The effect of Tempo and MitoTEMPO on oocyte maturation and subsequent embryo development in bovine model.

Reactive oxygen species (ROS) are one of the factors which reduces oocyte quality and viability of the in vitro produced embryos. Oocyte mitochondria are the major source of ROS production, hence, and the addition of mitochondrion-specific antioxidants could be suggested to minimize the damage caused by ROS during culture. MitoTEMPO, a targeted mitochondrial antioxidant, is formed by conjugating TEMPO to triphenylphosphonium and has an activity like that of superoxide dismutase. It can pass through lipid bilayers easily and accumulate selectively in mitochondria. The goal of this study was to investigate the effects of MitoTEMPO and its non-targeted form, TEMPO, on the developmental competence of bovine oocytes. Accordingly, oocytes were cultured in maturation medium supplemented with either five mM TEMPO (T5) or one µM MitoTEMPO (M1), or T5 + M1 (MT15), or without the antioxidants (C). Nuclear maturation to metaphase II (MII) stage, intracellular glutathione (GSH) content and ROS levels in matured oocytes were analyzed. In addition, cleavage after in vitro fertilization, and blastocyst rates, total cell number in blastocysts as well as the relative abundance of apoptosis-related genes (BAX and BCL2) in blastocysts were determined. Results revealed that the proportion of oocytes at the MII stage, embryos at the blastocyst stage and total cell number in blastocysts increased significantly in the M1 group compared to the C and T5 groups. The levels of intracellular GSH and ROS in oocytes decreased in the M1 group than in the C group (P < 0.05). The expression level of the pro-apoptotic gene (BAX) reduced in blastocysts from the M1 group in comparison to the C and T5 groups (P < 0.05). On the other hand, the expression level of anti-apoptotic gene (BCL2) in obtained blastocysts was not affected by TEMPO and MitoTEMPO. However, the ratio of BAX/BCL2 in blastocysts from the M1 and MT15 groups decreased significantly compared to the C group. These findings suggest that MitoTEMPO can mitigate the adverse effects of oxidative stress on the developmental competence of bovine oocytes.

Combined effect of probiotics and specific immunoglobulin Y directed against Escherichia coli on growth performance, diarrhea incidence, and immune system in calves.

Enterotoxigenic Escherichia coli (ETEC) K99 is one of the major pathogens associated with calf diarrhea. The induction of passive immunity in animals by immunoglobulin Y and using probiotics are inexpensive alternatives to antibiotics for the prevention and treatment of a number of bacterial infections, including diarrhea. Hence, the aim of this research was to evaluate the impact of dietary probiotics and ETEC K99-specific egg yolk antibody supplements, alone and in combination with each other, on health and growth parameters, diarrhea incidence and immune stimulation in newborn Holstein calves. One hundred and twenty neonatal calves were allocated randomly into 4 dietary groups (n = 30 per group) received colostrum/milk without any additives (control group), or supplemented with egg yolk powder contained E. coli K99-specific antibody (Ab group; 1 g/day), a commercial probiotic, Hypro-calves (Pro group; 3 g/day), and their combination (Ab+Pro group), from day (d) 1 to d28 of age. Analyses of the growth parameters, feed efficiency, fecal score, and microbiota and immune function were carried out on d0, 14, 21, and 28 of the experiment. Calves in Ab or Ab+Pro group had higher (P < 0.05) average daily gain compared to control and Pro groups during 0-14d. Feed efficiency of calves in Ab and Ab+Pro groups was significantly higher than that in control group during the period of 0-14d; however, no significant differences were observed in 0-28d period. Diarrhea prevalence and fecal score in Ab+Pro group were lower than control group (P < 0.05). Calves in Ab+Pro group had the lowest number of fecal E. coli in comparison to other groups on d28 (P < 0.05). Feeding Ab+Pro supplement increased (P < 0.05) concentrations of blood IgA and serum CD4 compared to the control group. Likewise, calves in Pro group had higher CD4 levels as compared to the control calves (P < 0.05). Serum concentration of interferon-gamma in control group was lower than other groups (P < 0.05). Overall, these data suggest that feeding a combination of probiotic and specific antibody against ETEC to neonate Holstein calves enhances feed efficiency, boosts immunity, and reduces diarrhea prevalence.

Immunolocalization of angiogenic growth factors in the ovine uterus during the oestrus cycle and in response to Steroids.

The vascular changes associated with endometrial maturation in preparation for embryo implantation depend on numerous growth factors, known to regulate key angiogenic events. Primarily, the vascular endothelial growth factor (VEGF) family promotes vascular growth, whilst the angiopoietins maintain blood vessel integrity. The aim was to analyse protein levels of VEGFA ligand and receptors, Angiopoietin-1 and 2 (ANG1/2) and endothelial cell receptor tyrosine kinase (TIE-2) in the ovine endometrium in the follicular and luteal phases of the oestrus cycle and in response to ovarian steroids. VEGFA and its receptors were localized in both vascular cells and non-vascular epithelium (glandular and luminal epithelium) and stroma cells. VEGFA and VEGFR2 proteins were elevated in vascular cells in follicular phase endometrium, compared to luteal phase, most significantly in response to oestradiol. VEGFR1 was expressed by epithelial cells and endothelial cells and was stimulated in response to oestradiol. In contrast, Ang-1 and Ang-2 proteins were elevated in luteal phase endometrium compared to follicular phase, and in response to progesterone, evident in vascular smooth muscle cells and glands which surround TIE-2-expressing blood vessels. Our findings indicate that VEGFA is stimulated by oestradiol, most predominantly in follicular phase endometrium, and Ang-1 and 2 are stimulated by progesterone and were increased during the luteal phase of the oestrus cycle, during the time of vascular maturation.

Sperm preparation through Sephadex™ filtration improves in vitro fertilization rate of buffalo oocytes.

Routinely, swim-up method is used to separate high-quality sperm; however, long processing time and close cell-to-cell contact during the centrifugation step are inevitable elements of oxidative stress to sperm. The objective was to evaluate Sephadex™ and glass wool filtration to separate motile, intact and viable sperm for in vitro fertilization in buffalo. The cumulus-oocyte complexes (COCs) were collected from ovaries of slaughtered buffaloes by aspiration and matured for 24 hr in CO2 incubator at 38.5°C and 5% CO2 . Matured COCs were rinsed twice in fertilization TALP and placed in the pre-warmed fertilization medium without sperm. Cryopreserved buffalo semen was thawed at 37°C for 30 s and processed through Sephadex™ , glass wool filtration and swim-up (control). Total and motile sperm recovery rates were assessed, resuspended in fertilization TALP and incubated for 15-20 min in CO2 incubator. Samples prepared by each method were divided into two aliquots: one aliquot was studied for sperm quality (progressive motility, membrane integrity, viability, liveability), while the other was subjected to co-incubation with sets of 10-15 in vitro matured oocytes. Data on sperm quality were analysed by ANOVA, while in vitro fertilizing rates were compared by chi-squared test using SPSS-20. Least significant difference (LSD) test was used to compare treatment means. Glass wool filtration yielded higher total and motile sperm recovery rate, while Sephadex™ filtration improved (p < .05) sperm quality (progressive motility, membrane integrity, viability, liveability). Sperm preparation through Sephadex filtration yielded higher in vitro fertilization rate in terms of cleavage rate compared to glass wool filtration and swim-up (control). In conclusion, cryopreserved Nili-Ravi buffalo sperm selected through Sephadex filtration showed improved quality and yielded better fertilization rates (cleavage rate) of in vitro matured/fertilized oocytes. Sephadex filtration could be a promising technique for use in in vitro fertilization in buffalo.

Localisation and endocrine control of hyaluronan synthase (HAS) 2, HAS3 and CD44 expression in sheep granulosa cells.

The aim of the present study was to investigate the hormonal regulation of hyaluronan (HA) components in sheep granulosa cells. HA components are present in the reproductive tract and have a range of physical and signalling properties related to reproductive function in several species. First, abattoir-derived ovaries of sheep were used to determine the localisation of HA synthase (HAS) 1-3 and CD44 proteins in antral follicles. Staining for HAS1-3 and CD44 proteins was most intense in the granulosa layer. Accordingly, the expression of HAS2, HAS3 and CD44 mRNA was measured in cultured granulosa cells exposed to 0-50ngmL(-1) of 17ß-oestradiol and different combinations of oestradiol, gonadotropins, insulin-like growth factor (IGF)-1 and insulin for 48-96h (1ngmL(-1) FSH, 10ngmL(-1) insulin, 10ngmL(-1) IGF-1, 40ngmL(-1) E2 and 25ngmL(-1) LH.). mRNA expression was quantified by real-time polymerase chain reaction using a fold induction method. The results revealed that the hormones tested generally stimulated mRNA expression of the genes of interest in cultured granulosa cells. Specifically, oestradiol, when combined with IGF-1, insulin and FSH, stimulated HAS2 mRNA expression. Oestradiol and LH had synergistic effects in increasing HAS3 mRNA expression. In conclusion, we suggest that the hormones studied differentially regulate HAS2, HAS3 and CD44 in ovine granulosa cells in vitro. Further work is needed to address the signalling pathways involved.

Effects of different polyunsaturated fatty acid supplementations during the postpartum periods of early lactating dairy cows on milk yield, metabolic responses, and reproductive performances.

In spite of the difficulties in delivering PUFA to ruminants, studies have generally indicated that the PUFA of the omega-6 (linoleic acid) and omega-3 [α-linolenic acid; eicosapentaenoic (EPA), C20:5 omega-3; docosahexaenoic (DHA), C22:6 omega-3] families are the most beneficial to improving reproduction in cows. The objectives were to determine if a diet enriched in α-linolenic acid (omega-3) or linoleic acid (omega-6) would influence milk production and composition, metabolic status, and reproductive performance in lactating dairy cows. High-yielding multiparous Holstein dairy cows (n = 120) with no overt clinical illnesses were blocked according to calving date and parity. Cows were assigned randomly to be fed 1) soybean whole roast (Soy, omega-6, n = 40) or 2) linseed (Lin, omega-3, n = 40) or 3) palm oil as a source of SFA (PO, n = 40) from calving until first heat after 40 d postpartum (dpp), and then half of the cows in each treatment group were switched to receive either Lin or SFA (PO) from first heat after d 40 to 120 dpp. Blood was collected from a subsample of cows. Blood was collected at 14 d intervals for 12 wk, starting on the day of calving. Results showed milk yield and DMI were not affected. Milk compositions were similar (P > 0.08) among diets, except concentration and yield of milk fat percentage, which was less in cows fed Lin (P < 0.05). Uterine involution in cows fed Soy occurred earlier (P < 0.05). Diets affected day to first estrus and day to first insemination in cows (P < 0.05). There were no differences among treatments for percent heat detection, percent pregnancy per first insemination, and percent conception per AI at estrus. Also, there is a trend of pregnancy by 120 d, which is 66.7% for the Lin group vs. 50.91% for the PO group (P < 0.08). Of the 4 pregnancy losses, 2 occurred in PO-PO group and 2 occurred in Soy-PO group, and none occurred in the other 4 treatments. In conclusion, our study showed feeding omega-6 fatty acids during 40 dpp could be a good treatment for early postpartum periods, and a shift to omega-3 fatty acids until 40 d after AI can be considered as a strategy for improving fertility in lactating dairy cows.

Role of hyaluronic acid in maturation and further early embryo development of bovine oocytes.

Hyaluronic acid (HA), an important component of the extracellular matrix, plays a crucial role for cumulus cell expansion. Genes and proteins involved in HA synthesis and its receptor CD44 are expressed in cumulus oocyte complexes (COCs) in different animal species and increase during maturation. Hyaluronidase enzymes (Hyal) degrade HA into smaller biologically active HA fragments. To investigate the effects of the molecular size and concentration of HA on oocyte maturation and further embryo development, bovine oocytes were matured in vitro in the presence or absence of HA, Hyal-2 or 4-methylumbelliferone (4-MU); an HA synthesis inhibitor. The rates of oocyte nuclear maturation to metaphase II stage and development of embryos to blastocyst stage and blastocyst quality were recorded. Hyal-2 inhibited cumulus cell expansion without affecting oocyte maturation and further embryo development. Whereas, 4-MU at 1 mm reduced cumulus cell expansion, oocyte maturation rate and further embryo development; an effect which was partially abrogated by exogenous HA supplementation. These data suggest that HA production by cumulus cells during maturation is essential not only for cumulus cell expansion, but also for oocyte maturation and further embryo development. This effect is not affected by HA-degradation by Hyal-2.

Effect of dietary-induced changes in plasma insulin concentrations during the early post partum period on pregnancy rate in dairy cows.

Dietary stimulation of insulin in post partum dairy cows has been found to enhance ovarian follicle development but to impair oocyte developmental competence. The objective of this study was to test the hypothesis that pregnancy rate would be improved by feeding a diet to stimulate higher insulin (H) until cows resumed ovarian cyclic activity after parturition, and then feeding a diet to lower insulin (L) during the mating period. Each diet was fed to 30 post partum dairy cows until their first rise in milk progesterone, when 15 cows in each group were transferred to the other diet (treatments HL and LH) and 15 cows in each group remained on their original diet (treatments HH and LL) until 120 days post partum. Treatments did not affect dry matter intake, milk yield and metabolisable energy balance. Plasma insulin concentration was elevated in cows fed on H compared with cows fed on L. Treatment did not affect days to first progesterone rise, first oestrus or first insemination. At 120 days post partum, 27% of cows on each of treatments HH, LL and LH were pregnant, but 60% of cows on treatment HL were pregnant (P=0.021). These findings support the concept that physiological relationships between insulin and the reproductive system vary according to stage of the reproductive cycle, and suggest that pregnancy rate can be enhanced by a two-diet strategy tailored to optimise responses before and after the first post partum ovulation.

Interleukin 1 signaling is regulated by leukemia inhibitory factor (LIF) and is aberrant in Lif-/- mouse uterus.

This study addresses the regulation of the interleukin 1 (IL1) system in the murine uterine luminal epithelium (LE) and stroma by leukemia inhibitory factor (LIF). Using RT-PCR we compared expression of Il1a, Il1b, Il1rn, Il1r1, and Il1r2 during the pre- and peri-implantation periods of pregnancy in wild-type (WT) and LIF-null LE and stroma. In WT LE, Il1a transcripts were down-regulated on Day 4 of pregnancy (D4), with renewed expression by the evening of D4 (D4 pm). In Lif(-/-) LE there was a gradual decrease in expression on D2, and expression became undetectable by D6. Il1b and Il1r1 expression were similar in WT and null mice, but Il1rn expression was almost completely lost during the peri-implantation period in Lif(-/-) LE. In the stroma, Il1a was sharply down-regulated on D4 and reappeared on D4 pm but was only expressed from D3 to D5 in the null mice. Stromal Il1r1 and Il1r2 were also misregulated. Il1rn showed constitutive expression in null stroma in contrast to the loss of expression on D4 in the WT mouse. In Lif-deficient mice, immunostaining indicated a reduction of endometrial IL1A at the time of implantation and of IL1B in stroma. LE-stromal coculture revealed that LIF stimulated the apical secretion of both IL1A and PTGES2 by LE cells without affecting basal secretion of IL1A and with only a small effect on basal PTGES2 secretion. We conclude that Il1a and Il1rn in LE and Il1a, Il1rn, and Il1r1 in stroma are regulated by LIF, which stimulates apical secretion of IL1A by LE.

Methotrexate induced differentiation in colon cancer cells is primarily due to purine deprivation.

The folate antagonist methotrexate (MTX) inhibits synthesis of tetrahydrofolate (THF), pyrimidines and purines, and induces differentiation in several cell types. At 1 microM, MTX reduced proliferation and induced differentiation in HT29 colon cancer cells; the latter effect was augmented (P < 0.001) by thymidine (100 microM) but was reversed (P < 0.001) by the purines, hypoxanthine (Hx; 100 microM) and adenosine (100 microM). In contrast 5-fluoro-uracil (5-FU), a specific thymidylate synthase (TS) inhibitor, had no effect on differentiation, suggesting that MTX-induced differentiation is not due to a reduction in thymidine but to the inhibition of purine biosynthesis. Inhibition of cyclic AMP (cAMP) by RpcAMP (25 microM) further enhanced (P < 0.001) MTX induced differentiation, whereas the cAMP activator forskolin (10 microM) reversed (P < 0.001) MTX induced differentiation. These observations implicate a central role of adenosine and cAMP in MTX induced differentiation. By combining Western blot analysis with liquid chromatography-mass spectrometry (LC-MS)and HPLC analyses we also reveal both the expression and activity of key enzymes (i.e. methionine synthase (MS), s-adenosylhomocysteinase, cystathionine beta-synthase and ornithine decarboxylase) regulating methyl cycle, transsulfuration and polyamine pathways in HT29 colon cancer cells. At 1 microM, MTX induced differentiation was associated with a marked reduction in the intracellular concentrations of adenosine and, consequently, S-adenosylmethionine (SAM), S-adenosylhomocysteine, polyamines and glutathione (GSH). Importantly, the marked reduction in methionine that accompanied MS inhibition following MTX treatment was non-limiting with respect to SAM synthesis. Collectively, these findings indicate that the effects of MTX on cellular differentiation and single carbon metabolism are primarily due to the intracellular depletion of purines.

Dissociation of oocyte nuclear and cytoplasmic maturation by the addition of insulin in cultured bovine antral follicles.

Follicles of 4-8 mm in diameter were dissected from ovaries and cultured in Waymouth culture medium in the presence or absence of insulin (5 mug/ml) at 39 degrees C in a humidified atmosphere of 45% O2, 5% CO2 and 50% N2 for 24 h. Following follicle culture, the oocytes were collected and examined for developmental potential, total protein profile and ultrastructural aspects. Oocytes aspirated directly from follicles of the same size were used as controls. Addition of insulin to the follicle culture medium significantly reduced expression of the low molecular weight insulin-like growth factor-binding proteins (IGFBPs) in the follicular fluid, and significantly reduced the cleavage rate of subsequently matured and fertilised oocytes (0.52 vs 0.61). However, there were no differences in the proportion of cleaved embryos which developed to the blastocyst stage (0.30 vs 0.28), nor embryo quality as assessed by total cell number (137 +/- 8.53 vs 124.6 +/- 6.95). The total protein profiles of immature oocytes recovered after 24 h of follicle culture were compared by PAGE. There were marked differences between the two groups, unmatured oocytes recovered from the insulin-positive follicle group showed a protein pattern similar to that of matured oocytes. In addition, examination of ultrastructural features by transmission electron microscopy indicated that oocytes from follicles cultured in the presence of insulin undergo many of the cytoplasmic changes associated with oocyte maturation. In conclusion, follicle culture in the presence of insulin is beneficial for follicular survival and significantly reduces cleavage but has no detrimental effects on the development of cultured embryos. However, many of the cytoplasmic changes associated with oocyte maturation occur prior to the induction of nuclear maturation.

Differential staining combined with TUNEL labelling to detect apoptosis in preimplantation bovine embryos.

Development of accurate laboratory methods to assess embryo quality will improve the efficiency of embryo production from in-vitro culture systems. Currently, the techniques of TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end (TUNEL) labelling for the detection of apoptosis, and differential staining for determining the ratio of inner cell mass (ICM) to trophectoderm (TE) cells, are used separately to assess embryo quality in a range of different species. This paper reports a unique, simple and fast method for the assessment of embryo quality using differential staining of TE and ICM, but combined with TUNEL labelling (DST staining). This technique was used to investigate the effect of serum supplementation on total cell number, ICM:TE ratio and apoptosis index after in-vitro production of bovine embryos. Serum supplementation increased total cell number (P < 0.01), but reduced the ratio of ICM:TE cells. No differences were observed in the number of apoptotic nuclei between treatments, or in the localization of the apoptotic nuclei. However, more apoptotic nuclei were observed in ICM than TE cells in both culture groups. In conclusion, using DST, it has been possible to carry out both a qualitative and quantitative analysis of embryos produced using the two different methods. DST provides a means of assessing the effect of culture conditions on cell number of both embryo compartments (ICM and TE), as well as providing information on the localization of apoptotic nuclei within the blastocyst.

Characterization of the uterine phenotype during the peri-implantation period for LIF-null, MF1 strain mice.

Leukemia inhibitory factor plays a major role in the uterus and in its absence embryos fail to implant. Our knowledge of the targets for LIF and the consequences of its absence is still very incomplete. In this study, we have examined the ultrastructure of the potential implantation site in LIF-null MF1 female mice compared to that of wild type animals. We also compared expression of proteins associated with implantation in luminal epithelium and stroma. Luminal epithelial cells (LE) of null animals failed to develop apical pinopods, had increased glycocalyx, and retained a columnar shape during the peri-implantation period. Stromal cells of LIF-null animals showed no evidence of decidual giant cell formation even by day 6 of pregnancy. A number of proteins normally expressed in decidualizing stroma did not increase in abundance in the LIF-null animals including desmin, tenascin, Cox-2, bone morphogenetic protein (BMP)-2 and -7, and Hoxa-10. In wild type animals, the IL-6 family member Oncostatin M (OSM) was found to be transiently expressed in the luminal epithelium on late day 4 and then in the stroma at the attachment site on days 5-6 of pregnancy, with a similar but not identical pattern to that of Cox-2. In the LIF-null animals, no OSM protein was detected in either LE or stroma adjacent to the embryo, indicating that expression requires uterine LIF in addition to a blastocyst signal. Fucosylated epitopes: the H-type-1 antigen and those recognized by lectins from Ulex europaeus-1 and Tetragonolobus purpureus were enhanced on apical LE on day 4 of pregnancy. H-type-1 antigen remained higher on day 5, and was not reduced even by day 6 in contrast to wild type uterus. These data point to a profound disturbance of normal luminal epithelial and stromal differentiation during early pregnancy in LIF-nulls. On this background, we also obtained less than a Mendelian ratio of null offspring suggesting developmental failure.

Relationship between low-molecular-weight insulin-like growth factor-binding proteins, caspase-3 activity, and oocyte quality.

Bovine follicular atresia is associated with the apoptosis of granulosa cells and the subsequent loss of oocyte competence through the reduction of cellular contact (e.g., gap junctions). Several components of the insulin-like growth factor (IGF) system are thought to affect follicular atresia. Whereas the IGF-binding proteins (IGFBPs) are present in varying quantities throughout follicular development, IGFBP-5 appears to be present only during atresia, in parallel with its regulation in other tissue remodeling systems. However, to our knowledge, no connection has yet been made between atresia, low-molecular-weight IGFBP content, and oocyte quality in the bovine ovary. Caspases are actively involved in ovarian follicular atresia, and apoptosis in antral follicles is caspase-3-dependent. Hence, the aim of the present study was to investigate the use of these factors in the assessment of oocyte quality and developmental potential. Oocytes were aspirated, morphologically classified, and individually matured in vitro. The follicular fluid and granulosa cells of these follicles were analyzed for IGFBP profile and caspase-3 activity, respectively. A significant correlation was found between the presence of low-molecular-weight IGFBPs in bovine follicular fluid and caspase-3 activity of granulosa cells isolated from individual follicles. The highest percentage of development to the blastocyst stage was observed in oocytes from slightly atretic follicles. This group of oocytes contained an equal proportion of oocytes at grades 1-3. These data demonstrate that low-molecular-weight IGFBP profile is a more reliable method than the traditional morphological assessment of oocytes and can be used as an effective marker of developmentally competent oocytes. Importantly, these results have implications for the use of noninvasive follicular fluid markers in the selection of competent oocytes to improve outcomes of in vitro fertilization.

Role of leukemia inhibitor factor (LIF) in decidualisation of murine uterine stromal cells in vitro.

Decidualisation of uterine stromal cells is a prerequisite for implantation of the embryo in mice. Here we have used an in vitro culture system in which stromal cells decidualise as indicated by a number of markers, including an increase in alkaline phosphatase (ALP) activity. The latter was used as a quantitative marker of decidualisation in the presence of low (2%) fetal calf serum. Prostaglandin E(2) (PGE(2)), which is known to induce decidualisation, increased ALP activity, and this effect was blocked in a dose-dependent manner by indomethacin. Leukemia inhibitory factor (LIF) was then examined, but it had no effect on PGE(2) secretion. However, LIF suppressed ALP activity in a dose-dependent manner in the presence of 2% serum, while an inhibitor of LIF that competes for binding to its receptor reversed the effect of LIF and increased ALP activity above the control level. In serum-free cultures, stromal cells differentiated rapidly, and no differences were observed between LIF-treated and untreated cultures. Stromal cells produce LIF during in vitro culture, and this peaked at 48 h. Freshly collected stromal cells from both day-2 and -4 pregnant mice expressed mRNA for the LIF receptor, and the transcript level was higher in cells isolated on day 4. However, no differences were observed in the relative levels of transcripts in cells from day 2 and day 4 after culture, nor were there differences between the LIF-treated cultures and controls. Therefore, in this study, we have shown that LIF suppresses decidualisation of murine uterine stromal cells in the presence of serum, this is not due to the regulation of PGE(2) secretion by stromal cells.

Maintenance of bovine oocytes in meiotic arrest and subsequent development In vitro: A comparative evaluation of antral follicle culture with other methods.

The frequency of development of bovine embryos produced by maturation, fertilization, and culture in vitro is lower than that observed in vivo. One factor that may affect both the frequency of development and the quality of the embryos produced is the developmental competence of the oocyte. In current in vitro production systems, oocyte maturation, characterized by the resumption of meiosis, occurs after oocyte aspiration from the follicle. The developmental competence of individual oocytes may be improved by inducing maturation after culturing under conditions that inhibit the resumption of meiosis. In order to test this hypothesis, a system has been established in which intact antral follicles (3-8 mm in diameter) are cultured in vitro. During this period the oocytes are maintained at the germinal vesicle (GV) stage under the inhibitory effects of the follicle. Culture of intact antral follicles was compared with two other "physiological" methods for the maintenance of GV arrest: oocytes were cultured attached to a small part of the follicle wall or within hemisections of follicles. It was found that 96.8% of oocytes recovered from intact antral follicles-as compared to 24.6% attached to a small part of the follicle wall and 62.7% within hemisections of follicles-were maintained at the GV stage after 24-h culture. The effects on GV arrest and subsequent maturation of the oocytes were evaluated after longer periods of antral follicle culture (2, 4, and 7 days). As the culture period increased, the number of GV-arrested oocytes decreased; the maximum percentage of GV arrest was observed after 24-h culture. The majority of these oocytes matured to metaphase II. A comparison of blastocyst production was made after fertilization and subsequent development of oocytes obtained following follicle culture and of control oocytes aspirated directly from antral follicles. The cleavage rate and percentage of blastocyst production in these two groups were 54.6 +/- 13.9%, 48.4 +/- 8.4% and 68.6 +/- 8.6%, 32.8 +/- 10.8%, respectively. Statistical analysis showed significant differences in both cleavage rate and blastocyst production between these two groups. Total cell numbers in the control group were 144.6 +/- 7.28 and 152.0 +/- 25.8 after follicle culture. It is concluded that culture of intact antral follicles for 24 h is an alternative method for the maintenance of bovine oocytes in meiotic arrest and that these oocytes acquire a greater developmental competence in vitro.