Earle's Balanced Salt Solution Research Articles

351 EFFECTS OF FETAL CALF SERUM, GROWTH FACTOR, AND HORMONE SUPPLEMENTATION DURING IN VITRO MATURATION ON PARTHENOGENETIC ACTIVATION AND EMBRYO DEVELOPMENT OF FOLLICULAR RABBIT OOCYTES

Improvement of the in vitro maturation (IVM) system for rabbit oocytes could play a role in rabbit biotechnology. Our goal was to improve IVM to have an efficient source of rabbit oocytes for further studies on nuclear transfer. The effects of FCS, growth factors, and hormone supplementation on oocyte maturation, activation, and embryo development rates were evaluated. Rabbit ovaries were transferred from the slaughterhouse to the laboratory in PBS. Oocytes were collected by aspiration and subjected to IVM in 3 types of media based on TCM-199. In one group, growth factors (50 ng mL−1 of insulin-like growth factor-I and 10 ng mL−1 of epidermal growth factor) and hormones (5 IU mL−1 of hCG and 5 IU mL−1 of pregnant mare serum gonadotropin) and BSA were added to the IVM medium (IVM+); in the other groups, IVM medium was supplemented with either 10% (IVM + 10% FCS) or 20% FCS (IVM + 20% FCS). Maturation was assessed by the presence of a polar body after 16 h. Matured oocytes were activated twice by electric stimuli (3 DC pulses, 1.6 kV cm−1, 60 µs) and twice by chemical activation (incubation with 2.5 mM 6-DMAP for 30 min, second time for 2 h) and cultured in vitro in Earle's balanced salt solution complete medium at 38.5°C under 5% CO2 in air. Cleavage rates were recorded 16 h after activation and the blastocyst rates were recorded at Day 5 of in vitro culture. Data were analyzed by ANOVA. Maturation rates did not differ between the treatment groups (Table 1). There was no significant difference in cleavage rates between the IVM+ and the IVM + 10% FCS groups; however, the cleavage rate of the IVM + 20% FCS group was significantly lower compared with the others (Table 1; P < 0.05). Development to the blastocyst did not differ significantly between the treatment groups (Table 1; P < 0.05). The results showed that high (20%) FCS supplementation during IVM had a detrimental effect on oocyte cleavage. Hormonal and growth factor supplementation had no beneficial effects on maturation, activation rates, or blastocyst formation, and in fact were not essential for in vitro embryo production in the rabbit. Table 1.Effect of hormonal and growth factor supplementation on embryo development in rabbit oocytes The project was supported by RABIOTECH OMFB-00330/2004, EU FP6 (MEXT-CT-2003-509582, and 518240), Wellcome Trust (Grant No. 070246), and TET CH-28/04.

Microtubules Support Production of Starvation-induced Autophagosomes but Not Their Targeting and Fusion with Lysosomes

Autophagy is a major catabolic pathway in eukaryotic cells whereby the lack of amino acids induces the formation of autophagosomes, double-bilayer membrane vesicles that mediate delivery of cytosolic proteins and organelles for lysosomal degradation. The biogenesis and turnover of autophagosomes in mammalian cells as well as the molecular mechanisms underlying induction of autophagy and trafficking of these vesicles are poorly understood. Here we utilized different autophagic markers to determine the involvement of microtubules in the autophagic process. We show that autophagosomes associate with microtubules and concentrate near the microtubule-organizing center. Moreover, we demonstrate that autophagosomes, but not phagophores, move along these tracks en route for degradation. Disruption of microtubules leads to a significant reduction in the number of mature autophagosomes but does not affect their life span or their fusion with lysosomes. We propose that microtubules serve to deliver only mature autophagosomes for degradation, thus providing a spatial barrier between phagophores and lysosomes.

Posttranslationally Modified Proteins as Mediators of Sustained Intestinal Inflammation

Oxidative and carbonyl stress leads to generation of N(epsilon)-carboxymethyllysine-modified proteins (CML-mps), which are known to bind the receptor for advanced glycation end products (RAGE) and induce nuclear factor (NF)-kappaB-dependent proinflammatory gene expression. To determine the impact of CML-mps in vivo, RAGE-dependent sustained NF-kappaB activation was studied in resection gut specimens from patients with inflammatory bowel disease. Inflamed gut biopsy tissue demonstrated a significant up-regulation of RAGE and increased NF-kappaB activation. Protein extracts from the inflamed zones, but not from noninflamed resection borders, caused perpetuated NF-kappaB activation in cultured endothelial cells, which was mediated by CML-mps including CML-modified S100 proteins. The resulting NF-kappaB activation, lasting 5 days, was primarily inhibited by either depletion of CML-mps or by the addition of sRAGE, p44/42 and p38 MAPKinase-specific inhibitors. Consistently, CML-mps isolated from inflamed gut areas and rectally applied into mice caused NF-kappaB activation, increased proinflammatory gene expression, and histologically detectable inflammation in wild-type mice, but not in RAGE-/- mice. A comparable up-regulation of NF-kappaB and inflammation on rectal application of CML-mps was observed in IL-10-/- mice. Thus, CML-mps generated in inflammatory lesions have the capacity to elicit a RAGE-dependent intestinal inflammatory response.

Intracellular Zinc Release and ERK Phosphorylation Are Required Upstream of 12-Lipoxygenase Activation in Peroxynitrite Toxicity to Mature Rat Oligodendrocytes

Peroxynitrite toxicity has been implicated in the pathogenesis of white matter injury. The mechanisms of peroxynitrite toxicity to oligodendrocytes (OLs), the major cell type of the white matter, are unknown. Using primary cultures of mature OLs that express myelin basic protein, we found that 3-morpholinosydnonimine, a peroxynitrite generator, caused toxicity to OLs. N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine, a zinc chelator, completely blocked peroxynitrite-induced toxicity. Use of FluoZin-3, a specific fluorescence zinc indicator, demonstrated the liberation of zinc from intracellular stores by peroxynitrite. Peroxynitrite caused the sequential activation of extracellular signal-regulated kinase 42/44 (ERK42/44), 12-lipoxygenase, and generation of reactive oxygen species, which were all dependent upon the intracellular release of zinc. The same cell death pathway was also activated when exogenous zinc was used. These results suggest that in addition to preventing the formation of peroxynitrite, useful strategies in preventing disease progression in pathologies in which peroxynitrite toxicity plays a critical role might include maintaining intracellular zinc homeostasis, blocking phosphorylation of ERK42/44, inhibiting activation of 12-lipoxygenase, and eliminating the accumulation of reactive oxygen species.

Calcium-induced Acrosomal Exocytosis Requires cAMP Acting through a Protein Kinase A-independent, Epac-mediated Pathway

Epac, a guanine nucleotide exchange factor for the small GTPase Rap, binds to and is activated by the second messenger cAMP. In sperm, there are a number of signaling pathways required to achieve egg-fertilizing ability that depend upon an intracellular rise of cAMP. Most of these processes were thought to be mediated by cAMP-dependent protein kinases. Here we report a new dependence for the cAMP-induced acrosome reaction involving Epac. The acrosome reaction is a specialized type of regulated exocytosis leading to a massive fusion between the outer acrosomal and the plasma membranes of sperm cells. Ca2+ is the archetypical trigger of regulated exocytosis, and we show here that its effects on acrosomal release are fully mediated by cAMP. Ca2+ failed to trigger acrosomal exocytosis when intracellular cAMP was depleted by an exogenously added phosphodiesterase or when Epac was sequestered by specific blocking antibodies. The nondiscriminating dibutyryl-cAMP and the Epac-selective 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate analogues triggered the acrosome reaction in the effective absence of extracellular Ca2+. This indicates that cAMP, via Epac activation, has the ability to drive the whole cascade of events necessary to bring exocytosis to completion, including tethering and docking of the acrosome to the plasma membrane, priming of the fusion machinery, mobilization of intravesicular Ca2+, and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti-alpha-SNAP, anti-NSF, and anti-Rab3A antibodies, to intra-acrosomal Ca2+ chelators, and to botulinum toxins but was resistant to cAMP-dependent protein kinase blockers. These experiments thus identify Epac in human sperm and evince its indispensable role downstream of Ca2+ in exocytosis.

A Role for Kif17 in Transport of Kv4.2

Although kinesins are known to transport neuronal proteins, it is not known what role they play in the targeting of their cargos to specific subcellular compartments in neurons. Here we present evidence that the K+ channel Kv4.2, which is a major regulator of dendritic excitability, is transported to dendrites by the kinesin isoform Kif17. We show that a dominant negative construct against Kif17 dramatically inhibits localization to dendrites of both introduced and endogenous Kv4.2, but those against other kinesins found in dendrites do not. Kv4.2 colocalizes with Kif17 but not with other kinesin isoforms in dendrites of cortical neurons. Native Kv4.2 and Kif17 coimmunoprecipitate from brain lysate, and introduced, tagged versions of the two proteins coimmunoprecipitate from COS cell lysate, indicating that the two proteins interact, either directly or indirectly. The interaction between Kif17 and Kv4.2 appears to occur through the extreme C terminus of Kv4.2 and not through the dileucine motif. Thus, the dileucine motif does not determine the localization of Kv4.2 by causing the channel to interact with a specific motor protein. In support of this conclusion, we found that the dileucine motif mediates dendritic targeting of CD8 independent of Kif17. Together our data show that Kif17 is probably the motor that transports Kv4.2 to dendrites but suggest that this motor does not, by itself, specify dendritic localization of the channel.

Bicarbonate and bovine serum albumin reversibly ‘switch’ capacitation-induced events in human spermatozoa

We have investigated the reversibility of biochemical and physiological changes that occur upon suspension of ejaculated human spermatozoa during in vitro capacitation. Cells were swum up in a simple HEPES-based saline [lacking bicarbonate and bovine serum albumin (BSA)], then resuspended either in supplemented Earle's balanced salt solution (sEBSS) (25 mM bicarbonate) with 0.3% BSA (for in vitro capacitation) or in medium-lacking bicarbonate and/or BSA. Progesterone-induced acrosome reaction (AR) developed during in vitro capacitation (6 h). A progesterone-induced [Ca2+]i signal was detectable in cells maintained in the simple HEPES-based saline, but upon transfer to sEBSS, the response increased three- to four-fold, saturating within <30 min. Serine/threonine phosphorylation saturated within minutes of resuspension, but tyrosine phosphorylation developed over 3 h. Return of cells to non-capacitating conditions caused reversal of all capacitation-dependent changes. The [Ca2+]i signal reverted to its 'uncapacitated' size within <30 min. Protein phosphorylation reversed gradually and could be reinduced (kinetics resembling the first response) upon resuspension in sEBSS. The ability of cells to undergo progesterone-induced AR fell to levels similar to those in uncapacitated cells within 1 h of resuspension in medium not supporting capacitation. Loss of protein phosphorylation occurred only in the absence of both bicarbonate and BSA, but effects on [Ca2+]i signalling and AR could be seen after removal of only one of these factors. We conclude that key events in the capacitation of human spermatozoa are both reversible and repeatable.

Amino Acid Deprivation Induces Both Apoptosis and Autophagy in Murine C2C12 Muscle Cells

Apoptosis and autophagy are closely interconnected types of programmed cell death. In the present study, mouse C2C12 muscle cells were starved in Earle's Balanced Salt Solution or treated with TNF-alpha and cycloheximide to induce autophagy and apoptosis, respectively. The majority of starved C2C12 cells underwent autophagy, as shown by LC3 processing, formation of autophagic vesicles and bulk degradation of long-lived proteins. However, some cells showed features of apoptosis including caspase-3 cleavage, chromatin condensation, DNA fragmentation and annexin V labeling. Caspase-3 cleavage was also induced in culture medium without serum, suggesting that serum withdrawal rather than amino acid deprivation triggered apoptosis. Starvation eliminated multiple pro-apoptotic proteins, but upregulated caspase-8, and rendered starved C2C12 cells much more susceptible to TNF-alpha/cycloheximide-induced apoptosis than non-starved cells. Our data suggest that amino acid deprivation of C2C12 cells induces a complex form of cell death with hallmarks of both apoptosis and autophagy.

Trafficking of the Plasma Membrane γ-Aminobutyric Acid Transporter GAT1: SIZE AND RATES OF AN ACUTELY RECYCLING POOL

Trafficking of the Plasma Membrane γ-Aminobutyric Acid Transporter GAT1: SIZE AND RATES OF AN ACUTELY RECYCLING POOL

Involvement of Adenosine Triphosphate-Sensitive Potassium Channels in the Response of Membrane Potential to Hyperosmolality in Cultured Human Aorta Endothelial Cells

The membrane potential of endothelial cells is an important determinant of endothelial functions, including regulation of vascular tone. We investigated whether adenosine triphosphate-sensitive potassium (K(ATP)) channels were involved in the response of membrane potential to hyperosmolality in cultured human aorta endothelial cells. The voltage-sensitive fluorescent dye, bis-(1,3-diethylthiobarbiturate)trimethine oxonol, was used to assess relative changes in membrane potential semiquantitatively. To investigate the effect of mannitol-, sucrose-, and NaCl-induced hyperosmolality on membrane potential, cells were continuously perfused with Earle's balanced salt solution (285 mOsm/kg H(2)O) containing 200 nM bis-(1,3-diethylthiobarbiturate)trimethine oxonol and exposed to 315 and 345 mOsm/kg H(2)O hyperosmotic medium sequentially in the presence and absence of 1 muM glibenclamide, a well-known K(ATP) channel blocker. Hyperosmotic mannitol significantly induced hyperpolarization of the endothelial cells, which was prevented by 1 microM glibenclamide (n = 6). Estimated changes of membrane potential at 315 and 345 mOsm/kg H(2)O were 13 +/- 8 and 21 +/- 8 mV, respectively. Hypertonic sucrose induced similar changes. However, although hypertonic saline also significantly induced hyperpolarization of the endothelial cells (n = 6), the hyperpolarization was not prevented by 1 muM glibenclamide. In conclusion, K(ATP) channels may participate in hyperosmotic mannitol- and sucrose-induced hyperpolarization, but not in hypertonic saline-induced hyperpolarization in cultured human aorta endothelial cells.

In vitro excystment of the metacercariae of Ribeiroia ondatrae

In vitro excystation studies were done on the metacercarial cysts of Ribeiroia ondatrae obtained from naturally infected leopard frogs, Rana pipiens. Studies were done in Petri dish cultures in various media maintained at 41 degrees C. The media consisted of 0.5% trypsin-0.5% bile salts in Earle's balanced salt solution at pH 7.8 (TB), 1% acid pepsin (AP) in 0.85% saline adjusted to pH 2 with HCl; acid saline (AS) adjusted to pH 2 with HCl, and saline (S). The TB medium, used successfully to excyst the metacercariae of numerous species of digeneans, did not induce excystation of Ribeiroia ondatrae. Some excystation occurred in AS but not in S. Excystation was 100% in AP with most organisms excysting within 30 s after placement in the medium at 41 degrees C. Excystation in vitro in AP at 41 degrees C is compatible with the fact that this organism establishes itself in the acid-pepsin environment of the proventriculus of avian hosts.

269 EFFECTS OF REPRODUCTIVE STATUS AND OOCYTE QUALITY ON THE DEVELOPMENTAL COMPETENCE OF OOCYTES FOLLOWING IN VITRO PRODUCTION IN DOMESTIC CAT

A recent study showed that the developmental competence of cat oocytes after IVM and IVF was affected by the estrous cycle stage of the donor (Freistedt et al. 2001 Biol. Reprod. 65, 9-13). This study was conducted to examine the effect of the cat reproductive cycle stage and the oocyte quality on the developmental competence of the oocyte following IVF production. Cat ovaries were collected at veterinary clinics and stored at 4�C for 24 h. Based on the presence or absence of follicles and corpora lutea, the ovaries were classified into the luteal, follicular, or inactive stage. Cumulus oocyte complexes (COCs) from the different stage ovaries were separated at recovery into three ranks (A, B, and C) according to pigmentation, uniformity and smoothness of ooplasm, and amount of surrounding cumulus cell mass and were cultured separately in 100 �L drops of maturation medium (TCM-199), supplemented with 0.4% bovine serum albumin, 0.1 IU/mL (human menopausal gonadotropin (HMG), 10 IU/mL HCG, 1 �g/mL 17�-estradiol, and 100 �g/mL gentamicin) for 24-h at 38�C, 5% CO2 in air. After 24-h in vitro culture, the oocytes were transferred into 100 �L sperm microdrops of Brackett-Oliphant medium (2 � 106 sperm/mL) for fertilization and were co-incubated for 12 h. Subsequently, presumptive zygotes were transferred into a modified Earle's balanced salt solution (MK-1) supplemented with 4 mg/mL BSA and 50 �g/mL gentamicin. Three days after insemination, all embryos were transferred into culture medium of MK-1 supplemented with 5% (v/v) fetal bovine serum and 50 �g/mL gentamicin. The cleaved embryos were further cultured for 5 days to evaluate their ability to develop to the blastocyst stage. Data were analyzed by ANOVA. The percentages of COCs of A (35.5%, 31.8%, 27.7%), B (41.5%, 39.9%, 42.9%), and C (23.0%, 28.3%, 29.4%) ranks did not show significant differences (P &gt; 0.05) among the reproductive stages of ovaries (luteal, follicular, and inactive, respectively). There were significant differences in the percentages of cleavage (P &lt; 0.05) among the A, B, and C ranks of oocytes from ovaries classified as luteal and follicular stages (50%, 35%, 1.8%; 52%, 35%, 3.9%, respectively, P &lt; 0.05). However, there were no significant differences between the A and B ranks of oocytes obtained from inactive stages of ovaries (47% vs. 44%, respectively; P &gt; 0.05) but there was a significant difference for C rank (1.9%). The percentages of blastocyst formation were significantly different (P &lt; 0.05) among the ranks of oocytes obtained from luteal, follicular, and inactive stages (27%, 14.6%, and 0% for A; 24%, 11.7%, and 0.6% for B; 35%, 21.6%, and 2.9% for C). However, there were no significant differences (P &gt; 0.05) among the reproductive cycle stages of ovaries in the three oocyte ranks with respect to the percentages of cleavage and blastocyst formation.These results indicate that the reproductive stage of donor cat ovaries stored at 4�C for 24 h has no apparent effect on the developmental competence of the oocyte following IVF, but development is affected by oocyte quality.

Long-term exposure to new peritoneal dialysis solutions: Effects on the peritoneal membrane

Chronic exposure to peritoneal dialysis fluid (PDF) affects the peritoneum, but precise causative factors are incompletely understood. We examined the effects of standard and new PDF on peritoneal function and structure. Female Wistar rats received twice daily intraperitoneal infusions of a standard lactate-buffered 3.86% glucose PDF at pH 5.5 (Dianeal) (N= 12), a low glucose degradation product (GDP) containing bicarbonate/lactate-buffered 3.86% glucose PDF at pH 7.4 (Physioneal) (N= 12), a lactate-buffered amino acid-based PDF at pH 6.7 (Nutrineal) (N= 12) or Earle's Balanced Salt Solution at pH 7.4 (EBSS) (N= 12) during 12 weeks. Net ultrafiltration was lower after treatment with standard PDF, but not with low-GDP bicarbonate/lactate-buffered and amino acid-based PDF, compared to EBSS. Peritonea exposed to standard PDF were characterized by an increased expression of vascular endothelial growth factor (VEGF), microvascular proliferation as well as submesothelial fibrosis, which were not observed in other groups. Staining for methylglyoxal adducts was prominent in the standard PDF-exposed group, mild in the low GDP bicarbonate/lactate-buffered group and absent in the other groups. Standard PDF induced accumulation of advanced glycation end products (AGEs) and up-regulation of the receptor for AGE (RAGE). AGEs accumulation was absent and RAGE expression was only modestly increased in low-GDP bicarbonate/lactate-buffered and amino acid-based PDF. Long-term in vivo exposure to standard PDF adversely affects peritoneal function and structure. A low-GDP bicarbonate/lactate-buffered and amino acid-based PDF better preserved peritoneal integrity and may thus improve the longevity of the peritoneal membrane. GDPs and associated accelerated AGE formation are the main causative factors in PDF-induced peritoneal damage.

Effect of propofol on hypotonic swelling-induced membrane depolarization in human coronary artery smooth muscle cells.

Stretch (mechanical stress)-induced membrane depolarization of smooth muscle may contribute to basal vascular tone and myogenic control. Propofol induces vasodilation and inhibits myogenic control. Hypotonic swelling was used as a model of mechanical stress. The authors investigated the effects of propofol and 5-nitro-2-(3-phenylpropylamino)benzoic acid, a chloride channel and nonselective cation channel blocker, on hypotonicity-induced membrane depolarization in cultured human coronary artery smooth muscle cells. A voltage-sensitive fluorescent dye, bis-(1,3-diethylthiobarbiturate)trimethine oxonol, was used to assess relative changes in membrane potential semiquantitatively. The cells were continuously perfused with Earle's balanced salt solution containing 200 nM bis-(1,3-diethylthiobarbiturate)trimethine oxonol and exposed sequentially to isotonic and hypotonic medium. In a second series of experiments, the cells were exposed to hypotonic media in the presence and absence of 5-nitro-2-(3-phenylpropylamino)benzoic acid or propofol. The relative fluorescence values at 10, 20, and 30% hypotonicity were 147 +/- 29, 214 +/- 74, and 335 +/- 102% of baseline, respectively. The changes were all significantly different from the isotonic time control group. In the presence of 200 microM 5-nitro-2-(3-phenylpropylamino)benzoic acid or 0.1, 1, 10, or 100 microg/ml propofol, the relative fluorescence values at 30% hypotonicity were 87 +/- 17, 194 +/- 27, 160 +/- 18, 130 +/- 18, and 84 +/- 15%, respectively. These changes were significantly less than the 30% for the hypotonic control (246 +/- 23%). These results suggest that volume-sensitive chloride channels and nonselective cation channels may participate in hypotonicity-induced membrane depolarization and that propofol inhibits hypotonicity-induced membrane depolarization in coronary artery smooth muscle.

Determination of Diclofenac Sodium in Eagle's Minimum Essential Medium with Earle's Balanced Salt Solution

A validated method was developed for determination of diclofenac sodium, considered a model hydrophilic drug for in vitro permeation studies, in Eagle's Minimum Essential Medium (MEM) with Earle's balanced salt solution. Liquid chromatography was used to determine diclofenac sodium. This method was developed with a reversed-phase column Supercosil LC 18, DB 25 cm x 4.5 mm; the mobile phase was methanol with 3% (v/v) acetic acid-Milli-Q water (74 + 26), and detection was at 283 nm. The detection and quantitation limits were 2.41 x 10(-8) and 3.31 x 10(-5) microg/microL, respectively. The accuracy within-day (n = 3) and day-to-day (n = 7) was 98.83%; the mean variation coefficient for inter- (n = 7) and intraday precision (n = 3) was 12.20%, thus, not exceeding 15%. This method can be used as an analytical procedure for the determination of diclofenac sodium in MEM for in vitro permeation studies.

Binding of Zona Binding Inhibitory Factor-1 (ZIF-1) from Human Follicular Fluid on Spermatozoa

Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.

Effects of serum-free culture media on in vitro development of domestic cat embryos following in vitro maturation and fertilization.

This study was conducted to determine the adequate medium for a serum-free culture system of domestic cat embryos produced by in vitro maturation (IVM) and fertilization (IVF). Cumulusoocyte complexes recovered from cat ovaries were matured in vitro for 24 h, and then inseminated in vitro for 12 h. After insemination, the oocytes were cultured in five media [Ham's F10, Waymouth 752/1 (Waymouth), TCM199, modified Earle's balanced salt solution (MK-1) and CR1aa], each of which contained 0.4% bovine serum albumin. There were no significant differences among the rates of fertilization of oocytes cultured in five media following IVF. The rate of oocytes/embryos developed to at least the morula stage was significantly lower (p < 0.05) in Waymouth than in MK-1, TCM199 and CR1aa. Moreover, none of the embryos cultured in Ham's F10 and Waymouth developed to the blastocyst stage. There were no differences among the rates of development to the blastocyst stage of oocytes/embryos cultured in MK-1, TCM199 and CR1aa. These results indicate that the type of serum-free medium has a major impact on in vitro development of domestic cat embryos derived from IVM/IVF, and MK-1, TCM199 and CR1aa media are suitable for in vitro culture of cat embryos in a serum-free culture system.

Maintenance of the inner cell mass in human blastocysts from fragmented embryos.

The degree of fragmentation during early cleavage is universally used as an indicator of embryo quality during human in vitro fertilization treatment. Extensive fragmentation has been associated with reduced blastocyst formation and implantation. We examined the relationship between early fragmentation and subsequent allocation of cells to the trophectoderm and inner cell mass in the human blastocyst. We retrospectively analyzed data from 363 monospermic human embryos that exhibited varying degrees of fragmentation on Day 2. Embryos were cultured from Day 2 to Day 6 in Earle balanced salt solution with 1 mM glucose and human serum albumin. Rates of development and blastocyst formation were measured. The number of cells in the trophectoderm and inner cell mass and the incidence of apoptosis were assessed following differential labeling with polynucleotide-specific fluorochromes. Increasing fragmentation resulted in reduced blastocyst formation and lower blastocyst cell numbers. For minimal and moderate levels of fragmentation, the reduction in cell numbers was confined largely to the trophectoderm and a steady number of inner cell mass cells was maintained. However, with extensive fragmentation of more than 25%, cell numbers in both lineages were reduced in the few embryos that formed blastocysts. Apoptotic nuclei were present in both the trophectoderm and inner cell mass, with the lowest incidence in blastocysts that had developed from embryos with minor (5-10%) fragmentation. Paradoxically, higher levels of apoptosis were seen in embryos of excellent morphology, suggesting a possible role in regulation of cell number.

Rapid actions of 17beta-oestradiol on a subset of lactotrophs in the rat pituitary.

Increasingly the role of rapid mechanisms of steroid action in physiological regulation are being recognised. We have investigated rapid effects of 17beta-oestradiol (E) on prolactin (PRL) release in vitro. Pituitary segments from male rats were incubated for 5, 10 or 20 min in Earle's balanced salt solution containing 1.2 mM tannic acid (to enable visualisation of exocytosed secretory granules by electron microscopy) either alone (control) or containing 10(-10)-10(-8) M E conjugated to bovine serum albumin (E-BSA). PRL and leuteinising hormone (LH) release from pituitary segments were also determined in response to E and E-BSA by radioimmunoassay. Within 10 min E-BSA and E (10(-12)-10(-6) M) stimulated a significant (P < 0.05) concentration-dependent release of PRL but not LH. After exposure to experimental media for 5 min, only occasional exocytosis from type I lactotrophs (characterised by large polymorphic secretory granules) was observed in either control or E-BSA treated tissue. In contrast, E-BSA (10(-10)-10(-8) M) induced a significant (P < 0.05) increase in the number of exocytotic profiles from type II lactotrophs (characterized by smaller, spherical granules). This effect was not inhibited by removal of extracellular calcium, or by pre-treatment of cells with the RNA synthesis inhibitor actinomycin-D (0.5 microg ml(-1)), the protein synthesis inhibitor cycloheximide (1 microg ml(-1)) or the anti-oestrogen ICI 182,780 (1 microM). FACS analysis demonstrated binding of E-BSA-fluorescein isothiocyanate (FITC) (10(-10)-10(-7) M) to a subpopulation of anterior pituitary cells. The E-BSA-FITC binding sites assumed a patchy distribution across the cell surface. In conclusion, we report for the first time a rapid, non-genomic effect of E on PRL secretion in normal pituitary tissue.

Amino acids promote human blastocyst development in vitro.

Supplementation of culture media with amino acids has been shown to benefit preimplantation embryo development in several species. This randomized study analysed the in-vitro development of human embryos obtained after IVF in the presence or absence of a combination of amino acids from the 2- to 4-cell stage to the blastocyst stage. A total of 129 human embryos was randomly distributed between three serum-free chemically defined sequential media: (i) glucose-free Earle's balanced salt solution (EBSS) with glutamine (Gln) prior to morula stage, supplemented with glucose for blastocyst formation; (ii) glucose-free EBSS with glutamine and non-essential amino acids (AA) for cleavage stage development, and supplemented with all 20 AA for blastocyst formation (Earle's+AA); and (iii) a sequential commercial medium containing amino acids (K-SCIM). Embryos were individually cultured for successive periods of 24 h. On day 6 of development, blastocysts were differentially labelled and the numbers of trophectoderm and inner cell mass cells, mitoses and dead cells were examined. Blastocyst development was similar for the three sequential media. The mixture of AA significantly increased total blastocyst cell numbers from 61.8 +/- 4.2 with Earle's+Gln to 99.3 +/- 8.4 with Earle's+AA and 100.2 +/- 9.4 with K-SCIM (P = 0.005). This increase was present in both the trophectoderm and inner cell mass lineages (P < 0.02). Furthermore, the dead cell index was significantly lower with Earle's+AA (P = 0.047).