Susannah Varmuza

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. Mol. Reprod. Dev. 51:243–253, 1998.

Recent acquisition of imprinting at the rodent Sfmbt2 locus correlates with insertion of a large block of miRNAs

BackgroundThe proximal region of murine Chr 2 has long been known to harbour one or more imprinted genes from classic genetic studies involving reciprocal translocations. No imprinted gene had been identified from this region until our study demonstrated that the PcG gene Sfmbt2 is expressed from the paternally inherited allele in early embryos and extraembryonic tissues. Imprinted genes generally reside in clusters near elements termed Imprinting Control Regions (ICRs), suggesting that Sfmbt2 might represent an anchor for a new imprinted domain.ResultsWe analyzed allelic expression of approximately 20 genes within a 3.9 Mb domain and found that Sfmbt2 and an overlapping non-coding antisense transcript are the only imprinted genes in this region. These transcripts represent a very narrow imprinted gene locus. We also demonstrate that rat Sfmbt2 is imprinted in extraembryonic tissues. An interesting feature of both mouse and rat Sfmbt2 genes is the presence of a large block of miRNAs in intron 10. Other mammals, including the bovine, lack this block of miRNAs. Consistent with this association, we show that human and bovine Sfmbt2 are biallelic. Other evidence indicates that pig Sfmbt2 is also not imprinted. Further strengthening the argument for recent evolution of Sfmbt2 is our demonstration that a more distant muroid rodent, Peromyscus also lacks imprinting and the block of miRNAs.ConclusionsThese observations are consistent with the hypothesis that the block of miRNAs are driving imprinting at this locus. Our results are discussed in the context of ncRNAs at other imprinted loci.Accession numbers for Peromyscus cDNA and intron 10 genomic DNA are [Genbank:HQ416417 and Genbank:HQ416418], respectively.

The imprinted polycomb group gene Sfmbt2 is required for trophoblast maintenance and placenta development

Imprinted genes play important roles in placenta development and function. Parthenogenetic embryos, deficient in paternally expressed imprinted genes, lack extra-embryonic tissues of the trophoblast lineage. Parthenogenetic trophoblast stem cells (TSCs) are extremely difficult to derive, suggesting that an imprinted gene(s) is necessary for TSC establishment or maintenance. In a candidate study, we were able to narrow the list to one known paternally expressed gene, Sfmbt2. We show that mouse embryos inheriting a paternal Sfmbt2 gene trap null allele have severely reduced placentae and die before E12.5 due to reduction of all trophoblast cell types. We infected early embryos with lentivirus vectors expressing anti-Sfmbt2 shRNAs and found that TSC derivation was significantly reduced. Together, these observations support the hypothesis that loss of SFMBT2 results in defects in maintenance of trophoblast cell types necessary for development of the extra-embryonic tissues, the placenta in particular.

Imprinting and extraembryonic tissues-mom takes control.

Genomic imprinting is an epigenetic mechanism that silences one parental allele of a small subset of genes. Many imprinted genes exhibit this property only in extraembryonic tissues-placenta and yolk sac. This has led to the idea that imprinting in mammals coevolved with some aspect of placentation. Nevertheless, many studies of imprinting have ignored the extraembryonic tissues, the yolk sac and its precursor, the primitive endoderm, in particular. The primitive endoderm is involved in very early signaling events during a critical stage in development, gastrulation, during which body plan axes and head process neuroectoderm are established. Improper signaling from primitive endoderm as a result of abnormal expression of imprinted genes has the capacity to effect long-term defects in embryonic/fetal tissues that might hitherto have been overlooked. We discuss these gaps in the knowledge, propose a mechanism for genomic imprinting based on current data, and suggest a line of investigation that will expand our understanding of this unique regulatory mechanism and its impact on development.

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 PPP1CC2s 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.

Loss of protein phosphatase 1cγ (PPP1CC) leads to impaired spermatogenesis associated with defects in chromatin condensation and acrosome development: an ultrastructural analysis

Human male infertility affects approximately 5% of men, with one-third suffering from testicular failure, likely the result of an underlying genetic abnormality that disrupts spermatogenesis during development. Mouse models of male infertility such as the Ppp1cc knockout mouse display very similar phenotypes to humans with testicular failure. Male Ppp1cc mutant mice are sterile due to disruptions in spermatogenesis that begin during prepubertal testicular development, and continue into adulthood, often resulting in loss of germ cells to the point of Sertoli cell-only syndrome. The current study employs light and electron microscopy to identify new morphological abnormalities in Ppp1cc mutant seminiferous epithelium. This study reveals that germ cells become delayed in their development around stages VII and VIII of spermatogenesis. Loss of these cells likely results in the reduced numbers of elongating spermatids and spermatozoa previously observed in mutant animals. Interestingly, Ppp1cc mutants also display reduced numbers of spermatogonia compared with their wild-type counterparts. Using electron microscopy, we have shown that junction complexes in Ppp1cc mutants are ultrastructurally normal, and therefore do not contribute to the breakdown in tissue architecture seen in mutants. Electron microscopy revealed major acrosomal and chromatin condensation defects in Ppp1cc mutants. Our observations are discussed in the context of known molecular changes in Ppp1cc mutant testes.

What does genetics tell us about imprinting and the placenta connection

Genomic imprinting is an epigenetic gene silencing phenomenon that is specific to eutherians in the vertebrate lineage. The acquisition of both placentation and genomic imprinting has spurred interest in the possible evolutionary link for many years. In this review we examine the genetic evidence and find that while many imprinted domains are anchored by genes required for proper placenta development in a parent of origin fashion, an equal number of imprinted genes have no apparent function that depends on imprinting. Examination of recent data from studies of molecular and genetic mechanisms points to a maternal control of the selection and maintenance of imprint marks, reinforcing the importance of the oocyte in the healthy development of the placenta and fetus.

The application of proteomic approaches to the study of mammalian spermatogenesis and sperm function.

Spermatogenesis is the process by which terminally differentiated sperm are produced from male germline stem cells. This complex developmental process requires the coordination of both somatic and germ cells through phases of proliferation, meiosis, and morphological differentiation, to produce the cell responsible for the delivery of the paternal genome. With infertility affecting ~ 15% of all couples, furthering our understanding of spermatogenesis and sperm function is vital for improving the diagnosis and treatment of male factor infertility. The emerging use of proteomic technologies has played an instrumental role in our understanding of spermatogenesis by providing information regarding the genes involved. This article reviews existing proteomic literature regarding spermatogenesis and sperm function, including the proteomic characterization of spermatogenic cell types, subcellular proteomics, post‐translational modifications, interactomes, and clinical studies. Future directions in the application of proteomics to the study of spermatogenesis and sperm function are also discussed.

Development to Blastocyst Is Impaired When Intracytoplasmic Sperm Injection Is Performed with Abnormal Sperm from Infertile Mice Harboring a Mutation in the Protein Phosphatase 1cγ Gene1

Abstract Idiopathic azoospermia, characterized by abnormal spermatogenesis, is commonly treated by performing intracytoplasmic sperm injection (ICSI) with sperm retrieved from testicular biopsies. However, no controlled experiments have been performed using an animal model to assess the efficacy or safety of the procedure. We have performed ICSI with testicular sperm obtained in a similar manner from testes of male mice homozygous for a null mutation in the protein phosphatase 1cγ gene (PP1cγ) or those of their wild-type littermates. PP1cγ mutant testicular sperm are less resistant to sonication than are wild-type sperm and display a range of morphological abnormalities, similar to those reported for testicular sperm from idiopathic azoospermic men. PP1cγ mutant sperm are unable to support development to the blastocyst stage, resulting in arrested development either before or just after compaction. A comparison of testicular and epididymal sperm from wild-type males revealed that the epididymal sperm caused embryos to fragment at an elevated rate. These results suggest that ICSI with any kind of testicular sperm carries an increased risk of embryo fragmentation and that abnormal testicular sperm has an added risk of embryo wastage at later preimplantation stages.

Identification of Potentially Damaging Amino Acid Substitutions Leading to Human Male Infertility

Abstract There are a number of known genetic alterations found in men with nonobstructive azoospermia, or testicular failure, such as Y microdeletions and cytogenetic abnormalities. However, the etiology of nonobstructive azoospermia is unknown in the majority of men. The aim of this study was to investigate the possibility that unexplained cases of nonobstructive azoospermia are caused by nonsynonymous single-nucleotide polymorphisms (SNPs) in the coding regions of autosomal genes associated with sperm production and fertility. Using a candidate gene approach based on genetics of male infertility in mice, we resequenced nine autosomal genes from 78 infertile men displaying testicular failure using custom-made next-generation resequencing chips. Analysis of the data revealed several novel heterozygous nonsynonymous SNPs in four of nine sequenced genes in 14 of 78 infertile men. Eight SNPs in SBF1, three SNPs in LIMK2, two SNPs in LIPE, and one SNP in TBPL1 were identified. All of the novel mutations were in a heterozygous configuration, suggesting that they may be de novo mutations with dominant negative properties.