An EORTC international multicenter randomized trial (EORTC number 19923) comparing two dosages of liposomal amphotericin B for treatment of invasive aspergillosis

Experiments were performed to determine the actions of recombinant bovine interleukin-1beta (IL-1beta) on the growth of preimplantation embryos. In the first series of studies, IL-1beta was added at 8-10 h after insemination, and the percentage of oocytes developing to the blastocyst stage was evaluated. IL-1beta increased development to the blastocyst stage when embryos were cultured at high density ( approximately 25-30 embryos/drop) but decreased or had no effect on development when cultured at low density ( approximately 10 embryos/drop). Thus, the positive effect of IL-1beta depends upon some other embryo-derived product. The effect of IL-1beta on embryonic development was maintained in completely denuded embryos, indicating that cumulus cells do not mediate the actions of IL-1beta. Maximum development of embryos cultured at approximately 25-30/drop occurred at 0.1-1 ng/ml; 10 ng/ml was less effective. Addition of IL-1beta to groups of approximately 25-30 embryos/drop at 8-10 h after insemination also increased embryo cell number at Day 5 postinsemination by increasing the proportion of embryos that reached the 9- to 16-cell stage. However, IL-1beta had no effect on the proportion of blastocysts when added at Day 5 postinsemination. Thus, IL-1beta probably acts to increase blastocyst numbers by exerting actions on embryo growth before Day 5. In contrast to its effect on embryos, addition of IL-1beta during oocyte maturation did not affect cumulus expansion, cleavage rate of oocytes, or subsequent development to the blastocyst stage. In conclusion, IL-1beta can modulate growth of bovine embryos at early stages of development in a manner dependent upon embryo density.     

Free innervated latissimus dorsi muscle flap for reconstruction of full-thickness abdominal wall defects

The aim of this study was to develop a maturation protocol for immature oocytes and assess the protocol with cryopreserved oocytes. Nuclear maturation (mature spindle and aligned chromosomes) occurred irrespective of the treatment regime: 71-89% of oocytes matured in vitro had a normal spindle and chromosomes compared with 87% matured in vivo. Fertilization rates were not significantly different from those of in-vivo matured oocytes. Of the maturation treatment regimes investigated, the initial treatment producing best development to blastocyst (cytoplasmic maturation) involved a 2 h incubation in standard maturation medium (SMM) containing 7.5 IU follicle stimulating hormone (FSH) followed by 14 h in SMM plus 7.5 IU FSH:luteinizing hormone with follicular cells [62% (range 49-69)]. The addition of 1 ng/ml epidermal growth factor (EGF) in this protocol resulted in development [75% (range 71-81)] that was not significantly different from in-vivo matured oocytes [82% (range 73-90)]. Exposure of the oocytes to 1.5 M dimethylsulphoxide (DMSO) did not affect fertilization or development rates. Following a slow-cool/thaw freezing regime, 81% (range 74-89) of the oocytes were morphologically normal, i.e. had a spherical shape with an intact zona and oolemma; they had, however, lost their previously attached cumulus and corona cells. Maturation of frozen-thawed oocytes in the presence of EGF gave good fertilization rates but poor development rates [80% (range 77-86) and 37% (range 33-40) respectively]. In conclusion, the best maturation, both nuclear and cytoplasmic, occurred in the presence of a combination of gonadotrophins, EGF and follicular cells. Oocytes cryopreserved using a slow-cool/thaw regime can be matured to produce blastocysts after in-vitro fertilization.     

Effects of glycosaminoglycans on the development of in vitro-matured and -fertilized porcine oocytes to the blastocyst stage in vitro

We examined the effects of four glycosaminoglycans (GAGs) on the development of in vitro-matured (IVM) and -fertilized (IVF) porcine oocytes to the blastocyst stage. IVM and IVF oocytes were cultured in Whitten's medium supplemented with hyaluronic acid, chondroitin sulfate A, dermatan sulfate, or heparin at 38.5 degrees C in an atmosphere of 5% CO2 in humidified air for up to 6 days. After 2 days in culture, 28-34% of the inseminated oocytes cleaved to the 2- to 8-cell stage, and the GAGs showed no significant effect on development. After 6 days in culture, blastocysts were observed in all groups. The percentage of blastocysts was significantly higher in hyaluronic acid-supplemented medium (14%) than in dermatan sulfate-supplemented (5%), heparin-supplemented (2%), or nonsupplemented (2%) media. In addition, the percentage of blastocysts was significantly higher in chondroitin sulfate A-supplemented medium (11%) than in heparin-supplemented and nonsupplemented media, although the number of blastocysts in chondroitin sulfate A was not significantly different from that in hyaluronic acid- and dermatan sulfate-supplemented media. There were no significant differences in the mean number of nuclei per blastocyst cultured in any group. The effects of hyaluronic acid and chondroitin sulfate A on development to the blastocyst stage was examined at various concentrations. After 6 days in culture, development of IVM and IVF oocytes to the blastocyst stage was best supported in 0.5 mg/ml hyaluronic acid-supplemented (17%) and in 0.1 or 0.5 mg/ml chondroitin sulfate A-supplemented (10% or 9%, respectively) media. It is concluded from these results that hyaluronic acid and chondroitin sulfate A supported the development of porcine oocytes matured and fertilized in vitro to the blastocyst stage.     

Antigens recognized by T-lymphocytes on human tumours

Some of the factors influencing the success of a nuclear transfer procedure are the quality of the recipient oocyte and the efficiency of the method of artificial activation. In this study we evaluated the ability of an electrical pulse to stimulate in vitro-matured porcine oocytes to develop. Maturation in Waymouth's medium resulted in significantly greater development than maturation in TCM-199 (p < 0.05), while there was no significant difference between degrees of development in Waymouth's medium and Whitten's medium. Oocytes matured in Waymouth's medium and electrically stimulated at 36 h (young oocytes) developed to the same degree as oocytes stimulated at 48 h (aged oocytes). Oocytes matured in Waymouth's medium and treated with cytochalasin B showed significantly greater development (p < 0.10) in response to electrical activation than controls. Staurosporine activation of oocytes resulted in significantly (p < 0.05) fewer morulae and blastocysts when compared to electrical stimulation. Development of parthenogenic embryos to the elongated filamentous stage (10% development beyond blastocyst) was obtained by maturing oocytes in Waymouth's medium and electrically stimulating them to develop. By obtaining development of porcine parthenotes beyond the blastocyst stage, we have identified an efficient method of oocyte maturation and oocyte activation for use in a system for nuclear transfer.     

Development of pig oocytes activated by stimulation of an exogenous G protein-coupled receptor

This study determined whether stimulation of a G protein-coupled receptor could initiate the events that occur at fertilization in pig oocytes and, if so, whether the activated oocytes were competent to form blastocysts. After maturation for 30 h, oocytes received microinjections of mRNA encoding the rat M1 muscarinic receptor, a G protein-coupled acetylcholine (ACh) receptor. Oocytes were then incubated for an additional 15 h to complete maturation of oocytes and translation of microinjected mRNA, and they were subsequently cultured in the presence of ACh. ACh treatment of these oocytes triggered pronuclear formation (50.4%) as well as cortical granule exocytosis. SDS-PAGE showed that mRNA-microinjected oocytes treated with ACh were activated (61.1%), as characterized by the appearance of the 22-kDa polypeptide derived from dephosphorylation of the 25-kDa precursor. Furthermore, after being cultured in a ligated pig oviduct for 6 days, 17.4% of treated oocytes developed to the compact morula or blastocyst stage. Transmission electron microscopy revealed that blastocysts recovered from ligated oviducts contained reticulated nucleoli with fibrillar cores surrounded by fibrillar and granular components. In addition, mitochondria in the blastocysts were dispersed throughout the cytoplasm and contained numerous transverse cristae. These results show that pig oocyte activation mediated by a G protein-coupled signal transduction system can signal a series of intracellular changes that lead to activation events associated with fertilization. Furthermore, oocytes activated through this pathway showed preimplantation development consistent with normal development.     

Current and future methodology for monitoring sleep

The present study examined the effect of follicular shell pieces (FSP) during in vitro maturation (IVM) of porcine oocytes on 1) in vitro fertilization (IVF) parameters, 2) subsequent embryo development, 3) oocyte glutathione (GSH) concentration, and 4) viability after embryo transfer. Cumulus-oocyte complexes were cultured in North Carolina State University (NCSU) 23 medium containing porcine follicular fluid, cysteine, and hormonal supplements and with or without FSP for 20-22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20-22 h. After culture, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 5-6 h. Putative zygotes were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 h. In comparisons between the presence and absence of FSP, no differences were observed in fertilization parameters. At 48 h, no mean differences were found in cleavage rates. However, at 144 h, the proportion of embryos that developed to the blastocyst stage was significantly (p < 0.01) higher (18% vs. 36%) for oocytes cocultured with FSP. A significantly (p < 0.05) higher GSH concentration was found in oocytes matured with FSP as determined by dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Transfer of embryos to 9 recipients resulted in 5 pregnancies with the birth of 18 live piglets. The results provide clear evidence of the beneficial effect of FSP during IVM of pig oocytes cultured in the presence of cysteine on subsequent embryo development to the blastocyst stage. The birth of piglets confirms the viability of IVM-IVF-derived embryos.     

Effect of free amino acids and vitamins on cleavage and developmental rate of bovine zygotes in vitro

Due to the complicated media used for culturing bovine embryos, most of the nutrient requirements are unknown. Recently, we developed a simple, serum-free medium (CR1) that allows bovine embryos to develop in vitro. Therefore, our objective was to determine whether development of bovine embryos would be improved by the addition of free amino acids and vitamins to CR1. Oocytes were recovered from slaughterhouse ovaries and matured 22 +/- 2 h, following which the oocytes were randomly allotted to treatment. The experiment was a randomized block design with a 2 x 5 factorial treatment structure. The oocytes were fertilized with or without cumulus cells intact. The five fertilization media were 1) Control (CR1 +/- 10 micrograms/mL of phenol red); 2) control + basal medium Eagle (BME) essential amino acids (EAA) + minimum essential medium (MEM) nonessential amino acids (NEA) + MEM vitamins (VIT); 3) control + EAA + NEA; 4) control + EAA + VIT; and 5) control + NEA + VIT. Cleavage rate was greater (P < .001) when cumulus cells remained on the oocytes during fertilization (51.7 vs 73.2% without and with cumulus cells, respectively). The frequency of blastocysts was increased (P < .001) when EAA or NEA were added to CR1; however, adding VIT had no effect or tended (P = .12) to decrease the frequency of embryos attaining the blastocyst stage. This experiment demonstrates that development of bovine embryos in vitro can be improved by the addition of free amino acids to a simple medium. Contrary to work in rodents, the mixture of vitamins in MEM was not beneficial for bovine embryos.     

Stimulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability

Glutathione (GSH) has been shown to play an important role in embryo development. In a previous study, we demonstrated that cysteamine supplementation of in vitro maturation (IVM) medium increased the intracellular GSH content in bovine oocytes and improved subsequent embryo development to the blastocyst stage. The present study was carried out to evaluate the effect of inhibition by buthionine sulfoximide (BSO) of GSH synthesis during IVM in the presence of cysteamine, on subsequent embryo development, and the effect of cysteamine during IVM on the survival of blastocysts following freezing. The effect of beta-mercaptoethanol and cysteine added to the maturation medium on GSH levels in bovine oocytes, as well as the effect of these compounds on de novo GSH synthesis by oocytes during in vitro maturation, was also studied. The inhibitory effect of BSO during in vitro maturation on GSH synthesis was also evaluated. Evidence was found confirming that GSH synthesis occurs intracellularly during IVM of oocytes and is stimulated by cysteamine, beta-mercaptoethanol and cysteine. Moreover, the present results suggest that the increase in the rate of embryo development exerted by cysteamine, when present during IVM, was due to its stimulatory effect on GSH synthesis. This increase in GSH levels during IVM improves embryo development and quality, producing more embryos reaching the blastocyst stage on day 6, those most suitable for freezing.     

Stress proteins and stress tolerance in an Antarctic, psychrophilic yeast, Candida psychrophila

This study evaluated whether trophoblastic tissue derived in vitro secretes factors that support bovine embryonic development in vitro. The embryotrophic activity of these secretions was analysed in three different culture conditions based on TCM-199: (1) in a routine culture system using cumulus cells and 10% oestrous cow serum; (2) without cells but with 10% oestrous cow serum; and (3) under serum-free conditions. Rates of development to the 5-8-cell and blastocyst stages, as well as numbers of inner cell mass and trophectoderm cells of blastocysts were determined. In the absence of cumulus cells, cleavage rates of 5-8-cell embryos were significantly (P < 0.05) higher in trophoblastic vesicle-conditioned medium than in TCM-199 in both the presence (71% versus 49%) and absence (70% versus 49%) of serum. Trophoblastic vesicle-conditioned medium had a significant (P < 0.05) positive effect on the rate of development to the blastocyst stage when compared with TCM-199 in the presence of cumulus cells and serum (39% versus 33%), only serum (26% versus 19%), or in the absence of cells and serum (21% versus 5%). The numbers of inner cell mass and trophectoderm cells, and total number of cells in blastocysts produced in the cumulus cell coculture system in serum-free trophoblastic vesicle-conditioned medium or TCM-199 supplemented with serum were greater than those of blastocysts produced without cumulus cells or serum. Fractionation of serum-free trophoblastic vesicle-conditioned medium by ultrafiltration (10 kDa cut off) confined the embryotrophic activity mainly to the low molecular mass fraction. This study shows that serum-free trophoblastic vesicle-conditioned medium contains potent embryotrophic factors which act in a complementary manner to those secreted by cumulus cells and those supplemented with serum and result in reliably high blastocyst rates in the range of 40%. Since contamination of trophoblastic vesicle-conditioned medium with serum proteins can be avoided, this medium may be a reasonable source for the purification of specific embryotrophic factors.     

Characterization of the embryotrophic activity of exogenous protein-free oviduct-conditioned medium used in culture of cattle embryos

Bovine zygotes, obtained after in vitro maturation and fertilization of oocytes from slaughtered cow ovaries, were cultured in droplets of nonconditioned or conditioned medium on bovine oviduct cell monolayers. The media tested were Medium 199 alone and Medium 199 supplemented with 10% fetal calf serum (FCS). Oviduct conditioning increased both early cleavage and development to blastocysts. Only the effect on early cleavage was mimicked by FCS. The blastocysts obtained in serum-free conditioned medium (SFCM) appeared morphologically normal and had the same cell number as those produced in conditioned medium containing serum. Their hatching rates did not differ. Transfer of 16 blastocysts developed in SFCM to 16 synchronized recipients resulted in five pregnancies (31%), indicating good embryonal viability. Boiling of SFCM resulted in a total loss of activity, while heating at 56 degrees C for 30 min had no deleterious effect. A 10-kDa ultrafiltration of SFCM removed the blastocyst development-supporting activity from the filtrate but not the early cleavage-favoring activity. This allows us to conclude that at least two different factors are present in SFCM: one of low molecular mass (< approximately 10 kDa), needed to obtain the 5-8 cell stage and mimicked by FCS, and another of higher molecular mass allowing embryos to develop from the 8-cell to blastocyst stage.     

Development of embryos produced by intracytoplasmic sperm injection of cat oocytes

Development of cat oocytes following intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) was compared in two experiments. Domestic cat donors (used as a model for wild felids) were treated with 150 IU equine chorionic gonadotrophin (eCG) on treatment day 1 or a total of 10-15 IU of follicle-stimulating hormone (FSH) over four days, followed by 100 IU human chorionic gonadotrophin (hCG) on day 5 and follicular aspiration 24-26 h later. A jaguarundi (Herpailurus yaguarondi) female was stimulated twice with FSH (20 IU) or eCG (300 IU) and hCG (250 or 300 IU) before oocyte recovery. After storage at 4 degrees C, domestic cat semen was washed and processed. For ICSI, denuded oocytes were each injected with an immobilised spermatozoon. IVF oocytes were co-incubated with 5 x 10(4) motile spermatozoa/0.5 ml for 4-6 h. Noncleaving oocytes were fixed and stained 24-28 h after injection or insemination. Presumptive zygotes were cultured before transfer on day 5 (experiment I only) or evaluation on day 7 (experiments I and II). In experiment I, fertilization frequency was 67.9% (72/106) and 58.1% (122/210) for IVF and ICSI oocytes, respectively (P > 0.05). Most noncleaving ICSI oocytes (71/88, 80.7%) at 24 h were at metaphase II, of which half (35/71, 49.3%) had an activated spermatozoon (n=4) or premature chromatin condensation (PCC, n=31) of the sperm head. All 69 day 7 IVF embryos developed to morulae (> 16-cells, 46.7%) or blastocysts (53.3%), and 59/63 (93.7%) ICSI embryos reached the morula (50.8%) or blastocyst (42.9%, P > 0.05) stage. Mean cell number in IVF and ICSI embryos was 136 and 116 (P > 0.05); morulae had 77 and 46 (P < 0.05) and blastocysts had 187 and 209 (P > 0.05) cells, respectively. After transfer of 10 or 11 day 5 ICSI morulae to each of four recipients, a total of three kittens were born to two dams at 66 or 67 days. Of 18 fair-to-good quality oocytes recovered from a jaguarundi on two occasions, 10 (55.6%) embryos were produced by ICSI with fresh (n=5) or frozen (n=5) conspecific spermatozoa, but no jaguarundi kittens were born after transfer of these embryos to domestic cat recipients. In experiment II, cleavage frequency following IVF (15/17, 88.2%) and ICSI (31/38, 81.6%) was higher (P < 0.05) than following sham ICSI (13/35, 37.1%). Mean cell number (27 cells) and blastocyst development (0%) on day 7 was lower (P < 0.05) in the sham ICSI group than in the ICSI group (45 cells, 15.6% blastocysts) which, in turn, was lower (P < 0.05) than the IVF group (94 cells, 46.7% blastocysts). We have demonstrated that ICSI can be applied successfully in domestic felids and suggest that the technique will effectively augment other biotechniques being developed for enhancing reproduction in endangered felids.     

Cytokine induction of heat shock protein expression in human oligodendrocytes: an interleukin-1-mediated mechanism

The role of the cumulus cells in initiating the resumption of meiosis after exposure to forskolin and dbcAMP was studied in the mouse. The resumption of meiosis was monitored by the percentage of germinal vesicle breakdown (GVBD) and polar body formation (PB). The cumulus-enclosed oocytes (CEO) and denuded oocytes (DO) were cultured with and without hypoxanthine (HX) in the culture medium. Three types of experiments were performed: (1) Effect of forskolin on spontaneous resumption of meiosis, i.e. cultures without HX, and two experiments in which HX is present throughout the culture: (2) Effect of transient exposure to forskolin or dibutyric-cyclic adenosinemonophosphate (dbcAMP) on GVBD prior to continued culture without forskolin or dbcAMP (oocyte priming). (3) Priming of CEO with forskolin for 2 hr, separation of cumulus cells and oocytes, followed by coculture of rejoined cumulus cells and oocytes, or coculture of the cumulus cells and new, unprimed DO. (1) Forskolin inhibited a spontaneous resumption of meiosis in a dose-dependent manner during the first 5 hr of culturing. After 22 hr all controls and CEO resumed meiosis, whereas only half of the DO did. (2) At least 1 hr of priming the CEO with forskolin is needed to induce GVBD and PB formation, but forskolin inhibited the resumption of meiosis when present for 24 hr. Similar results were obtained with a high concentration of dbcAMP. (3) A separation and rejoining of oocytes and cumulus cells after priming induced the resumption of meiosis in a significantly greater number of oocytes than in the control oocytes which were not primed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preimplantation development of in vitro-matured and in vitro-fertilized ovine zygotes: comparison between coculture on oviduct epithelial cell monolayers and culture under low oxygen atmosphere

The roles of medium composition, serum source, embryo coculture, and culture under low O2 conditions on the development of in vitro-matured and in vitro-fertilized (IVMF) ovine zygotes were investigated in three separate experiments. In the first experiment, the proportion of cocultured IVMF zygotes developing to the blastocyst stage was significantly higher (38.0% vs. 3.5%; p < 0.05) than that of non-cocultured zygotes treated within three embryo culture media (TCM-199 + 10% fetal bovine serum [FBS]; bicarbonate-buffered, glucose-free synthetic oviduct fluid medium [mod-SOFM] + 10% FBS; and bicarbonate-buffered BSA-free Tyrode's salt solution [mod-TALP] + 10% FBS) under a 5% CO2 atmosphere in air. In a second experiment, a significantly higher (p < 0.05) proportion of cocultured zygotes placed in TCM-199 medium survived to the blastocyst stage (37.4% blastocysts vs. 23.4% in mod-SOFM). No significant effect of serum (FBS vs. human serum [HS]) was observed on embryonic development, but coculture was confirmed to exert a significant influence on development to the blastocyst stage. In the final experiment, survival of the embryo under a reduced oxygen (5% CO2:5% O2:90% N2) atmosphere was investigated. In contrast to results in the initial experiments, embryonic survival was significantly higher (p < 0.05) in the non-cocultured treatment groups (21.9% blastocysts vs. 0.4% for cocultured zygotes). Serum source also had a significant (p < 0.05) influence upon the development of non-cocultured zygotes: 32.3% of zygotes cultured with HS progressed to the blastocyst stage vs. 11.5% of zygotes cultured in FBS-supplemented medium. These results have characterized two distinct culture environments, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro.     

Amino acids in maturation medium and presence of cumulus cells at fertilization promote male pronuclear formation in porcine oocytes matured and penetrated in vitro

The present study was conducted to examine the ability of porcine oocytes to achieve male pronuclear (MPN) formation when they are matured and penetrated in vitro under various culture conditions. When cumulus-enclosed oocytes were cultured for 24-48 h in modified Whitten's medium (pH 7.4) supplemented with 10% porcine follicular fluid, 10 IU eCG/ml, and 10 IU hCG/ml (designated mWM-FG), nuclear maturation of oocytes reaching metaphase II was completed by 36 h after the start of culture. However, there were no differences in the proportions (94-95%) of oocytes penetrated in vitro by cryopreserved ejaculated spermatozoa or in the rates (35-45%) of MPN formation between oocytes cultured for 36 and 48 h. When cumulus-enclosed oocytes were cultured for 36 h in mWM-FG supplemented with 2% (v:v) minimal essential medium (MEM) essential amino acids (EAA) with the addition of 0.1 mM glutamine and/or 1% (v:v) MEM nonessential amino acids (NEAA) and inseminated in vitro, 93-97% of oocytes were penetrated regardless of the presence of amino acids during maturation, but the rates of MPN formation were higher in the presence (79-84%) than in the absence (51%) of any amino acids. The addition of EAA+NEAA and/or 0.57 mM cysteine to mWM-FG also did not affect sperm penetration in vitro, while it promoted MPN formation (76-83%) in penetrated oocytes as compared with those matured in the absence of amino acids and cysteine (53%). When oocytes were freed from cumulus cells after culture in mWM-FG, sperm penetration rates were not different between cumulus-enclosed (100%) and cumulus-free (92%) oocytes, but the rate of MPN formation was higher in cumulus-enclosed (53%) than in cumulus-free (28%) oocytes. When EAA+NEAA+cysteine was added to mWM-FG, MPN formation was not improved in cumulus-free oocytes but was much improved (78%) in cumulus-enclosed oocytes. These results indicate that MPN formation in porcine oocytes is promoted by the addition of amino acids and/or cysteine in simple maturation medium and by the presence of cumulus cells at fertilization in vitro.     

In vitro fertilization, culture, and transfer of rabbit ova

Ovulated rabbit oocytes were fertilized in vitro in chemically defined media supplemented with bovine serum albumin and either cultured up to the expanding blastocyst stage or transferred to recipients after varying periods of culture. Embryos transferred after up to 72 hours of in vitro culture were born as viable young. Oocytes from young virgin does were superior to oocytes from nonvirgin does for the purpose of in vitro fertilization (54% versus 26% fertilized, P less than 0.01). Capacitated sperm from artificially inseminated capacitators resulted in fertilization rates slightly lower than those from naturally mated does (46% versus 57% fertilized, P less than 0.025). Removal of cumulus and corona cells from oocytes with hyaluronidase and repeated aspiration through a fine pipette resulted in lowered fertilization rates (51% versus 73%, P less than 0.025). Linbro Disposo Tray wells were as good as glass tissue-culture dishes for the in vitro mixing of gametes and were more convenient to use. Modified Ham's F10 medium was used to culture the in vitro-fertilized embryos. However, when a modified Brackett's medium was used instead of modified Ham's F10 for the initial 4-hour period after mixing gametes, more oocytes were fertilized (52% versus 28%, P less than 0.01).     

The Listeria monocytogenes iap gene as an indicator gene for the study of PrfA-dependent regulation

In this study the effects of hypoxanthine (HX) on meiotic maturation were compared using oocytes from mice possessing a hypoxanthine phosphoribosyltransferase null mutation (HPRT-) and from the corresponding HPRT-competent background strain (HPRT+). Oocyte-cumulus cell complexes and cumulus cell-enclosed oocytes (oocytes cultured while enclosed by cumulus cells) from HPRT+, but not HPRT-, mice took up HX and contained significant levels of HPRT activity. In addition, FSH increased, and HX suppressed, the de novo synthesis of purines in HPRT+ complexes, whereas de novo synthesis was elevated in HPRT complexes and was unaffected by FSH or HX. After 3 h of HX treatment, lower frequencies of germinal vesicle breakdown (GVB) were observed in cumulus cell-enclosed than in denuded HPRT+ oocytes; however, identical frequencies of maturation were observed in denuded and cumulus cell-enclosed HPRT oocytes. This demonstrates a direct inhibitory action of HX on the oocyte that does not depend on salvage, plus an additional action of the cumulus cells that requires HPRT activity. Nevertheless, cumulus cells from HPRT- mice are capable of exerting an additional inhibitory action of dibutyryl cAMP (dbcAMP) on the oocyte. A kinetics analysis of FSH action on HX-arrested cumulus cell-enclosed HPRT+ and HPRT- oocytes revealed, first, that the inhibitory effect of the cumulus cells is transient and, second, that HPRT activity is not required for FSH induction of GVB in HX-arrested oocytes. When dbcAMP- or HX-arrested oocytes were treated with FSH, GVB was blocked to the same extent in HPRT- oocytes with the purine de novo synthesis inhibitor, azaserine, but this drug was less effective in HX-treated HPRT+ oocytes. These results confirm the importance of the de novo pathway in hormone-induced maturation and also support a role for purine salvage as an alternative source of nucleotide in this process.     

Therapeutic drug monitoring: antiarrhythmic drugs

Two experiments were carried out to test the hypothesis that follicles recovered from Meishan animals may provide a more favourable environment for oocyte maturation in vitro than follicles recovered from Large-White hybrid animals. In Experiment 1, all follicles > or = 4 mm were recovered from six Meishan and seven Large-White hybrid gilts in the late follicular phase and healthy oocyte cumulus complexes recovered. Cumulus oocyte complexes were randomly divided into two groups, and each group cultured for 27 or 34 h (62 and 64; 56 and 56 for Meishan and Large-White hybrid, respectively) in defined medium in the presence of either of the two largest follicle shells per animal. Subsequent examination of oocyte nuclear maturation showed that although maturation did not differ significantly between the breeds after 27 h, more (P < 0.01) Meishan oocytes co-cultured with Meishan follicles developed to metaphase II stage than Large-White hybrid oocytes co-cultured with Large-White hybrid follicles after 34 h. The next eight largest follicles per animal were cultured for 34 h to produce conditioned media. In Experiment 2, oocytes recovered from the slaughterhouse were matured for 46 h in the presence of conditioned media from Meishan (612 oocytes) or Large-White hybrid (731 oocytes) follicles, or in fresh medium in the presence of a follicle shell from slaughterhouse ovaries. Oocytes were then inseminated and 12 h later examined for penetration and male pronuclear formation. A higher (P < 0.05) percentage of oocytes cultured in Meishan follicle conditioned medium underwent sperm penetration and male pronuclear formation than oocytes cultured in conditioned media from Large-White hybrid animals. Concentration of oestradiol and progesterone in the conditioned media did not differ between the breeds (P > 0.1). In conclusion, these results suggest that (1) Meishan oocytes have advanced maturational capacity when cultured with Meishan preovulatory follicle shells and (2) differences in follicle maturation in the Meishan compared to the Large-White hybrid pig may result in an improved ability of the follicles, via conditioned media, to support oocyte maturation and fertilization in vitro.     

Complete activation of porcine oocytes induced by the sulfhydryl reagent, thimerosal

Thimerosal (200 microM) triggered Ca2+ oscillations in 56 of 56 mature porcine oocytes. The Ca2+ oscillations were blocked by the sulfhydryl-reducing agent dithiothreitol (DTT), thus supporting the hypothesis that thimerosal acts by oxidizing critical sulfhydryl groups on intracellular Ca2+-release proteins. Thimerosal treatment alone arrested the oocytes in metaphase, probably by oxidizing tubulin sulfhydryl groups and thus destroying the spindle. However, a 10-min exposure to 200 microM thimerosal followed by a 30-min incubation in 8 mM DTT induced complete activation, as 73.8% of the oocytes formed pronuclei. The second polar body was visible in 73.3% (55 of 75) of the activated oocytes. Combined thimerosal/DTT treatment of the oocytes also induced cortical granule exocytosis, as revealed by confocal microscopy, and the subsequent hardening of the zona pellucida. After activation, some oocytes were incubated in vitro, or in vivo in a ligated porcine oviduct, for 6 days. When cultured in vitro, 42.0% (37 of 88) of the oocytes developed to the compact morula or blastocyst stage; the average number of inner cell mass (ICM) and trophectoderm (TE) nuclei in the blastocysts was 8.6 +/- 0.7 and 20.1 +/- 1.3, respectively. Culture in a ligated oviduct resulted in 42.9% development to the compact morula or blastocyst stage, with the blastocysts having a mean number of 12.5 +/- 1.0 ICM and 63.6 +/- 9.2 TE nuclei.