The antioxidants superoxide dismutase (SOD), catalase (CAT), cytochrome c (CHc) and glutathione peroxidase (GP) were added at various concentrations to Tris-glucose-yolk diluent (TGY), and their effects on motility, acrosome integrity and fertility of ram spermatozoa were assessed after extension and liquid storage. All the antioxidants improved the motility and acrosome integrity of spermatozoa, and a combination of SOD and CAT had an additive effect on the survival of spermatozoa stored at 5 degrees C but not at 25 degrees C. There was a linear improvement in survival of spermatozoa with increasing dose of antioxidants except for CAT for which doses higher than 200 U mL-1 were toxic. The proportion of oocytes fertilized in vitro declined with time of semen storage (P < 0.001), and was better for semen diluted with TGY containing SOD or CAT than TGY without antioxidants when stored for 7 days (116/246, 47% v. 25/79, 32%; P < 0.05) but not for 14 days (23/174, 13% v. 8/66, 12%). Fertilization rates were unaffected by the presence or absence in the diluent of CHc or GP. The proportions of ewes with fertilized ova and of recovered ova fertilized were better after insemination with semen diluted in TGY containing SOD and CAT than TGY without antioxidants when stored for 14 days (9/18, 50% and 20/40, 50% v. 2/13, 15% and 5/32, 16%; P < 0.05) but not for 7 days (9/20, 45% and 16/48, 33% v. 8/16, 50% and 24/41, 59%). Pregnancy rates were better after intrauterine insemination of ewes with fresh semen than stored semen (11/18, 61% v. 21/75, 28%; P < 0.01), and with semen stored in TGY containing SOD and CAT than in TGY without antioxidants (15/37, 41% v. 6/38, 16%; P < 0.05).

Growth of preimplantation embryos is influenced byThe cells of the mammalian preimplantation embryo (from the time of fertilization until the implantation of the blastocyst into the uterus) form the progenitor cells for all other cell lineages. The regulation of the growth and survival of the cells of the early embryo is, however, poorly understood. Mammalian preimplantation embryos develop in vitro with simple medium requirements and have no absolute requirement for exogenous vitamins, hormones, or growth factors. This contrasts with the absolute requirement of normal somatic cells for exogenous mitogens and survival factors. The continued mitoses of preimplantation embryo cells in the absence of exogenous growth factors implicates a role for endogenous, autocrine trophic factors, or the constitutive activation of signaling pathways in the early embryo. Several lines of evidence support a role for the former: (i) the rate of embryo development in vitro is density-dependent, with embryos growing in relatively small volumes (or in large groups) developing more successfully than those grown in large volumes (or individually) (1, 2); (ii) the synthesis by the preimplantation embryo of a number of growth factor ligands and their receptors (3-8); and (iii) the capacity of some exogenous growth factors to enhance embryo metabolism in vitro and to compensate for the adverse effects of culture in large medium volumes (1, 2, 9).Experimental partial deprivation of released autocrine trophic factors did not arrest the cell-cycle at given checkpoints (9). Rather, there was progressive loss of viability with increased cell death as embryos progressed past the 8-cell stage. This finding suggests that the autocrine factors may act as survival factors rather than classical growth factors (triggering progression through specific cell-cycle checkpoints). While several autocrine factors have been implicated, platelet-activating factor (PAF) 1 seems to be one of the first produced, being synthesized de novo by the embryo soon after fertilization (10, 11). Its actions are required by the mid-2-cell stage for normal rates of embryo survival (9).Despite this range of supportive data, there is limited direct evidence for the action of autocrine trophic factors in early embryo development. Transgenic and recombinant knock-out models have not generally been informative of the growth requirements of the early embryo prior to implantation. This may be due to extensive redundancy of regulatory pathways.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by an expansion of cytosine-adenine-guanine (CAG) repeats in the Huntingtin gene Htt. To facilitate research into HD, we have derived 4 human embryonic stem cell (hESC) lines containing ≥ 40 CAG repeats in exon 1 of Htt: SIVF017-HD (CAG₄₀), SIVF018-HD (CAG₄₆), SIVF020-HD (CAG₄₈), and SIVF046-HD (CAG₄₅). Additionally, we have derived a normal sibling-matched control for SIVF020-HD, cell line SIVF019. All 5 hESC lines had a normal karyotype, expressed pluripotency markers including Oct4, SSEA3, and Tra-1-81, and could be maintained in culture for multiple (>40) passages. Teratoma studies revealed that the hESC lines were capable of differentiating into cells representative of the 3 germ layers. Furthermore, in vitro neuronal differentiation experiments have confirmed that the hESC lines were able to generate MAP2-positive neuronal cells that express the Htt protein. Combined, these experiments confirm that the cell lines represent pluripotent stem cell lines. These HD-affected hESC lines will be made available to biomedical research laboratories and will provide a valuable tool to investigate the mechanisms and potential treatments for HD.

Protein biomarkers are fundamental tools for the characterization of stem cells and for tracking their differentiation and maturation down developmental lineages. Technology development allowing increased coverage of difficult cellular proteomes should allow for the discovery of new and novel membrane protein biomarkers for use by the stem cell research community. The amphipathic and highly hydrophobic nature and relative low abundance of many membrane proteins present significant analytical challenges. These difficulties are amplified when the source material (tissue or cells) is only available in limited quantities (e.g., embryonic stem cells). Recent advances in enrichment for purer membrane fractions, the enzymatic and chemical digestion of membrane proteins in the presence of solvents or chaotropes, and the use of "shotgun" proteomics methodologies have gradually resulted in increased membrane proteome coverage with numbers of predicted integral membrane proteins now in excess of 1000 being routinely reported. We have recently demonstrated the advantages of using peptide isoelectric focusing in the first dimension on immobilized pH gradients (peptide IPG-IEF) followed by reversed phase chromatography and tandem MS to increase membrane proteome coverage. This study looked at achieving a similar level of membrane proteome coverage using modifications to reported methodologies while restricting the number of characterized human embryonic stem cells to 10(7) cells. Two-thousand two-hundred and ninety-two (2292) nonredundant proteins were identified with two or more high accuracy peptide matches from 260 mug of a human embryonic stem cell membrane enriched fraction with a false discovery rate of 0.32%. Gene Ontology (GO) mapping predicted 1279 (44.9%) of this list to be membrane proteins of which 395 proteins were predicted to be derived from the plasma membrane compartment. The TMHMM algorithm predicted 904 integral membrane proteins with up to 16 transmembrane helices. Collectively, we assert that the substantial membrane proteome coverage achieved using these procedures will enable rapid advances in the identification and quantitation of novel membrane proteins as markers of differentiation status and/or genetic mutation from relatively low numbers of cultured embryonic stem cells.

Mammalian spermatozoa require a maturational event after ejaculation that allows them to acquire the capacity for fertilization. This process, known as capacitation, occurs spontaneously in simple defined medium implicating a potential role of autocrine induction. This study shows that the ether phospholipid 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (PAF) meets the criteria for an autocrine mediator of capacitation. Sperm released PAF after their dilution into capacitation medium and expressed a receptor for PAF on their membranes. PAF stimulated changes in the motility of sperm and enhanced fertilization in vitro. These actions were inhibited by a PAF receptor antagonist (UR-12519) and by extracellular recombinant PAF:acetylhydrolase (an enzyme that degrades PAF to a biologically inert form). Seminal plasma contained an acid-labile PAF:acetylhydrolase, whereas capacitation was inhibited by an acid-labile factor within seminal plasma, implicating this factor as a potential decapacitation factor within seminal plasma. Sperm from a PAF receptor knock-out mouse strain failed to express the receptor and displayed a significantly (p < 0.01) reduced rate of capacitation, as assessed by the spontaneous onset of the acrosome reaction in vitro. When used for in vitro fertilization, sperm from PAF receptor knock-out mice gave a significantly lower rate of fertilization (21.5%) than did wild-type sperm (66.7%). The study shows for the first time the operation of an autocrine loop that induces capacitation in sperm in vitro and shows that this loop acts in concert with other mediators of capacitation to promote efficient fertilization.