IFPA Meeting 2013 Workshop Report III: maternal placental immunological interactions, novel determinants of trophoblast cell fate, dual ex vivo perfusion of the human placenta.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2013 there were twelve themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of placental function, cell turnover and immunology: 1) immunology; 2) novel determinants of placental cell fate; 3) dual perfusion of human placental tissue.

New candidate targets of protein phosphatase-1c-gamma-2 in mouse testis revealed by a differential phosphoproteome analysis.

Reversible phosphorylation has been implicated in many developmental processes. Dephosphorylation is mediated by several families of phosphatases, including type 1 serine/threonine phosphatases (protein phosphatase-1 or PP1). The loss of the murine Ppp1cc gene causes male infertility as a result of impaired spermatogenesis. Ppp1cc encodes two splice isoforms, PPP1CC1 and PPP1CC2, with the latter being the most abundant isoform in the testis. However, the details of PPP1CC2's involvement in spermatogenesis are still unknown. As a phosphatase has been removed from the mutant mouse, a search for hyperphosphorylated proteins in the mutant testis may reveal the direct downstream targets of PPP1CC2. Using a whole tissue proteomics approach to identify testis-specific dephosphorylation targets of PPP1CC2, we found that two-dimensional electrophoresis identified 10 potential targets in the Ppp1cc null testis several of which are factors known to be important for spermatogenesis, such as HSPA2. Another potential target, tubulin, was found to be misregulated during Ppp1cc(-/-) spermatogenesis, disrupting manchette development. This work represents the first survey of the testicular phosphoproteome under pathological conditions.

The rate of aneuploidy is altered in spermatids from infertile mice.

BACKGROUND: It is now possible for infertile males to father their own genetic children through the technique of ICSI. This prospect has consequently prompted several investigations into the quality of sperm being retrieved from infertile males. One potential risk is the use of aneuploid sperm or spermatids, which might then be transferred to the fertilized oocyte. METHODS: In this investigation, aneuploidy of spermatids was assessed through immunocytochemistry using antibodies directed against chromosome centromeric regions and complexes. Three different types of infertile male mice with phenotypes closely resembling those described in human non-obstructive azoospermia [PP1cgamma-deficient mice, CREM-deficient mice and C57BL/6J.MAC-17(0--23) mice] were examined for chromosome numbers by counting the number of kinetochores in round spermatids using a CREST antiserum. RESULTS: PP1cgamma(-/-) and CREM(-/-) spermatids from infertile mice showed highly significant elevated levels in the rate of aneuploidy compared with wild-type animals (P < 0.0001). Thus infertile males with independent genetic mutations resulting in different histopathologies showed a high risk in the level of aneuploidy in their spermatids. CONCLUSIONS: These results suggest that impaired spermatogenesis may lead to production of aneuploid gametes. Analysis of aneuploidy in gametes from infertile men, coupled with appropriate genetic counselling, is recommended prior to ICSI.

Xenopus adenine nucleotide translocase mRNA exhibits specific and dynamic patterns of expression during development.

We report the isolation and characterization of the Xenopus homolog to human T1 ANT (adenine nucleotide translocase). The 1290-nucleotide sequence contains initiation and termination signals, and encodes a conceptual protein of 298 amino acids. The sequence shares high amino acid identity with the mammalian adenine translocases. The transcript is present in unfertilized eggs, and it is expressed at higher levels during formation of the antero-posterior dorsal axis in embryos. Although low levels are expressed constitutively except in endodermal cells, adenine nucleotide translocase (ANT) expression is dynamically regulated during neurulation. At this stage, expression in ectoderm rapidly diminishes as the neural folds form, and then ANT expression increases slightly in mesoderm. At the culmination of neurulation, the neural tube briefly expresses ANT, and thereafter its expression predominates in the somitic mesoderm and also the chordoneural hinge. In addition, ANT expression is particularly high in the prosencephalon, the mesencephalon, the branchial arches, eye, and the otic vesicle. Treatment of embryos with retinoic acid has the effect of diminishing constitutive expression of ANT, but microinjection studies demonstrate that immediate and local repression cannot be induced in dorsal structures.

LiCl disrupts axial development in mouse but does not act through the beta-catenin/Lef-1 pathway.

Chimera and cell marking studies suggest that axial determination in mouse embryos occurs at postimplantation stages. In contrast, Xenopus laevis axes are determined early due to the asymmetric distribution of maternally derived factors in the one-cell zygote. In our earlier study we used lithium chloride (LiCl) to perturb development of mouse axes. Here we investigate whether the lithium induced axial defects in mouse are being mediated by the beta-catenin/Lef-1 pathway as in Xenopus laevis. In lithium treated embryos we did not observe any changes in the amount or localization of beta-catenin protein. Furthermore, the lack of Lef-1 mRNA in treated and untreated embryos indicates the LiCl induced axial defects in the mouse are not mediated by the beta-catenin/Lef-1 pathway.

DNA damage in round spermatids of mice with a targeted disruption of the Pp1cgamma gene and in testicular biopsies of patients with non-obstructive azoospermia.

Non-obstructive azoospermia accounts for a considerable proportion of male factor infertility. Current therapies for treatment of this kind of infertility include procedures such as intracytoplasmic sperm injection (ICSI), round spermatid injection (ROSI), round spermatid nucleus injection (ROSNI) and elongated spermatid injection (ELSI). All involve injection of haploid germ cells retrieved from testicular biopsies into recipient oocytes. We have investigated a mouse model of azoospermia for quality of haploid germ cell genomes, based on 4,6-diamidino-2-phenylindole (DAPI)/TdT-mediated dUTP nick-end labelling (TUNEL) labelling. The mouse model, a targeted mutation in the protein phosphatase 1cg gene, results in severe depletion of haploid germ cells from the round spermatid stage on. Mice homozygous for the mutation are completely infertile, and produce only the occasional spermatozoon. Spermatozoa and round spermatids retrieved from either the epididymides or the testes of mutant mice displayed very high rates of DNA fragmentation. In contrast, similar cells retrieved from heterozygous or wild-type littermates displayed low levels of DNA fragmentation. In some cases, the high rates of DNA fragmentation in mutant cells could be lowered by inclusion of antioxidants in the retrieval media. High rates of DNA fragmentation were also observed in round spermatids retrieved from testicular biospies of human patients with non-obstructive azoospermia. These results suggest that one of the features of the pathology associated with azoospermia is fragmented DNA in haploid germ cells. This raises questions about the suitability of using these cells for fertility treatment.

Spermiogenesis is impaired in mice bearing a targeted mutation in the protein phosphatase 1cgamma gene.

Type 1 protein phosphatases (PP1) are involved in diverse cellular activities, ranging from glycogen metabolism to chromatin structure modification, mitosis, and meiosis. The holoenzymes are composed of two or more subunits, including a catalytic subunit (PP1c) and one or more regulatory subunits. Many eukaryotes possess several catalytic subunit genes which encode highly conserved isoforms. In rodents, one of these isoforms, PP1cgamma2, appears to be expressed predominantly in testes. Whether PP1cgamma2 performs a testis-specific function is unclear. To address this and other questions, the PP1cgamma gene was disrupted by targeted insertion in murine embryonic stem cells. Mice derived from these cells were viable, and homozygous females were fertile. However, males homozygous for the targeted insertion were infertile. Histological examination revealed severe impairment of spermiogenesis beginning at the round spermatid stage. In addition, defects in meiosis were inferred from the presence of polyploid spermatids. Immunohistochemistry revealed the presence of PP1calpha protein on condensing spermatids in both wild-type and mutant testes, suggesting that this closely related isoform is unable to compensate for the loss of PP1cgamma. These defects are discussed in the light of known functions of protein phosphatase 1.

Expression and regulation of genes associated with cell death during murine preimplantation embryo development.

The newly fertilized preimplantation embryo depends entirely on maternal mRNAs and proteins deposited and stored in the oocyte prior to its ovulation. If the oocyte is not sufficiently equipped with maternally stored products, or if zygotic gene expression does not commence at the correct time, the embryo will die. One of the major abnormalities observed during early development is cellular fragmentation. We showed previously that cellular fragmentation in human embryos can be attributed to programmed cell death (PCD). Here, we demonstrate that the PCD that occurs during the 1-cell stage of mouse embryogenesis is likely to be regulated by many cell death genes either maternally inherited or transcribed from the embryonic genome. We have demonstrated for the first time the temporal expression patterns of nine cell death regulatory genes, and our preliminary experiments show that the expression of these genes is altered in embryos undergoing fragmentation. The expression of genes involved in cell death (MA-3, p53, Bad, and Bcl-xS) seems to be elevated, whereas the expression of genes involved in cell survival (Bcl-2) is reduced. We propose that PCD may occur by default in embryos that fail to execute essential developmental events during the first cell cycle.

Expression of SC1 is associated with the migration of myotomes along the dermomyotome during somitogenesis in early mouse embryos.

SC1 is a secreted glycoprotein with a high amino acid sequence similarity to SPARC (Secreted Protein, Acidic, Rich in Cysteine). SC1 transcripts were first detected in mouse embryos after day 8.5 post coitus (p.c.) in somites at the medial lip of the dermomyotome. Expression of SC1 transcripts by the progenitor cells continued as they began involuting under the dermomyotome and during their migration along the lateral wall of the dermomyotome. After myotome migration was completed, SC1 mRNA expression was downregulated in the trunk region. The data indicate that SC1 expression is restricted to the initial stages of epaxial myotome differentiation and migration, undergoing rapid downregulation prior to myotome emigration from the somitic environment.

Defects in regulation of apoptosis in caspase-2-deficient mice.

During embryonic development, a large number of cells die naturally to shape the new organism. Members of the caspase family of proteases are essential intracellular death effectors. Herein, we generated caspase-2-deficient mice to evaluate the requirement for this enzyme in various paradigms of apoptosis. Excess numbers of germ cells were endowed in ovaries of mutant mice and the oocytes were found to be resistant to cell death following exposure to chemotherapeutic drugs. Apoptosis mediated by granzyme B and perforin was defective in caspase-2-deficient B lymphoblasts. In contrast, cell death of motor neurons during development was accelerated in caspase-2-deficient mice. In addition, caspase-2-deficient sympathetic neurons underwent apoptosis more effectively than wild-type neurons when deprived of NGF. Thus, caspase-2 acts both as a positive and negative cell death effector, depending upon cell lineage and stage of development.

Effect of maternal age and conditions of fertilization on programmed cell death during murine preimplantation embryo development.

One of the major morphological anomalies observed in many human pre-embryos is extensive cellular fragmentation. Previously we confirmed that embryo fragmentation seemed to be associated with the activation of programmed cell death (PCD). The purpose of our experiments was to establish a rate for murine embryo fragmentation in vivo after hormonal stimulation in young versus older females and to compare it with the rate of embryo fragmentation during in-vitro fertilization (IVF). While murine maternal age beyond 40 weeks increased the rate of embryo fragmentation following in-vivo fertilization (P = 0.001), oocytes from females of all ages had a uniformly high rate of fragmentation when fertilized in vitro (33%). None of the fragmented murine embryos proceeded further in development. In the mouse, fragmentation occurs exclusively during the first cell cycle. Furthermore, IVF significantly reduced the rate of blastocyst formation (P = 0.0001) and decreased the mean cell number at the blastocyst stage in comparison with embryos produced in vivo (P < 0.0001). The cell death index was significantly affected by both maternal age (P = 0.005) and IVF (P = 0.0001). Identification of specific factors which trigger PCD, especially those associated with IVF, may enable us to lower the rates of fragmentation in preimplantation embryos and thereby increase pregnancy rates after human IVF.

Genomic organization and functional analysis of the murine protein phosphatase 1c gamma (Ppp1cc) gene.

Protein phosphatase 1 holoenzymes are composed of catalytic subunits in combination with various regulatory subunits. In rodents, four different catalytic isoforms are known, PP1c alpha, -delta, -gamma 1, and -gamma 2. Here we describe the genomic organization of the murine Ppp1cc gene that encodes the PP1c gamma 1 and PP1c gamma 2 isoforms. We determined that Ppp1cc maps to F1.2-G1.2 on chromosome 5 by FISH mapping. Southern hybridization and analysis of cross-hybridizing genomic clones revealed four Ppp1cc-related pseudogenes in the mouse genome. The authentic Ppp1cc gene encodes two isoforms, PP1c gamma 1 and PP1c gamma 2, that arise from alternative splicing and differ by retention of the last intron. The introns of Ppp1cc are flanked by short direct repeats, the significance of which is not clear. Both isoforms retain phosphatase function since they are able to complement the cold-sensitive PP1 defect caused by the dis2-11 mutation in the fission yeast Schizosaccharomyces pombe.

Paternal transmission of the mouse Thp mutation is lethal in some genetic backgrounds.

Thp is a large deletion on chromosome 17 which includes the maternal lethal gene Tme. Documentation of inheritance patterns suggests that Tme is an imprinted gene which is required for viability; maternal deletion is lethal while paternal deletion is viable. However, paternal transmission of Thp is rarely the expected 50%. We show here that paternally inherited Thp is lethal in some strains, providing evidence of an incompletely penetrant, dosage sensitive lethal allele of a locus that probably maps to the hairpin tail region of chr. 17. Interpretation of the various phenotypes associated with loss of the putative Tme gene, Igf2r, may need to be revised in view of these observations.

Epigenetic alterations brought about by lithium treatment disrupt mouse embryo development.

The commonly accepted mechanism by which LiCl dorsalizes amphibian embryos is a respecification of ventral blastomeres, presumably through realignment of dorsal positional information in the embryo. An alternative mechanism, however, is an epigenetic change in the competence of cells to respond to cues they may be normally exposed to without effect. In order to test this hypothesis, we treated mouse preimplantation embryos, which do not possess any axial positional information, with LiCl, and observed axial abnormalities which must have been elaborated several days after treatment. We interpret this as support for the hypothesis that cellular competence rather than positional information is altered by LiCl, and suggest that this competence may be altered through the action of lithium sensitive enzymes that interact with chromatin.

Programmed cell death and human embryo fragmentation.

The quality of embryos produced by in-vitro fertilization (IVF) is variable. Many embryos contain unequal sized blastomeres and multiple cellular fragments. Embryos with excessive fragmentation have limited developmental potential both in vitro and in vivo. Histologically, some blastomeres of fragmented embryos resemble cells undergoing apoptosis as a result of programmed cell death (PCD). The objective of the present study was to determine if the morphological features of apoptosis are observed in fragmented human preimplantation embryos, supporting the possible involvement of PCD in early human embryo arrest and demise. Using combined nuclear and terminal transferase-mediated DNA end labelling (TUNEL) on arrested, fragmented human embryos, we were able to detect extensive condensation and degradation of chromatin, compatible with apoptosis. Electron microscopy confirmed the typical morphological features of apoptosis. No such abnormalities were observed in spare embryos with regular sized blastomeres without fragmentation. The high incidence of condensed chromatin, TUNEL detection of degraded DNA, cell corpses and apoptotic bodies in fragmented human embryos strongly suggest that PCD is triggered in human embryos at a stage prior to blastocyst formation. At such early stages, occurrence of apoptosis seemed to be detrimental, leading to preimplantation embryo death.

Recombinant human leukemia inhibitory factor does not enhance in vitro human blastocyst formation.

OBJECTIVE: To assess the effect of human recombinant leukemia inhibitory factor in different doses on human blastocyst formation. SETTING: A university-based tertiary referral center (The Toronto Hospital). INTERVENTIONS: Nontransferable human embryos (n = 473) at the two- to six-cell stage were obtained from patients undergoing IVF and were split randomly into five groups. Embryos in group A (n = 164) were cultured as the control group in Ham's F-10 (GIBCO-BRL, Grand Island, NY) + 10% human sera. Embryos in groups B, C, D, and E (n = 54, 78, 87, and 80, respectively) were cultured in the same medium supplemented with human recombinant leukemia inhibitory factor in four different concentrations (5, 7.5, 10, and 20 ng/mL, respectively). Morphological assessment of embryo development was recorded daily. MAIN OUTCOME MEASURE: Human blastocyst formation. RESULTS: No significant difference was detected in the rate of blastocyst formation of embryos in the study groups when compared with embryos in group A. CONCLUSIONS: This study shows that 5 to 20 ng/mL of recombinant leukemia inhibitory factor in standard medium does not enhance in vitro human blastocyst formation. It is possible that recombinant leukemia inhibitory factor may play a role at later stages of human embryogenesis and during implantation.

Involvement of programmed cell death in preimplantation embryo demise.

Fragmentation is frequently observed in animal and human embryos obtained via in-vitro fertilization (IVF), and is known to be associated with decreased pregnancy rates and poor survival following cryopreservation. We postulate that embryo fragmentation is a consequence of activated programmed cell death (PCD) and subsequent apoptosis and discuss evidence of morphological, histological and biochemical features compatible with the occurrence of PCD in preimplantation embryos. If PCD is an underlying cause of the high incidence of the fragmentation seen in human pre-embryos, it remains to be determined whether this is reflective of the natural incidence of lethal chromosomes in the human population or due to the IVF procedure and culture conditions.

Identification of genes showing altered expression in preimplantation and early postimplantation parthenogenetic embryos.

Uniparental embryos have been instrumental in studying imprinting because contributions from the parental genomes can be determined unambiguously. In this study, we set out to identify imprinted genes showing differential expression between parthenogenetic and fertilized embryos during preimplantation and early postimplantation stages of development. We identified three genes--apolipoprotein E, pyruvate kinase-3, and protein phosphatase 1 gamma--that represent excellent candidates for imprinted genes, based on the results of the differential screen, their function in differentiation and the cell cycle, and their location within imprinted chromosomal regions. In addition, two novel genes expressed in trophoblast were identified, 1661 and RA81. These genes, together with four known imprinted genes, H19, Igf2r, Igf2, and Snrpn, showed evidence of expression from both parental alleles in early stage embryos, indicating a role for postfertilization processes in regulating imprinted gene function.

Two isoforms of Xenopus retinoic acid receptor gamma 2 (B) exhibit differential expression and sensitivity to retinoic acid during embryogenesis.

We report the isolation of two retinoic acid receptor isoforms (RAR gamma), which differ only in the 5'untranslated and putative N-terminus A regions. The two isoforms appear to serve as early markers for the presumptive neural axis; however, their expression patterns differ. RAR-gamma 2.1 is first expressed at gastrulation at the dorsal lip and subsequently along the presumptive neural axis. RAR- gamma 2.2 represents the full-length sequence of a receptor cDNA already partially characterized and present as a maternal transcript [Ellinger-Ziegelbauer and Dreyer (1991); Genes Dev 5:94-104, (1993): Mech Dev 41:31-46; Pfeffer and DeRobertis, (1994) Mech Dev: 45:147-153]. Unlike RAR-gamma 2.2, the 2.1 variant is not expressed either in pre-somitic mesoderm or notochord. RAR-gamma 2.1 is strongly expressed in branchial arches and to a lesser extent in the neural floor plate. The two isoforms also exhibit differential sensitivity to retinoic acid. Constitutive expression of RAR gamma 2.2 following neurulation appears to be depressed by treatment with retinoic acid, but domains of highest expression, namely, the head and tail, remain relatively unaffected, as do patterns of expression prior to late neurulation. By contrast, RAR-gamma 2.1 is not transcribed in retinoid-inhibited structures. Using microinjection techniques, we show that changes of RAR-gamma 2.1 expression in presumptive head structures occur as an early and local consequence of retinoic acid administration. Since RAR-gamma 2.1 expression is inhibited by retinoic acid, we tested to see if other treatments that perturb axis formation had any effect. Surprisingly, UV irradiation did not suppress that its inhibition by retinoic acid is not due solely to inhibition of anterior neural development. These experiments demonstrate a new subdivision of isoforms that undergo differential expression during development and that exhibit differential sensitivity to retinoic acid and to UV. This sensitivity and the presence of this isoform variant in regions that are known to exhibit polarizing activity strengthen the hypothesis that these receptors play a primary role during morphogenesis.

Genomic imprinting--defusing the ovarian time bomb.

Why do mammals imprint their parental genomes? Imprinting is seen in many phyla, but that in mammals is by far the most dramatic. Is there something peculiar to mammals that calls for such a striking phenomenon? We propose that imprinting is a device that protects female mammals from the potential ravages of ovarian trophoblast disease. Without imprinting, the ovarian teratomas that frequently arise from parthenogenetically activated oocytes in situ might be capable of forming malignant trophoblast. An allele that favored imprinting would spread rapidly because of the great increase in fitness associated with suppressing a lethal cancer of females.