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1.
PLoS One ; 6(6): e21597, 2011.
Article in English | MEDLINE | ID: mdl-21738724

ABSTRACT

Oocyte cryopreservation is extremely beneficial for assisted reproductive technologies, the treatment of infertility and biotechnology and offers a viable alternative to embryo freezing and ovarian grafting approaches for the generation of embryonic stem cells and live offspring. It also offers the potential to store oocytes to rescue endangered species by somatic cell nuclear transfer and for the generation of embryonic stem cells to study development in these species. We vitrified mouse oocytes using a range of concentrations of trehalose (0 to 0.3 M) and demonstrated that 0.1 and 0.3 M trehalose had similar developmental rates, which were significantly different to the 0.2 M cohort (P<0.05). As mitochondria are important for fertilisation outcome, we observed that the clustering and distribution of mitochondria of the 0.2 M cohort were more affected by vitifrication than the other groups. Nevertheless, all 3 cohorts were able to develop to blastocyst, following in vitro fertilisation, although developmental rates were better for the 0.1 and 0.3 M cohorts than the 0.2 M cohort (P<0.05). Whilst blastocysts gave rise to embryonic stem-like cells, it was apparent from immunocytochemistry and RT-PCR that these cells did not demonstrate true pluripotency and exhibited abnormal karyotypes. However, they gave rise to teratomas following injection into SCID mice and differentiated into cells of each of the germinal layers following in vitro differentiation. The transfer of 2-cell embryos from the 0.1 and 0.3 M cohorts resulted in the birth of live offspring that had normal karyotypes (9/10). When 2-cell embryos from vitrified oocytes underwent vitrification, and were thawed and transferred, live offspring were obtained that exhibited normal karyotypes, with the exception of one offspring who was larger and died at 7 months. We conclude that these studies highlight the importance of the endometrial environment for the maintenance of genetic stability and thus the propagation of specific genetic traits.


Subject(s)
Fertilization in Vitro/methods , Oocytes/cytology , Vitrification/drug effects , Animals , Cryopreservation , Embryo, Mammalian , Female , Male , Mice , Oocytes/drug effects , Trehalose/pharmacology
2.
Endocrinology ; 151(5): 2331-42, 2010 May.
Article in English | MEDLINE | ID: mdl-20219979

ABSTRACT

The glioma pathogenesis-related 1 (GLIPR1) family consists of three genes [GLIPR1, GLIPR1-like 1 (GLIPR1L1), and GLIPR1-like 2 (GLIPR1L2)] and forms a distinct subgroup within the cysteine-rich secretory protein (CRISP), antigen 5, and pathogenesis-related 1 (CAP) superfamily. CAP superfamily proteins are found in phyla ranging from plants to humans and, based largely on expression and limited functional studies, are hypothesized to have roles in carcinogenesis, immunity, cell adhesion, and male fertility. Specifically data from a number of systems suggests that sequences within the C-terminal CAP domain of CAP proteins have the ability to promote cell-cell adhesion. Herein we cloned mouse Glipr1l1 and have shown it has a testis-enriched expression profile. GLIPR1L1 is posttranslationally modified by N-linked glycosylation during spermatogenesis and ultimately becomes localized to the connecting piece of elongated spermatids and sperm. After sperm capacitation, however, GLIPR1L1 is also localized to the anterior regions of the sperm head. Zona pellucida binding assays indicate that GLIPR1L1 has a role in the binding of sperm to the zona pellucida surrounding the oocyte. These data suggest that, along with other members of the CAP superfamily and several other proteins, GLIPR1L1 is involved in the binding of sperm to the oocyte complex. Collectively these data further strengthen the role of CAP domain-containing proteins in cellular adhesion and propose a mechanism whereby CAP proteins show overlapping functional significance during fertilization.


Subject(s)
Glycoproteins/metabolism , Oocytes/metabolism , Spermatozoa/metabolism , Testis/metabolism , Amino Acid Sequence , Animals , Blotting, Northern , Blotting, Western , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Female , Fertilization in Vitro , Gene Expression Profiling , Glycoproteins/genetics , Glycoproteins/physiology , Glycosylation , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Molecular Sequence Data , Oocytes/cytology , Phylogeny , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sperm Maturation/physiology , Sperm-Ovum Interactions/physiology , Spermatozoa/cytology , Spermatozoa/growth & development , Testis/cytology , Zona Pellucida/metabolism
3.
Am J Pathol ; 174(1): 184-95, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19095948

ABSTRACT

Activin A is a potent growth and differentiation factor whose synthesis and bioactivity are tightly regulated. Both follistatin binding and inhibin subunit heterodimerization block access to the activin receptor and/or receptor activation. We postulated that the activin-beta(C) subunit provides another mechanism regulating activin bioactivity. To test our hypothesis, we examined the biological effects of activin C and produced mice that overexpress activin-beta(C). Activin C reduced activin A bioactivity in vitro; in LNCaP cells, activin C abrogated both activin A-induced Smad signaling and growth inhibition, and in LbetaT2 cells, activin C antagonized activin A-mediated activity of an follicle-stimulating hormone-beta promoter. Transgenic mice that overexpress activin-betaC exhibited disease in testis, liver, and prostate. Male infertility was caused by both reduced sperm production and impaired sperm motility. The livers of the transgenic mice were enlarged because of an imbalance between hepatocyte proliferation and apoptosis. Transgenic prostates showed evidence of hypertrophy and epithelial cell hyperplasia. Additionally, there was decreased evidence of nuclear Smad-2 localization in the testis, liver, and prostate, indicating that overexpression of activin-beta(C) antagonized Smad signaling in vivo. Underlying the significance of these findings, human testis, liver, and prostate cancers expressed increased activin-betaC immunoreactivity. This study provides evidence that activin-beta(C) is an antagonist of activin A and supplies an impetus to examine its role in development and disease.


Subject(s)
Inhibin-beta Subunits/metabolism , Animals , Blotting, Western , Humans , Immunohistochemistry , In Vitro Techniques , Infertility, Male , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Male , Mice , Mice, Transgenic , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Smad2 Protein/metabolism , Testicular Neoplasms/genetics , Testicular Neoplasms/metabolism , Tissue Array Analysis , Transfection
4.
Mol Reprod Dev ; 75(1): 48-59, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17546630

ABSTRACT

There are five methyl binding domain (MBD) proteins characterized by a methyl CpG-binding domain. Four MBD proteins (MeCP2 and MBDs 1-3) are linked to transcriptional repression and one (MBD4), to DNA repair. During preimplantation development, the embryo undergoes global demethylation following fertilization and selective remethylation following the maternal to zygotic transition (MZT). This study characterized changes in MBD mRNA expression and protein localization during both murine and bovine preimplantation development. These species were selected because they undergo MZT at different developmental stages. Gene expression profiling during preimplantation development detected the presence of all MBDs examined, although stage and species-specific differences were observed. MBD2 was not expressed in murine or bovine oocytes and MeCP2 was not detected in murine blastocysts, subcellular protein localization was found to vary at time points critical in development. Most MBDs showed species-specificity in localization patterns and differences were found between individual MBDs. MBD1 localization is consistent with a novel role during MZT for both species. MBD3, known to play a crucial role in murine embryogenesis, was highly localized to the nucleus before and after, but not during the MZT in the bovine. MBD2, MBD4, and MeCP2 show varying patterns of localization which indicate possible roles in the early cleavage stages and in inner cell mass differentiation. Further experiments are currently underway to define discreet functional roles for specific MBDs during bovine preimplantation embryogenesis.


Subject(s)
Blastocyst/chemistry , Blastocyst/metabolism , Cattle/embryology , CpG Islands , DNA-Binding Proteins/analysis , Embryonic Development/genetics , Animals , Cattle/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/analysis , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Female , Gene Expression Profiling , Methyl-CpG-Binding Protein 2/analysis , Methyl-CpG-Binding Protein 2/genetics , Methyl-CpG-Binding Protein 2/metabolism , Mice , Mice, Inbred Strains , RNA, Messenger/metabolism , Transcription Factors/analysis , Transcription Factors/genetics , Transcription Factors/metabolism
5.
Reproduction ; 132(5): 799-810, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17071781

ABSTRACT

Maternal-fetal communications are critical for the establishment of pregnancy. Embryonic growth and differentiation factors produced by the oviduct and uterus play essential roles during the pre- and early post-implantation phases. Although several studies indicate roles for activin in embryonic development, gene-knockout studies have failed to identify a critical role in mammalian embryogenesis. We hypothesized that activin is produced by maternal tissues during the establishment of pregnancy, and thus maternally derived activin could compensate for the absence of embryonic activin in null homozygotes during critical developmental stages. We investigated the expression of inhibin alpha, activin betaA, and betaB subunits in the mouse oviduct and uterus during the estrous cycle and early pregnancy, and in the early conceptus. Inhibin alpha subunit was weakly expressed, while activin betaA and betaB subunits were strongly expressed in oviduct and uterus at estrous, and dramatically upregulated in the uterus on each day of pregnancy between days 3.5 and 8.5 post coitum. Prior to implantation, activin betaA and betaB subunits were immunolocalized to oviductal and uterine epithelial cells; following implantation they were expressed in the stroma, in a wave preceding decidualization. Later in pregnancy, activin betaA and betaB subunits were present in decidua basalis, trophoblast giant cells, and labyrinth zone of the developing placenta. Expression of activin betaA subunit was also detected in blastocysts and early post-implantation embryos. These data are consistent with a role for maternally derived activins in the support of the pre-implantation embryo, and during gastrulation and embryogenesis.


Subject(s)
Activins/metabolism , Maternal-Fetal Exchange , Pregnancy, Animal/metabolism , Uterus/metabolism , Animals , Corpus Luteum Maintenance , Estrous Cycle , Fallopian Tubes/chemistry , Fallopian Tubes/metabolism , Female , Immunohistochemistry , Inhibins/analysis , Inhibins/metabolism , Mice , Placenta/chemistry , Placenta/metabolism , Pregnancy , Reverse Transcriptase Polymerase Chain Reaction , Uterus/chemistry
6.
J Cell Sci ; 119(Pt 1): 75-84, 2006 Jan 01.
Article in English | MEDLINE | ID: mdl-16352663

ABSTRACT

Cloning of the fibroblast growth factor receptor (FGFR) adaptor Snt-2 cDNA and the identification of FGFR-1 protein in association with sperm tails, suggested that FGFR-1 signaling was involved in either sperm tail development or function. This hypothesis was tested by the creation of transgenic mice that specifically expressed a dominant-negative variant of FGFR-1 in male haploid germ cells. Mating of transgenic mice showed a significant reduction in pups per litter compared with wild-type littermates. Further analysis demonstrated that this subfertility was driven by a combination of reduced daily sperm output and a severely compromised ability of those sperm that were produced to undergo capacitation prior to fertilization. An analysis of key signal transduction proteins indicated that FGFR-1 is functional on wild-type sperm and probably signals via the phosphatidylinositol 3-kinase pathway. FGFR-1 activation also resulted in the downstream suppression of mitogen activated protein kinase signaling. These data demonstrate the FGFR-1 is required for quantitatively and qualitatively normal spermatogenesis and has a key role in the regulation of the global tyrosine phosphorylation events associated with sperm capacitation.


Subject(s)
Receptor, Fibroblast Growth Factor, Type 1/metabolism , Signal Transduction/physiology , Sperm Capacitation/physiology , Spermatogenesis/physiology , Animals , Female , Fertility/physiology , Fibroblast Growth Factor 2/metabolism , Male , Mice , Mice, Transgenic , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptor, Fibroblast Growth Factor, Type 1/genetics , Spermatozoa/cytology , Spermatozoa/physiology , Transgenes
7.
Biol Reprod ; 69(5): 1683-9, 2003 Nov.
Article in English | MEDLINE | ID: mdl-12890731

ABSTRACT

Re-establishment of mouse strains used for mutagenesis and transgenesis has been hindered by difficulties in freezing sperm. The use of intracytoplasmic sperm injection (ICSI) enables the production of embryos for the restoration of mouse lines using sperm with reduced quality. By using ICSI, simplified sperm-freezing methods such as snap freezing can be explored. We examined the capacity of embryos from the inbred C57Bl/6J and 129Sv/ImJ mouse strains, commonly used for transgenic and N-ethyl-N-nitrosourea mutagenesis purposes to develop to blastocysts in vitro and to term following ICSI with sperm frozen without cryoprotectant. The results were compared to F1 (C57BlxCBA) hybrid embryos. Following freezing, sperm were immotile but could fertilize oocytes at similar rates to fresh sperm. However, embryo development in vitro to the blastocyst stage was reduced in all three strains. No pups were born from C57Bl/6J or 129Sv/ImJ embryos obtained from frozen sperm following transfer to foster females, and only a limited number of F1 embryos developed to term. Activation of oocytes injected with frozen sperm with 1.7 mM Sr2+ (SrCl2) did result in the birth of pups in all three strains. We conclude that the inability of sperm frozen without cryoprotectants to effectively activate oocytes may affect embryo development to term and can be overcome by strontium activation. This may become an effective strategy for sperm preservation and the restoration of most popular strains used for genetic modifications.


Subject(s)
Oocytes/physiology , Semen Preservation , Sperm Injections, Intracytoplasmic , Spermatozoa/physiology , Animals , Chromosomes/genetics , Cryoprotective Agents/pharmacology , Embryo Transfer , Embryonic and Fetal Development , Female , Fertilization in Vitro , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Oocytes/drug effects , Parthenogenesis/drug effects , Parthenogenesis/physiology , Pregnancy , Strontium/pharmacology
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