Liquid storage of ram semen in the absence or presence of some antioxidants.

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).

Flow cytometric sorting of fresh and frozen-thawed spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla).

We adapted flow cytometry technology for high-purity sorting of X chromosome-bearing spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla). Our objectives were to develop methodologies for liquid storage of semen prior to sorting, sorting of liquid-stored and frozen-thawed spermatozoa, and assessment of sorting accuracy. In study 1, the in vitro sperm characteristics of gorilla ejaculates from one male were unchanged (P > 0.05) after 8 hr of liquid storage at 15 degrees C in a non-egg yolk diluent (HEPES-buffered modified Tyrode's medium). In study 2, we examined the efficacy of sorting fresh and frozen-thawed spermatozoa using human spermatozoa as a model for gorilla spermatozoa. Ejaculates from one male were split into fresh and frozen aliquots. X-enriched samples derived from both fresh and frozen-thawed human semen were of high purity, as determined by fluorescence in situ hybridization (FISH; 90.7%+/-2.3%, overall), and contained a high proportion of morphologically normal spermatozoa (86.0%+/-1.0%, overall). In study 3, we processed liquid-stored semen from two gorillas for sorting using a modification of methods for human spermatozoa. The sort rate for enrichment of X-bearing spermatozoa was 7.3+/-2.5 spermatozoa per second. The X-enriched samples were of high purity (single-sperm PCR: 83.7%) and normal morphology (79.0%+/-3.9%). In study 4 we examined frozen-thawed gorilla semen, and the sort rate (8.3+/-2.9 X-bearing sperm/sec), purity (89.7%), and normal morphology (81.4%+/-3.4%) were comparable to those of liquid-stored semen. Depending on the male and the type of sample used (fresh or frozen-thawed), 0.8-2.2% of gorilla spermatozoa in the processed ejaculate were present in the X-enriched sample. These results demonstrate that fresh or frozen-thawed gorilla spermatozoa can be flow cytometrically sorted into samples enriched for X-bearing spermatozoa.

Ontogeny of expression of a receptor for platelet-activating factor in mouse preimplantation embryos and the effects of fertilization and culture in vitro on its expression.

Platelet-activating factor (PAF; 1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent ether phospholipid. It is one of the preimplantation embryo's autocrine growth/survival factors. It may act via a G protein-linked receptor on the embryo; however, the evidence for this is conflicting. The recent description of the intracellular form of the PAF:acetlyhydrolase enzyme as having structural homology with G proteins and Ras also suggests this as a potential intracellular receptor/transducer for PAF. This study used reverse transcription-polymerase chain reaction to examine the ontogeny of expression of the genes for these proteins in the oocyte and preimplantation-stage embryo. Transcripts for the G protein-linked PAF receptor were detected in the late 2-cell-stage embryo and in all stages from the 4-cell stage to blastocysts. They were also present in unfertilized oocytes and newly fertilized zygotes but only at relatively low levels. The incidence of expression was generally low and variable in late zygotes and early 2-cell embryos. Expression past the 2-cell stage was alpha-amanitin sensitive. The results indicated that mRNA for this receptor is a maternal transcript that was degraded during the zygote-2-cell stage. New expression of the receptor transcript required activation of the zygotic genome. Fertilization of embryos in vitro caused this transcript not to be expressed in the zygote. Culture of zygotes (irrespective of their method of fertilization) caused expression from the zygotic genome to be retarded by more than 24 h. This retardation did not occur if culture commenced at the 2-cell stage. The transcripts for the subunits of intracellular PAF:acetylhydrolase were not detected in oocytes or at any stage of embryo development examined, despite their being readily detected in control tissue. This study confirms the presence of the G protein-linked PAF receptor in the 2-cell embryo and describes for the first time its normal pattern of expression during early development. The adverse effects of in vitro fertilization (IVF) and embryo culture on the expression of this transcript may be a contributing factor for the poor viability of embryos produced in this manner. The reduced expression of PAF-receptor mRNA following IVF predicts that such embryos may have a deficiency in autocrine stimulation and also suggests that supplementation of growth media with exogenous PAF would be only partially beneficial. The effect of IVF and culture may also explain the conflicting literature.

In vitro fertilization causes epigenetic modifications to the onset of gene expression from the zygotic genome in mice.

The effect of in vitro fertilization (IVF) and culture of mouse preimplantation embryos in vitro on the onset of expression of insulin-like growth factor 1 (IGF-1) ligand and receptor, insulin ligand and receptor, alpha-transforming growth factor (alpha-TGF) ligand, PAF:acetylhydrolase 1b (Pafah1b; alpha(1), alpha(2), and beta subunits of the enzyme), and the transcription requiring complex proteins (TRC) was examined. The IGF-1 ligand was detected in preimplantation embryos by immunofluorescence at all developmental stages tested. However, IVF and culture significantly reduced the amount of protein detected in the 8-cell embryo and blastocyst (P: < 0.001), and this was due to a delayed onset of expression of the mRNA for IGF-1 ligand from the zygotic genome. The expression of the alpha(1) subunit of Pafah1b was first detected at the 2-cell stage in fresh embryos, but expression was significantly retarded (P: < 0.001) when IVF and ISF (in situ-fertilized) zygotes were cultured in vitro. In vitro fertilization or ISF did not delay the onset of expression of TRC nor mRNA for the IGF-1 receptor, insulin receptor, alpha(2) or beta subunit of Pafah1b, nor did they effect alpha-TGF protein synthesis. Thus, IVF causes epigenetic modification in the normal pattern of expression of some but not all genes involved in normal embryo growth and survival.

In-vitro fertilization and culture of mouse embryos in vitro significantly retards the onset of insulin-like growth factor-II expression from the zygotic genome.

In this study, the effect of in-vitro fertilization (IVF) and culture of mouse embryos in vitro on the normal expression of insulin-like growth factor-II (IFG-II) ligand and receptor was examined. The expression of IGF-II increased in a linear fashion at least up to the 8-cell stage of development. IGF-II expression in embryos collected fresh from the reproductive tract was significantly (P < 0.001) greater than in embryos fertilized in the reproductive tract and cultured in vitro (in-situ fertilized: ISF), and its expression was further reduced (P < 0.001) in IVF embryos at all development stages tested. The expression of IGF-II was significantly (P < 0.001) lower when embryos were cultured individually in 100 microl drops compared with culture in groups of 10 in 10 microl drops of medium. The addition of platelet activating factor to culture medium partially overcame this density-dependent decline of expression. Culture of ISF and IVF zygotes also caused the onset of new IGF-II mRNA transcription from the zygotic genome to be significantly (P < 0.001) retarded, until at least the 8-cell stage of development. This effect was greater (P < 0.05) for IVF than for ISF embryos. Neither IVF nor culture had any obvious effect on IFG-II/mannose-6-phosphate receptor (IGF-IIr) mRNA expression.

Evidence for the autocrine induction of capacitation of mammalian spermatozoa.

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.

Characterization and functional significance of calcium transients in the 2-cell mouse embryo induced by an autocrine growth factor.

Growth of preimplantation embryos is influenced by autocrine trophic factors that need to act by the 2-cell stage, but their mode of action is not yet described. This report shows that late zygote and 2-cell stage mouse embryos responded to embryo-derived platelet-activating factor (PAF) with transient increases in intracellular calcium concentration ([Ca(2+)](i)). [Ca(2+)](i) transients were single global events and were specifically induced by embryo-derived PAF. They were blocked by inhibition of phospholipase C (U 73122) and an inositol trisphosphate (IP(3)) receptor antagonist (xestospongin C), indicating the release of calcium from IP(3)-sensitive intracellular stores. Transients were also inhibited by the absence of calcium from extracellular medium and partially inhibited by treatment with dihydropyridine (nifedipine, 10 micrometer), but not pimozide (an inhibitor of an embryonic T-type calcium channel). (+/-)BAY K8644 (an L-type channel agonist) induced [Ca(2+)](i) transients, yet these were completely inhibited by nifedipine (10 micrometer). The complete inhibition of BAY K8644, but only partial inhibition of PAF by nifedipine shows that L-type channels were only partly responsible for the calcium influx. Depolarization of 2-cell embryos by 50 mm K(+) did not inhibit PAF-induced calcium transients, showing that the influx channels were not voltage-dependent. Depletion of intracellular calcium stores by thapsigargin revealed the presence of store-operated channels. The interdependent requirement for IP(3)-sensitive internal calcium stores and extracellular calcium in the generation of PAF-induced transients may be explained by a requirement for capacitative calcium entry via store-operated channels. A functionally important role for the PAF-induced transients is supported by the observation that inhibition of [Ca(2+)](i) transients by a PAF-antagonist (WEB 2086) or an intracellular calcium chelator (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; BAPTA-AM) caused marked inhibition of early embryo development. Growth inhibition by BAPTA-AM was relieved by addition of exogenous PAF.