the conceptus trophectoderm and uterine epithelia, but not in the uterine stroma or myometrium. In Study 2, Day 14 conceptuses were cultured ex vivo for 24 h and found to release EVs into the culture medium. Proteomics analysis of the Day 14 conceptus-derived EVs identified 231 proteins that were enriched for extracellular space and several protein classes, including proteases, protease inhibitors, chaperones and chaperonins. RNA sequencing of Day 14 conceptus-derived EVs detected expression of 512 mRNAs. The top-expressed genes were overrepresented in ribosomal functions and components. Isolated EVs from conceptuses were fluorescently labeled with PKH67 and infused into the uterine lumen of cyclic sheep for 6 days using an osmotic pump. On Day 14, labeled EVs were observed in the uterine epithelia, but not in the uterine stroma or myometrium. Labeled EVs were not observed in the ovary or in other maternal tissues. These studies support the ideas that EVs emanate from both the conceptus trophectoderm and uterine epithelia, and are involved in intercellular communication between those cells during the establishment of pregnancy in sheep.

Peroxisome proliferator activator receptor gamma (PPARG) regulates conceptus elongation in sheep.

The ovine blastocyst hatches from the zona pellucida by Day 8 and develops into an ovoid or tubular conceptus (embryo and associated extraembryonic membranes) that grows and elongates into a filamentous form between Days 12 and 16. The trophectoderm of the elongating conceptus synthesizes and secretes interferon tau (IFNT) as well as prostaglandins (PGs) via prostaglandin synthase two (PTGS2). Intrauterine infusion of a PTGS2 inhibitor prevents conceptus elongation in sheep. Although many PGs are secreted, PGI2 and PGJ2 can activate nuclear peroxisome proliferator activator receptors (PPARs) that heterodimerize with retinoic X receptors (RXRs) to regulate gene expression and cellular function. Expression of PPARD, PPARG, RXRA, RXRB, and RXRG is detected in the elongating ovine conceptus, and nuclear PPARD and PPARG are present in the trophectoderm. Consequently, PPARD and PPARG are hypothesized to have essential roles in conceptus elongation in ruminants. In utero loss-of-function studies of PPARD and PPARG in the ovine conceptus trophectoderm were conducted using morpholino antisense oligonucleotides (MAOs) that inhibit mRNA translation. Elongating, filamentous-type conceptuses were recovered from ewes infused with a control morpholino or PPARD MAO. In contrast, PPARG MAO resulted in severely growth-retarded conceptuses or conceptus fragments with apoptotic trophectoderm. In order to identify PPARG-regulated genes, PPARG chromatin immunoprecipitation sequencing and RNA sequencing were conducted using Day 14 ovine conceptuses. These analyses revealed candidate PPARG-regulated genes involved in biological pathways, including lipid and glucose uptake, transport, and metabolism. Collectively, results support the hypothesis that PTGS2-derived PGs and PPARG are essential regulators of conceptus elongation, with specific roles in trophectoderm survival and proliferation.