Follicle-stimulating hormone administration affects amino acid metabolism in mammalian oocytes†.

Culture media used in assisted reproduction are commonly supplemented with gonadotropin hormones to support the nuclear and cytoplasmic maturation of in vitro matured oocytes. However, the effect of gonadotropins on protein synthesis in oocytes is yet to be fully understood. As published data have previously documented a positive in vitro effect of follicle-stimulating hormone (FSH) on cytoplasmic maturation, we exposed mouse denuded oocytes to FSH in order to evaluate the changes in global protein synthesis. We found that dose-dependent administration of FSH resulted in a decrease of methionine incorporation into de novo synthesized proteins in denuded mouse oocytes and oocytes cultured in cumulus-oocyte complexes. Similarly, FSH influenced methionine incorporation in additional mammalian species including human. Furthermore, we showed the expression of FSH-receptor protein in oocytes. We found that major translational regulators were not affected by FSH treatment; however, the amino acid uptake became impaired. We propose that the effect of FSH treatment on amino acid uptake is influenced by FSH receptor with the effect on oocyte metabolism and physiology.

Crocin supplementation during oocyte maturation enhances antioxidant defence and subsequent cleavage rate.

The purpose of the present study was to assess the effect of crocin supplementation during oocyte maturation on the antioxidant defence and anti-apoptotic ability and subsequent developmental competence of porcine oocytes. Oocytes were cultured in media containing 0, 300, 400 or 500 µg/ml of crocin. Upon maturation, the maturation rates, reactive oxygen species (ROS) and glutathione (GSH) levels, mRNA expression of genes (SOD, CAT, GPx, Bcl-2, BAX and Caspase3), expression of cleaved caspase3 and subsequent embryo cleavage rates were measured. Results indicated that the maturation rate of the 400 µg/ml group was 86.80% (p < 0.01). The ROS concentration of the 500 µg/ml group was the lowest (p < 0.01). The GSH concentration of the 400 µg/ml group was the highest (p < 0.01). The SOD, CAT and GPx mRNA expression levels were the highest in the 300, 400 and 500 µg/ml groups, respectively, with the expression levels of all genes being significantly higher than that of the control group (p < 0.01). The Bcl-2/BAX mRNA expression ratio in 400 and 500 µg/ml groups significantly higher than other groups and significantly decreased caspase3 expression level (p < 0.01). The expression level of cleaved caspase3 in the 500 µg/ml treatment group was the lowest, significantly lower than that of the control group (p < 0.01). The cleavage rate of the 400 µg/ml group was 62.50% (p < 0.01). These experimental results show that the supplementation of in vitro culture medium with 400 µg/ml of crocin significantly enhanced the antioxidant defence and anti-apoptotic ability and subsequent cleavage rate of porcine embryo.

Calcium-sensing receptor (CASR) is involved in porcine in vitro fertilisation and early embryo development.

It has been demonstrated that extracellular calcium is necessary in fertilisation and embryo development but the mechanism is still not well understood. The present study mainly focussed on the extracellular calcium effector called the calcium-sensing receptor (CASR) and examined its expression in porcine gametes and embryos and its function during fertilisation and early embryo development. By using reverse transcription polymerase chain reaction, CASR was found to be expressed in porcine oocytes, spermatozoa and embryos at different developmental stages. Functionally, medium supplementation with a CASR agonist or an antagonist during in vitro fertilisation (IVF) and in vitro culture (IVC) was tested. During fertilisation, the presence of a CASR agonist increased sperm penetration rate and decreased polyspermy rate leading to an increased normal fertilisation rate. During embryo development, for the IVF embryos, agonist treatment during IVC significantly increased cleavage rate and blastocyst formation rate compared with the control group. Furthermore, parthenogenetically activated embryos showed similar results with lower cleavage and blastocyst formation rates in the antagonist group than in the other groups. It was concluded that CASR, as the effector of extracellular calcium, modulates porcine fertilisation and early embryo development.

The beneficial effect of repaglinide on in vitro maturation and development ability of immature mouse oocytes.

Repaglinide is a hypoglycemic drug, causing depolarization of the cell membrane, opening the voltage-gated calcium channels, and then increasing intracellular calcium in the pancreatic B cells by inhibition of the K-ATP-sensitive channels. Oocyte in vitro maturation (IVM) is influenced by different factors such as calcium signaling. In this study, we examined the effects of repaglinide on in vitro maturation and fertilization ability of mouse oocyte. Immature oocytes were isolated from female Naval Medical Research Institute mice which are 6-8 wk old mechanically and then cultured in 30 µl droplets of T6 medium with different concentrations of repaglinide. The control group did not receive repaglinide (R0). Treatment groups received different concentrations (5, 10, and 100 nM and 1 and 10 µM) of repaglinide (R1, R2, R3, R4, and R5, respectively). Oocyte in vitro maturation rate was assessed after 24 h. In vitro fertilization was performed using metaphase II oocytes obtained from R0 and R4 treatments. Embryo cleavage rate was calculated at 48 h post-IVF. Chi-square test was used for evaluating difference between control and treatment groups (p < 0.05). Oocyte maturation rate after 24 h in treatment groups R2, R3, R4, and R5 was significantly higher than that in the control (p < 0.05). Supplementation of medium with 1 µM of repaglinide (R4) during IVM significantly improved outcome of embryo cleavage rate than control at 48 h post-IVF (p < 0.05). In conclusion, repaglinide can be considered as an effective agent for in vitro oocyte maturation and embryo cleavage.

Supplementation of IVF medium with melatonin: effect on sperm functionality and in vitro produced bovine embryos.

Gamete co-incubation generates high free radical levels surrounding growing zygotes which may impair subsequent embryo viability. Melatonin eliminates a wide variety of free radicals; hence, we tried to improve in vitro embryo production by adding melatonin to in vitro fertilisation (IVF) media in high (Exp. 1) and low concentrations (Exp. 2), and we evaluated its effect on bull sperm function during IVF co-incubation time (Exp. 3). In Experiment 1, we supplemented IVF media culture with 0.01, 0.1 and 1 mmol of melatonin, along with a no melatonin control group. In Experiment 2, melatonin levels were reduced to 10, 100 and 1000 nmol, with a no melatonin control group. In Experiment 3, spermatozoa were incubated in IVF media with melatonin (as Exp. 2) and functional parameters were analysed at 0, 4 and 18 h. In Experiment 1, only 1 mmol melatonin showed lesser blastocyst rates than control (C: 23.2 ± 6.7% versus 1 mmol: 2.0 ± 1.7%). In Experiment 2, no statistical differences were found in cleavage percentage, blastocyst percentage and total cell count for any melatonin treatment. In Experiment 3, sperm samples with 1000 nmol melatonin had a significantly higher wobbler (WOB) coefficient, a lower percentage of intact acrosomes, a lower percentage of viable spermatozoa with ROS, greater DNA fragmentation and higher DNA oxidation than controls. Total fluorescence intensity for ROS at 10 nmol melatonin was significantly greater than controls (P < 0.05). IVF media with 1 mmol melatonin is deleterious for embryo development, and in lower concentrations, it modulated sperm functionality, but had no effects on embryo production.

L-carnitine supplementation during vitrification of mouse oocytes at the germinal vesicle stage improves preimplantation development following maturation and fertilization in vitro.

Oocyte cryopreservation is important for assisted reproductive technologies (ART). Although cryopreservation of metaphase II (MII) oocytes has been successfully used, MII oocytes are vulnerable to the damage inflicted by the freezing procedure. Cryopreservation of germinal vesicle stage oocytes (GV-oocytes) is an alternative choice; however, blastocyst development from GV-oocytes is limited largely due to the need for in vitro maturation (IVM). Herein, we evaluated the effects of l-carnitine (LC) supplementation during vitrification and thawing of mouse GV-oocytes, IVM, and embryo culture on preimplantation development after in vitro fertilization (IVF). We first compared the rate of embryonic development from the oocytes that had been collected at the GV stage from three mouse strains, (B6.DBA)F1, (B6.C3H)F1, and B6, and processed for IVM and IVF, as well as that from the oocytes matured in vivo, i.e. ovulated (IVO). Our results demonstrated that the rate of blastocyst development was the highest in the (B6.DBA)F1 strain and the lowest in the B6 strain. We then supplemented the IVM medium with 0.6 mg/ml LC. The rate of blastocyst development improved in the B6 but not in the (B6.DBA)F1 strain. Vitrification of GV-oocytes in the basic medium alone reduced the rate of blastocyst development in both of those mouse strains. LC supplementation to the IVM medium alone did not change the percentage of blastocyst development. However, LC supplementation to both vitrification and IVM media significantly improved blastocyst development to the levels comparable with those obtained from vitrified/thawed IVO oocytes in both of the (B6.DBA)F1 and B6 strains. We conclude that LC supplementation during vitrification is particularly efficient in improving the preimplantation development from the GV-oocytes that otherwise have lower developmental competence in culture.

Effect of 5-aza-2'-deoxycytidine on methylation of the putative imprinted control region of H19 during the in vitro development of vitrified bovine two-cell embryos.

OBJECTIVE: To determine the effect of vitrification and 5-aza-2'-deoxycytidine (5-aza-dC) on the methylation levels of the putative imprinted control region (ICR) of H19 and H19 expression in bovine two-cell embryos and their derived blastocysts. DESIGN: Experimental animal study. SETTING: Academic institution. ANIMAL(S): Abattoir-derived bovine ovaries. INTERVENTION(S): Vitrified two-cell embryos were cultured in vitro to blastocysts with 0.01 µM 5-aza-dC (5-aza-dC group) or without 5-aza-dC (vitrification group). Fresh embryos and their derived blastocysts were used as control. MAIN OUTCOME MEASURE(S): Putative ICR methylation of H19 was measured by bisulfate mutagenesis and sequencing, blastocyst development rate; total cell number were determined; and H19 expression was measured by real-time reverse transcriptase-polymerase chain reaction (PCR). RESULT(S): Vitrification significantly increased putative ICR methylation of H19 in two-cell embryos and their derived blastocysts; 5-aza-dC significantly reduced putative ICR methylation of H19 in vitrified two-cell embryos and their derived blastocysts. The H19 expression level was significantly higher in blastocysts from the 5-aza-dC group than the vitrification group. The blastocyst development rate and total cell number in the 5-aza-dC and vitrification groups were similar. CONCLUSION(S): Putative ICR methylation levels of H19 significantly increased in vitrified two-cell embryos and their derived blastocysts; 5-aza-dC significantly reduced putative ICR methylation of H19 and increased H19 expression in blastocysts derived from vitrified two-cell embryos.

Impact of linoleic acid on bovine oocyte maturation and embryo development.

Linoleic acid (LA; 18:2 n-6) is the most abundant fatty acid in bovine follicular fluid, and it was previously reported that LA concentration significantly decreases when follicle size increases. This suggests that LA may have a role in the regulation of oocyte maturation. The present study investigated the effect of LA supplementation on bovine oocyte maturation and early embryo development in vitro. Treatment of cumulus-oocyte complexes (COCs) with LA significantly inhibited cumulus cell expansion and retarded development of the oocytes to the metaphase II (MII) stage in a dose-dependent manner. This effect was reversible, and the oocytes developed to the MII stage after extended culture in the absence of LA. Treatment of COCs with LA also resulted in a significantly lower percentage of cleaved embryos and blastocyst yield. Furthermore, COCs treated with LA had significant effects compared with controls in i) increasing prostaglandin E(2) concentration in the medium, ii) decreasing intracellular cAMP at 6 and 24 h of maturation and iii) decreasing phosphorylation of the MAPK1 and 3 at 24 h, and AKT at 6 h of maturation. In conclusion, LA supplementation to bovine oocytes during maturation altered the molecular mechanisms regulating oocyte maturation and resulted in decreased percentage of oocytes at MII stage and inhibition of the subsequent early embryo development. These data provide evidence for adverse effects of LA on oocyte development, which can be associated with dietary increased level of LA in the follicular fluid and the decline in fertility in farm animals and human.

Effects of L-ascorbic acid, alpha-tocopherol and co-culture on in vitro developmental potential of porcine cumulus cells free oocytes.

The maturation and developmental potential on cumulus-cell-free oocytes is of great importance theoretically and practically. The present study was to investigate the effects of l-ascorbic acid, alpha-tocopherol and co-culture on in vitro developmental potential of porcine denuded oocytes (DOs). Porcine DOs were cultured in maturation medium supplemented with vitamin C (0, 50, 100, 250, 500, 750 microM) and vitamin E (0, 10, 20, 50, 100, 250 microm), respectively. And they were also co-cultured with dispersed cumulus cells (group CCscoculture), intact cumulus cells oocyte complexes (COCs) (group COCscoculture), and COCs whose oocytes were removed (group OOXcoculture), respectively. After 44 h incubation, the maturation rates, cleavage rates and blastocyst rates after parthenogenetic activation in three experiments mentioned above were collected and analysed, respectively. L-Ascorbic acid promoted porcine DOs in vitro maturation and blastocyt development after parthenogenetic activation while alpha-tocopherol did not increase the in vitro maturation rates, but improved the blastocyst rate. None of the three co-culture manner promoted the in vitro maturation and the cleavage of porcine DOs after parthenogenetic activation, but all the co-culture manners improved the blastocyst rates. Both Vitamin C and E enhance the in vitro developmental potential of porcine DOs. Co-culture increases the developmental potential of porcine DOs.

Effects of N, N-dimethylglycine on the development of in vitro produced bovine embryos.

This study investigated the effects of N, N-Dimethylglycine (DMG) on the development of in vitro produced (IVP) bovine embryos. IVP embryos were obtained by in vitro fertilization of in vitro matured oocytes for 6 h. In Experiment 1, IVP embryos were cultured in mSOFaa supplemented with bovine serum albumin but without glucose (SOF1) for 4 days, transferred to mSOFaa (with 5% fetal bovine serum and 1.5 mM glucose; SOF2) supplemented with 0 (control), 0.1,1 or 10 microM DMG and cultured for an additional 7 days (11 days in total) to assess their development in vitro. When cultured in the medium with 0.1 microM DMG, a significantly higher number of IVP embryos developed to the blastocyst and hatched blastocyst stages (40.3 and 40.8%, respectively) compared with the other groups (18.7-31.0% and 15.0-28.7%, respectively; P<0.05, analysis of variance). In Experiment 2, IVP embryos were cultured in SOF1 with or without 0.1 microM DMG for 4 days, transferred to SOF2 with or without 0.1 microM DMG and further cultured as in Experiment 1; DMG was added to either SOF1 or SOF2 and to both of them to assess its exposure effects on embryo development. When cultured continuously with DMG for 11 days, significantly higher rates of IVP embryos developed into blastocyst and hatched blastocyst stages (39.0 and 47.7%, respectively) compared with the other groups (31.0-32.2% and 29.5-31.0%, respectively; P<0.05). In Experiment 3, we examined developmental speed of IVP embryos cultured with or without addition of 0.1 microM DMG to IVC medium after 7 days of IVC. When DMG was added to IVC medium, the ratio of embryos developed to advanced developmental stages (No. of embryos developed to the blastocyst and expanded blastocyst stages/No. of embryos developed to the morula stage) was 28.7% (86/3) and 7 times higher than that of those cultured without DMG, 4.0% (52/13). These results suggest that addition of 0.1 microM DMG to mSOFaa during IVC of IVP bovine embryos has a promoting effect on their development.

Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development.

The purpose of this study was to evaluate whether enriching the oocyte in vitro maturation medium with cystine, in the presence of cysteamine, would improve the in vitro embryo production efficiency in buffalo by further increasing the GSH reservoir created by the oocyte during maturation. Cumulus-oocytes complexes were matured in vitro in TCM 199 + 10% FCS, 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 0.3mM cystine. In Experiment 1, glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in representative samples of oocytes matured in the four different experimental conditions. In Experiment 2, oocytes were fixed and stained to assess nuclear maturation and normal pronuclear development following IVM and IVF respectively. In Experiment 3, mature oocytes were in vitro fertilized and cultured to assess development to blastocysts. In all supplemented groups the intracytoplasmic GSH concentration was significantly higher than the control, with the highest GSH levels in oocytes matured in the presence of both thiol compounds (3.6, 4.7, 5.4 and 6.9 picomol/oocyte in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Cystine supplementation of IVM medium, both in the presence or absence of cysteamine, significantly increased the proportion of oocytes showing two normal synchronous pronuclei following fertilization. In all supplemented groups, cleavage rate was significantly improved compared to the control (55, 66.1, 73.5 and 78.4% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Similarly, blastocyst yield was also increased in the three enriched groups compared to the control (17.1, 23.8, 29.3, 30.9% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Overall, the addition of cystine to a cysteamine-enriched medium resulted in a significant increase of cleavage rate and transferable embryo yield compared to the medium supplemented with only cysteamine.

Production of dromedary (Camelus dromedarius) embryos by IVM and IVF and co-culture with oviductal or granulosa cells.

The general objective of this work was to produce dromedary embryos from cumulus-oocyte complexes (COCs) that were matured, fertilized and co-cultured in vitro. A total of 1598 COCs were recovered from 457 ovaries; 1308 were deemed suitable for IVM and were cultured at 38.5 degrees C, 5% CO2, and >95% humidity for 36 h in TCM-199 supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/ml epidermal growth factor (EGF), 1 microg/ml FSH, and 500 microM cysteamine. Matured COCs (n = 88) were denuded, fixed, and stained to determine nuclear status; 63% (56/88) had reached metaphase II (MII) at 36 h. Overall, 1135 COCs were inseminated with ejaculated fresh semen (0.5 x 10(6)spermatozoa/ml in modified TALP-solution). Inseminated oocytes (n = 155) were examined for evidence of fertilization; 68% (106/155) were penetrated by spermatozoa, including 52% (55/106) with two pronuclei and 34% (36/106) with polyspermy. Inseminated, denuded oocytes (n = 819) were co-cultured with dromedary oviductal epithelial or granulosa cells in TCM-199 supplemented with 10% heat-treated FCS. Although the rate of first cleavage (two to eight cells) was similar for the two co-culture systems (32 versus 33%, respectively), more embryos (two-cell to blastocyst stage) were obtained from oocytes co-cultured with oviductal versus granulosa cells (61 versus 45%; P < 0.05). The proportions of fertilized oocytes developing to the early morula stage were 19% (80/417) and 12% (48/402) for oocytes co-cultured for 7 days with oviductal or granulosa cells, respectively (P > 0.05). However, development to the blastocyst stage (10% of fertilized oocytes) occurred only in oocytes co-cultured with oviductal cells. In conclusion, dromedary embryos were produced in vitro using abattoir-derived oocytes, fresh (ejaculated) semen, and oviductal cell co-culture.

Consequences of exposure to serum, with or without vitamin E supplementation, in terms of the fatty acid content and viability of bovine blastocysts produced in vitro.

To determine whether serum supplementation influenced fatty acid content of bovine blastocysts and whether vitamin E addition to culture medium containing serum could improve development in vitro, cleaved eggs were cultured in synthetic oviduct fluid supplemented with bovine serum albumin (BSA, 0.4% w/v, fraction V) (SVBSA), fetal calf serum (FCS, 10% v/v) (SFCS) or FCS (10% v/v) plus 100 micro M vitamin E (SFCS + E). Blastocyst yields were recorded and fatty acid composition was determined by gas chromatography. Day 7 blastocysts were incubated with [2-(14)C] pyruvate for 3 h and then fixed for cell counts. Yields of good quality blastocysts were greatest from cleaved eggs cultured in serum-free conditions (P < 0.01). In the presence of serum, supplementation with vitamin E increased both total and good quality blastocyst yields (P < 0.01). Presence of serum increased fatty acid content (mean +/- SEM) of blastocysts (SVBSA v. SFCS = 57 +/- 2 v. 74 +/- 2 ng embryo(-1); P < 0.001). In contrast, pyruvate metabolism was greater in blastocysts produced without serum (27 +/- 3 v. 21 +/- 3 picomoles embryo(-1) 3h(-1); P < 0.01) but, on a per cell basis, no differences were detected. Addition of vitamin E to the serum-supplemented formulation did not alter either the fatty acid content (73 +/- 2 ng embryo(-1)) or pyruvate metabolism index (19 +/- 1 pmol embryo(-1) 3h(-1)) of SFCS + E blastocysts. Thus, despite lipid accumulation, supplementary vitamin E improved blastocyst yields in embryos exposed to serum.

Mitogenic and anti-apoptotic activity of insulin on bovine embryos produced in vitro.

Insulin improves development of mammalian preimplantation embryos and, in addition to the regulation of glucose transport, it exerts mitogenic and anti-apoptotic activities. The expression of glucose transporters (Glut) mediating the uptake of this essential energy substrate is critical for embryo survival. An impaired expression of Glut leads to an increase in apoptosis at the blastocyst stage and involves Bax. The various effects of insulin were unravelled by supplementing the in vitro culture medium with insulin (1.7 micromol l(-1)) and (i) the rates of cleavage and blastocyst development were recorded; (ii) mitogenic activity was studied by determining the total number of blastocyst cells and the ratio between trophectoderm and inner cell mass (ICM) cells; (iii) the frequency of apoptosis in blastocysts was determined by the TdT-mediated duTP nick-end labelling (TUNEL) assay and by quantification of the relative amounts of mRNA for Bax and Bcl-XL; and (iv) expression for Glut1, Glut3 and Glut8 transcripts was compared between embryos cultured in the presence or absence of insulin. Insulin increased rates of cleavage (81.2+/-2.2 (control) to 86.0+/-2.5) and blastocyst development (24.7+/-1.9 to 31.3+/-1.2), and number of blastocyst cells (123.7+/-6.0 to 146.3+/-6.6); the increase in the number of blastocyst cells was due to a significantly higher number of trophectoderm cells (82.3+/-5.0 versus 100.3+/-5.5). Blastocysts derived from cultures supplemented with insulin showed a significant decrease in apoptosis as determined by the TUNEL assay (14.8+/-0.9 to 12.2+/-0.7). No effects of insulin on the mRNA expression of Glut isoforms and Bax and Bcl-XL were found. These results demonstrate that the mitogenic and anti-apoptotic effects of insulin on bovine preimplantation embryos did not correlate with changes in the amounts of mRNA for the glucose transporter isoforms Glut1, -3 and -8, or transcripts for Bax and Bcl-XL.

Endogenous folic acid is essential for normal development of preimplantation embryos.

Preimplantation mammalian embryos develop with a high degree of autonomy. To date, there have been no unequivocal demonstrations of a requirement for vitamins in preimplantation embryo development. Reduced folic acid acts as an important methyl donor in many reactions including the synthesis of thymidine. Thymidine does not accumulate in cells so it might be expected that significant amounts of reduced folate would be required to support the exponential increase in DNA synthesis that occurs during early embryo development. The reduction of folate is catalysed by dihydrofolate reductase (EC 1.5.1.3) which is selectively inhibited by the anti-cancer drug methotrexate. Methotrexate caused a dose-dependent inhibition of cell division in 1-cell, 2-cell and 8-cell mouse embryos with 50% inhibition of division occurring at concentrations of 1-10 microM. At a concentration of 0.1 microM only minimal inhibition of the initial cell division occurred, but continuous culture in this concentration of methotrexate completely inhibited further cell divisions. This suggests that most of the exogenous store of reduced folates was used in the first round of cell division. The effects of methotrexate were apparently primarily due to thymidine starvation, since a 10-fold excess of thymidine over methotrexate in culture media reversed the inhibition of development. Supplementing media with folic acid had no beneficial effect on the rate at which zygotes produced by in-vitro fertilization developed to the blastocyst stage. It is concluded that the development of the early embryo has an absolute requirement for reduced folate for thymidine synthesis which is met entirely by endogenous sources.

Evidence for the presence of the platelet-activating factor receptor in the CFW mouse preimplantation two-cell-stage embryo.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF), a potent signaling phospholipid, has a significant role in preimplantation embryo development. CFW mouse embryos respond to PAF with improved development and implantation rates. PAF's signal transduction mechanism in other cell types is receptor mediated. However, embryonic mRNA for the PAF receptor has not been detected. The study objectives were to determine the presence of PAF receptor mRNA in CFW mouse two-cell embryos by reverse transcription (RT)-polymerase chain reaction and Northern blot analysis and to ascertain the effect of PAF on intracellular calcium levels (a receptor-mediated event). Total RNA was purified by acid-phenol extraction and ethanol precipitation. Complementary DNA was synthesized by RT. RNA was primed with oligo-dT plus PAF receptor-specific primer (3' to 5') at 42 degrees C for 60 min, 95 degrees C for 10 min, and 5 degrees C for 5 min. The RT product was amplified with Taq polymerase and PAF receptor-specific primer (5' to 3') at 94 degrees C for 5 min and 54 degrees C for 5 min for one cycle, and at 72 degrees C for 3 min, 93 degrees C for 90 sec, and 61 degrees C for 150 sec for 30 cycles followed by 72 degrees C for 10 min and then holding at 4 degrees C. The product was analyzed by agarose gel electrophoresis, producing a single band (610 base pairs [bp]), thus demonstrating the presence of PAF-receptor mRNA. Sequence analysis of the cloned 610-bp fragment confirmed that it is the PAF receptor. Northern blot analysis also confirmed the expression of the PAF receptor in the CFW mouse preimplantation two-cell-stage embryo. PAF treatment of the two-cell-stage CFW mouse embryo resulted in a fourfold increase in intracellular calcium over background levels.

Nonessential amino acids and glutamine decrease the time of the first three cleavage divisions and increase compaction of mouse zygotes in vitro.

PURPOSE: The objective of this study was to determine the effect of supplementing embryo culture media with amino acids on the duration of the first three cell cycles of mouse zygotes in vitro. METHODS: Zygotes were cultured in the presence of different groups of amino acids and cleavage assessed every 30 min. RESULTS: Culture of zygotes with Eagle's nonessential amino acids and glutamine significantly reduced the time of cleavage divisions to the eight-cell stage compared to culture without amino acids. Beneficial effects of amino acids were found to be cumulative over time. Nonessential amino acids and glutamine also increased the percentage of eight-cells that compacted after 57 hr of culture compared to embryos in medium devoid of amino acids. CONCLUSIONS: The present data suggest that media for the development of cleavage-stage embryos, such as in clinical IVF, should be supplemented with Eagle's nonessential amino acids and glutamine.

Effects of osmolarity and amino acids in a chemically defined medium on development of rat one-cell embryos.

Rat one-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (R1ECM) under different experimental conditions. When the osmolarity of the medium with a reduced concentration (63.8 mmol l-1) of NaCl was varied by adding different amounts of D-sorbitol, more (79-91%) of the one-cell embryos developed to the four-cell stage at 212-278 mosmol than at 306 mosmol (13%). The greatest proportions of morulae (74%) and blastocysts (60%) were obtained at 246 mosmol. When the medium was supplemented with amino acids in various combinations and the osmolarity adjusted to about 246 mosmol, more (80-98%) of the embryos developed to the morula stage. More blastocysts were obtained in medium supplemented with glutamine (Gln: 80%), minimal essential medium (MEM) essential amino acids (EAA) (90%), Gln+EAA (83%), EAA+MEM nonessential amino acids (NEAA) (83%) or EAA+Gln+NEAA (90%) than in medium without amino acids (59%). Few (3-10%) hatching or hatched blastocysts were observed 120 h after the start of culture in the medium with EAA plus Gln or NEAA. The mean number of cells in blastocysts developed in the medium with EAA+Gln+NEAA was 46.7 +/- 7.2. When a total of 82 morulae or early blastocysts that had developed in culture were transferred to eight pseudopregnant rats on day 4, six recipients into which 62 embryos were transferred maintained their pregnancies beyond day 23, although no deliveries had occurred by day 25 or 26. When the rats were killed, 42 (68%) implantation sites and eight (13%) full-term fetuses with no gross abnormality were observed in the uterine horns.

Bilirubin helps to overcome the two-cell block in mouse oocyte cultures.

PURPOSE: In vitro fertilization and culture of mouse oocytes, under normal atmospheric oxygen tension, subjects them to severe oxidative stress. Oocytes from some strains of mice lack the natural protective mechanism that guards them against this oxidative stress and fail to develop beyond the two-cell stage. METHODS: We could overcome the toxic effects of oxygen metabolites by adding 0.2-0.4 mg/dl bilirubin in a lactate-pyruvate culture medium defined by Whitten (1971). Six- to 8-week-old ICR (Institute of Cancer Research) female mice were super ovulated by intra peritoneal injection of 5 IU PMSG (pregnant mare serum gonadotropin) followed by 10 IU hCG 48 h later. The oocytes were collected from the distended fallopian tubes and inseminated with 1-2 million sperm from 3-4-month-old ICR male mice. The eggs were scored at 24, 48, and 72 h after the hCG injection. CONCLUSIONS: With 0.4 mg/dl bilirubin supplement, by the end of 72 h, 82% of the eggs progressed from the two-cell stage to the four-cell stage. Routine inclusion of bilirubin can improve embryo development in vitro.

Nicotinamide, a component of complex culture media, inhibits mouse embryo development in vitro and reduces subsequent developmental potential after transfer.

OBJECTIVE: To determine the effects of B-group vitamins present in culture media on mouse embryo development in vitro and subsequent viability. DESIGN: Mouse zygotes were cultured in the presence of B-group vitamins. Embryo morphology and cell numbers were determined at 96 and 120 hours after hCG. Viability was assessed by transfer of embryos after 3 days of culture to pseudopregnant recipients. Resultant pregnancy rates (PRs) and fetal weights were determined. RESULTS: Supplementation of an amino acid-free medium with minimal essential medium (MEM) B-group vitamins significantly decreased embryo cleavage rates, whereas the inclusion of Ham's F-10 medium B-group vitamins significantly reduced both cleavage rates and morphological development. Subsequent experiments determined that nicotinamide (5 microM) significantly reduced blastocyst cell number, implantation rate, viable PR, and fetal weight. CONCLUSION: The data indicate that nicotinamide inhibits mouse embryo development in culture and reduces viability. Nicotinamide is present at high levels in Ham's F-10 and MEM media that are used routinely in human embryo culture. The role of vitamins in human embryo development in vitro warrants investigation.