Enhancing oxygen delivery to ovarian follicles by three different methods markedly improves growth in serum-containing culture medium.

Invitro ovarian follicle culture systems are routinely used to study folliculogenesis and may provide solutions for infertility. Mouse follicles are typically cultured in standard gas-impermeable culture plates under gas phase oxygen concentrations of 5% or 20% (v/v). There is evidence that these conditions may not provide adequate oxygenation for follicles cultured as non-attached intact units in medium supplemented with serum and high levels of FSH. Three different methods of enhancing follicle oxygenation were investigated in this study: increasing the gas phase oxygen concentration, inverting the culture plates and using gas-permeable culture plates. Follicles cultured under 40% O2 were significantly larger (P P P 2 . These effects were associated with reduced secretion of vascular endothelial growth factor (P P P invivo -matured follicles (~500µm in diameter). Such follicular development is not possible under hypoxic conditions.

Corrigendum to: Hypoxia limits mouse follicle growth in vitro.

Ovarian follicle culture is useful for elucidation of factors involved in the regulation of follicular function. We examined the effects of gas phase oxygen concentration, an oil overlay, serum type and medium supplementation with FSH, insulin-transferrin-selenium (ITS) and I-ascorbic acid on cultured preantral mouse follicle growth in a spherical, non-attached follicle culture system. Follicle growth in 5% oxygen was significantly (P<0.01) inferior to growth in 20% oxygen in terms of follicle diameter. This was likely due to hypoxia, as evidenced by significantly (P<0.05) increased follicle secretion of vascular endothelial growth factor (VEGF), a marker of cell hypoxia. Follicular growth was not (P>0.05) affected by an oil overlay, ITS supplementation or serum type. Culture in medium with 5% mouse serum, 1 IU mL-1 FSH, 25 µgmL-1 l-ascorbic acid and 20% oxygen without an oil overlay supported the growth of follicles to a maximum diameter of 380 µm in 6 days. Compared with mature preovulatory mouse follicles in vivo that often have diameters >500 µm within the same time frame, in vitro-grown follicles clearly exhibit limited growth. Thus, adequate oxygenation is an essential factor in the process of optimising follicle growth.

Hypoxia limits mouse follicle growth in vitro.

Ovarian follicle culture is useful for elucidation of factors involved in the regulation of follicular function. We examined the effects of gas phase oxygen concentration, an oil overlay, serum type and medium supplementation with FSH, insulin-transferrin-selenium (ITS) and l-ascorbic acid on cultured preantral mouse follicle growth in a spherical, non-attached follicle culture system. Follicle growth in 5% oxygen was significantly (P < 0.01) inferior to growth in 20% oxygen in terms of follicle diameter. This was likely due to hypoxia, as evidenced by significantly (P < 0.05) increased follicle secretion of vascular endothelial growth factor (VEGF), a marker of cell hypoxia. Follicular growth was not (P > 0.05) affected by an oil overlay, ITS supplementation or serum type. Culture in medium with 5% mouse serum, 1 IU mL-1 FSH, 25 µg mL-1 l-ascorbic acid and 20% oxygen without an oil overlay supported the growth of follicles to a maximum diameter of 380 µm in 6 days. Compared with mature preovulatory mouse follicles in vivo that often have diameters >500 µm within the same time frame, in vitro-grown follicles clearly exhibit limited growth. Thus, adequate oxygenation is an essential factor in the process of optimising follicle growth.

Inositol transport in mouse embryonic stem cells.

The uptake of myo-inositol by mouse embryonic stem (ES) cells was measured using [2-(3)H]myo-inositol. Uptake of myo-inositol by ES cells occurred in a mainly saturable, sodium-, time- and temperature-dependent manner, which was inhibited by glucose, phloridzin and ouabain. Self inhibition by inositol was much greater than inhibition by glucose indicating that transport was not occurring via a sodium-dependent glucose transporter. Uptake rate was much greater than efflux rate indicating a mainly unidirectional transport mechanism. Estimated kinetics parameters for sodium-dependent inositol uptake were a K m of 65.1 +/- 11.8 micromol L(-1) and a V max of 5.0 +/- 0.59 pmol microg protein(-1) h(-1). Inositol uptake was also sensitive to osmolality; uptake increased in response to incubation in hypertonic medium indicating a possible role for inositol as an osmolyte in ES cells. These characteristics indicate that myo-inositol transport in mouse ES cells occurs by a sodium-dependent myo-inositol transporter protein.

Oxidative phosphorylation and the tricarboxylic acid cycle are essential for normal development of mouse ovarian follicles.

BACKGROUND: Mouse ovarian follicles are typically grown in upright drops of culture medium. Recently we found that culture of follicles at the medium-gas interface in inverted drops markedly improved follicular development, possibly due to improved access of oxygen to the follicle. In this study, we examined the importance of aerobic energy metabolism for follicle development by culturing mouse follicles (198 6 16.5 initial microm diameter, mean 6 SD) in the presence of phosphorylation and tricarboxylic acid (TCA) cycle inhibitors. METHODS: All inhibitors were tested in the inverted system using 100 microl medium drops in 96-well plates; certain inhibitors were also tested in upright drops with or without an oil overlay. RESULTS: The oxidative phosphorylation inhibitor rotenone (0.1, 0.5 and 1 micromol/l) totally abolished follicle growth in the inverted system; cyanide (1 mmol/l) totally abolished growth in the upright with oil system but not in the inverted system (possibly due to loss of cyanide gas due to the absence of an oil overlay). The mitochondrial uncoupler 2,4-dinitrophenol (0.5 and 1 mmol/l) also abolished growth in the inverted system. The TCA cycle inhibitor monofluoroacetate (10 mmol/l), significantly inhibited growth in all three culture systems (P < 0.01) but malonate (10 mmol/l) had no effect. CONCLUSIONS: Aerobic metabolism and an adequate oxygen supply are essential for normal follicular development.

Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct 4 gene expression: effects of lif, serum-free medium, retinoic acid, and dbcAMP.

In this study we examined the interplay between serum, leukemia inhibitory factor (LIF), retinoic acid, and dibutyrl cyclic adenosine monophosphate (dbcAMP) in affecting IOUD2 embryonic stem cell self-renewal and differentiation as assessed by Oct 4 expression, and cell proliferation as measured by total cell protein. Removal of LIF, reduced levels of fetal calf serum (FCS), and addition of retinoic acid all induced embryonic stem cell differentiation as measured by reduced Oct 4 expression. Lower levels of retinoic acid (0.1-10 nM) promoted the formation of epithelial-like cells, whereas higher levels (100-10,000 nM) favored differentiation into fibroblastic-like cells. The effects of dbcAMP varied with the presence or absence of FCS and LIF and the concentration of dbcAMP. In FCS-containing media, a low level of dbcAMP (100 microM) increased self-renewal in the absence of LIF, but it had no effect in its presence. In contrast, at higher concentrations (1,000 microM dbcAMP), regardless of LIF, differentiation was promoted. A similar effect of dbcAMP was seen in the presence of retinoic acid. In media without FCS but with serum replacement supplements, there was no effect of dbcAMP. This study shows that the Oct 4 expression system of IOUD2 cells provides a novel, simple method for quantifying cellular differentiation.

A novel follicle culture system markedly increases follicle volume, cell number and oestradiol secretion.

This study reports a novel, simple method for culture of mouse follicles which results in follicles with cell numbers similar to in vivo fully grown follicles. Using this method, follicles (180-240 microm in diameter) were cultured in a 100 microl inverted drop of medium without oil and compared with culture in upright drops with and without a mineral oil overlay. Follicles, isolated from C57BL/6 x CBA/ca crossbred and MF1 inbred mice, were cultured individually at 37 degrees C in 96-well round-bottomed suspension cell tissue culture plates for 6 days. Follicles grown in the inverted drop culture system reached a markedly higher final diameter (means+/-s.e.m.; 471 +/- 6.0 microm) as compared with the upright with oil (363 +/- 2.7 microm) and without oil (358 +/- 4.0) systems. There was no significant effect of mouse strain on follicle diameter. Follicular secretion of oestradiol and lactate into the medium was measured on days 2, 4 and 6 of culture. Secretion of oestradiol per follicle on day 6 was 2.49 +/- 0.45 ng in the inverted and 0.90 +/- 0.17 ng in the upright without oil system (P < 0.001). Follicular secretion of lactate on a per unit of follicle volume basis remained constant in the inverted system over days 2, 4 and 6 and was less (P < 0.001) than secretion in both the upright with and without oil systems. Follicle cell proliferation was markedly increased in the inverted as compared with the upright with oil system; the increases in cell numbers were significant on day 3 (P < 0.01) and on all subsequent days (P < 0.001). These results are discussed in relation to the supply of oxygen to the follicle in culture.

In situ oviduct and uterine pH in cattle.

Knowledge of oviduct and uterine pH in cattle is lacking mainly because of the difficulty of accessing these reproductive tissues, which for the oviduct at least necessitates anesthesia. Because halothane anesthesia is known to depress respiratory function and thus increase blood CO2 and decrease pH, oviduct and uterine pH was measured both in the presence and absence of halothane. Using short-term anesthesia with thiopentone only, oviduct pH was measured on days 2-4 of the estrous cycle and uterine pH on days 6 and 8; there was no significant effect of day of the cycle but oviduct pH ( 7.60+/-0.010 ) was greater ( P<0.001 ) than uterine pH ( 6.96+/-0.009 ). Oviduct pH was higher ( P<0.001 ) and uterine pH lower ( P<0.001 ) than venous blood pH ( 7.41+/-0.007 ). Using thiopentone/halothane anesthesia, oviduct pH was measured on days 0, 2, 3, 4 and 6, and uterine pH on days 6, 8 and 14; there was no effect of day of cycle but oviduct pH values were generally higher than uterine values and significantly so ( P<0.001 ) on day 6 where direct comparison was possible. To our knowledge these are the first published in situ measurements of oviduct pH in cattle.

A review of in vitro gamete maturation and embryo culture and potential impact on future animal biotechnology.

This review considers the relationship of in vitro gamete maturation and embryo culture to the future development of animal biotechnology. The areas reviewed are oocyte maturation in vitro and embryo culture and their importance for successful in vitro embryo production. The rapidly developing area of spermatogonial cell transplantation and culture is also reviewed. The scientific milestones leading to the development of each area, the problems and prospects for future development and the possible significance of major advances in each area are discussed.

Phospholipase C and protein kinase C involvement in mouse embryonic stem-cell proliferation and apoptosis.

Activation of the phosphatidylinositol (PtdIns) signal transduction system involves stimulation of phospholipase C (PLC) by hormones and other agonists to produce two second messengers, the inositol phosphate, Ins(1,4,5)P3 which releases calcium from intracellular stores, and diacylglycerol which activates protein kinase C (PKC). This study, using activators or inhibitors of PLC and PKC and a calcium ionophore, examined the role of the PtdIns system in mouse embryonic stem (ES) cells. The PLC inhibitor, U-73122, inhibited ES-cell proliferation and also inhibited PLC activation as evidenced by a decrease in inositol phosphate formation in response to fetal calf serum stimulation. The two PKC activators, the diacylglycerol analogue 1,2, dioctanoyl-sn-glycerol (DOG) and the phorbol ester 12-O-tetra-decanoyl phorbol 13-acetate (TPA), increased cell proliferation in a dose-dependent manner, as did the calcium ionophore, ionomycin. However, co-stimulation with either ionomycin and DOG or ionomycin and TPA resulted in a reduced number of cells. The PKC inhibitor, bisindolylmaleimide II (Bis II), significantly decreased the number of ES cells, mainly due to increased apoptosis. The possible feedback effect of PKC on PLC was examined by preincubating ES cells with either the PKC inhibitor Bis II or the activator TPA before stimulation of inositol phosphate production with fetal calf serum; preincubation with Bis II increased inositol phosphate formation whereas preincubation with TPA decreased inositol formation. These results indicate that the PtdIns system is involved in the control of ES-cell proliferation and apoptosis.

Inositol transport in preimplantation rabbit embryos: effects of embryo stage, sodium, osmolality and metabolic inhibitors.

The preimplantation period in the rabbit consists of a 3 day cleavage stage during which the number of cells increases with little change in embryo size, followed by a 3-4 day blastocyst stage during which the inner cell mass, the blastocoel and the trophectodermal layer are formed and the embryo grows rapidly in size and protein content. This study used [3H]inositol to investigate the transport of inositol, an essential component of the phosphatidylinositol signal transduction system, over the 6 days of preimplantation development by rabbit embryos. In the presence of 15 micromol inositol-1 in the incubation medium, there was a small linear increase in inositol uptake from 0.07 pmol per embryo per h at the one-cell stage (day 1) to 0.135 pmol at the late morula (day 3) stage. Inositol uptake increased to 0.58 pmol per embryo per h for early blastocysts (day 4) and 23.7 pmol for late blastocysts (day 6). There was a significant linear relationship between inositol uptake and blastocyst diameter and surface area. Efflux of inositol from early morulae was minimal (about 1.25% of embryo content per h), whereas efflux from mid-blastocysts (day 5) was much greater (about 15.6% of embryo content per h). Efflux of inositol from both early morulae and mid-blastocysts was increased by decreasing the osmolality of the incubation medium. Varying the osmolality had no effect on inositol uptake up to 2 h. Inositol uptake was dependent on sodium in cleavage-stage embryos but independent of sodium in blastocyst stages. In early morulae, inositol uptake was inhibited by glucose and the sodium-dependent hexose transport inhibitor, phloridzin, but not by the facilitated transport inhibitor, phloretin. Inositol uptake in early morulae was saturable; estimates of 0.227 and 0.288 pmol per morula per h for V(max) and 0.045 and 0.038 mmol-1 [corrected] for Km were obtained for sodium-dependent transport in two separate experiments. All of these results are consistent with the hypothesis that transport in cleavage stages occurs via a sodium myo-inositol transporter (SMIT) protein. Uptake in blastocysts was non-saturable. Uptake into blastocysts appeared to take place by a transcellular rather than a paracellular route.

Inositol transport in mouse oocytes and preimplantation embryos: effects of mouse strain, embryo stage, sodium and the hexose transport inhibitor, phloridzin.

The uptake of myo-inositol by mouse oocytes and preimplantation embryos of a crossbred (DBA x C57BL/6) and a purebred outbred strain (MF1) was measured using [2-(3)H]myo-inositol. Uptake in crossbred embryos increased about 15-fold between the one- and two-cell stages and increased again by about sixfold at the blastocyst stage compared with the morula stage. Uptake in purebred embryos increased about 42-fold between the one- and two-cell stages and increased more than threefold at the blastocyst stage compared with the morula stage. In all stages examined, except two-cell crossbred embryos, inositol uptake was, depending on the stage, either largely or partly sodium dependent and could be inhibited by the sodium-dependent hexose transport inhibitor, phloridzin. This is consistent with the hypothesis that transport occurs via a sodium myo-inositol transporter (SMIT) protein. In addition, there was strong evidence that a sodium-independent mechanism of uptake, possibly a channel, was switched on at the two-cell stage coincident with zygotic gene activation which resulted in 141-fold and 71-fold increases in sodium-independent uptake from the one-cell to two-cell stages in crossbred and purebred embryos, respectively. This mechanism was either abolished or drastically downregulated at the blastocyst stage, whereas sodium-dependent uptake was markedly upregulated. In two-cell crossbred embryos, there was a complete abolition of sodium-dependent uptake, again possibly regulated by zygotic gene activation. The hypothesis that the changes in mechanism of inositol uptake at about the two-cell stage are due to zygotic gene activation was supported by the finding that these changes did not occur in parthenogenetic two-cell embryos.

The phosphatidylinositol signalling system in elongating bovine blastocysts; formation of phosphoinositides, inositol phosphates and stimulation by growth factors.

The uptake of myo-inositol and its incorporation into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system by in vivo elongating cattle blastocysts was investigated using [3H]myo-inositol. Uptake was examined in 13-, 14- and 16-day-old blastocysts and was largely sodium-dependent throughout (P<0.001), indicating the presence of a sodium-dependent inositol transporter. Incorporation of inositol into the three phosphoinositides, PtdIns, PtdInsP and PtdInsP2, and the inositol phosphates of the phosphatidylinositol signal transduction system was examined at Days 14 and 16; incorporation into the three phosphoinositides and into the inositol phosphate species, InsP1, InsP2, InsP3 (including the second messenger, Ins(1,4,5)P3) and InsP4 was detected in both blastocyst stages. The effects of the peptide growth factor, epidermal growth factor (EGF), and the lipid growth factors, lysophosphatidic acid (LPA) and platelet activating factor (PAF), on the activity of the phosphatidylinositol signalling system in 14- and 16-day-old blastocysts were examined. All growth factors significantly stimulated phosphatidylinositol signalling activity. Epidermal growth factor was stimulatory (P<0.001) only in 16-day-old blastocysts, whereas LPA and PAF were active in both 14- (P<0.005 for LPA and P<0.001 for PAF) and 16-day-old blastocysts (P<0.001 for LPA and PAF). These results indicate that the phosphatidylinositol signalling system is present in cattle blastocysts at the elongation stage and is responsive to stimulation by growth factors.

Nature of glycosylphosphatidylinositols produced by mouse embryonic stem cells.

Incorporation of [(3)H]inositol into mouse embryonic stem cells of the CCE cell line leads to the labelling of the three common phosphoinositides, phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, and a fourth unknown lipid (lipid X). Incubation with [(3)H]glucosamine results in the labelling of lipid X and at least one other lipid that co-migrates with phosphatidylinositol (lipid Y), indicating that both of these lipids are putative glycosylphosphatidylinositols. In this study, the incorporation of other possible glycosylphosphatidylinositol precursors, ethanolamine, mannose and galactose, into lipids X and Y was examined. Galactose was incorporated into lipids X and Y, and ethanolamine and mannose into lipid Y only. Inhibitors of glycosylphosphatidylinositol biosynthesis pathways, mannosamine and 2-fluoro-2-deoxyglucose, both significantly inhibited ethanolamine incorporation into lipid Y. A high glucose concentration (25 mmol l(-1)) abolished the action of both inhibitors. Phospholipase C treatment of embryonic stem cells that had been labelled in culture with [(3)H]ethanolamine caused a large release of ethanolamine label into the incubation medium and markedly decreased the amount of ethanolamine-labelled lipid Y remaining in the cell membranes. These effects were almost totally abolished by incubation with mannosamine before ethanolamine labelling. These studies strongly indicate that lipid Y is a member of the protein anchor class of glycosylphosphatidylinositol, whereas lipid X is a member of the signal transduction inositol phosphoglycan class of glycosylphosphatidylinositol.

Uptake and incorporation of myo-inositol by bovine preimplantation embryos from two-cell to early blastocyst stages.

The uptake of myo-inositol and its incorporation into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system by in vivo preimplantation cattle embryos was investigated using [(3)H] myo-inositol. Uptake of inositol was examined in two-cell and four-cell embryos (day 2 after insemination), morulae (day 6) and early blastocysts (day 7). Uptake in all stages examined was largely sodium-dependent indicating the presence of a sodium-dependent inositol transporter. Uptake of inositol did not vary significantly from two-cell to early blastocyst stages when expressed either on a per embryo or a per microg of protein basis. Incorporation of inositol into the three phosphoinositides, PtdIns, PtdInsP, and PtdInsP(2), was detectable at all stages examined. In contrast, incorporation of inositol into inositol phosphates was not detected until blastocyst formation at day 7. The second messenger, Ins(1,4,5)P(3), was first detected in day 7 blastocysts.

Immunization of rats and sheep against granulosa cell-inhibitory factor from bovine follicular fluid increases the number of large follicles in rats and the ovulation rate in sheep.

Granulosa cell-inhibitory factor (GCIF), a low molecular weight factor from bovine follicular fluid, inhibits the proliferation of bovine granulosa cells in vitro and the growth of large follicles in rats in vivo. In this study the effects of (1) immunization of rats against GCIF on follicular growth and (2) immunization of sheep against GCIF on ovulation rate were studied. The ability of antiserum from sheep immunized against GCIF to reduce the inhibitory effect of GCIF on bovine granulosa cell proliferation in culture was also examined. Immunization of rats against GCIF increased the number of large follicles (P < 0.001) but decreased the number of small follicles (P < 0.05) per ovary. Ovarian mass (P < 0.05) and uterine wet (P < 0.05) and dry (P < 0.01) masses were increased in immunized rats. Immunization of sheep against GCIF, followed by boosting over two breeding seasons, increased ovulation rate (P < 0.01). Addition of antiserum from sheep immunized against GCIF reduced or abolished the inhibitory effect of GCIF on granulosa cell proliferation (P < 0.01). These data provide further evidence that GCIF has an important role in controlling follicle growth and ovulation in vivo.

Designing and Evaluating Standard-Setting Procedures for Licensure and Certification Tests.

Passing scores for licensure and certification tests are justified by showing that decisions based on the passing score achieve the purposes of the credentialing program while avoiding any serious negative consequences. The standard should be high enough to provide adequate protection for the public, and not so high as to unnecessarily restrict the supply of qualified practitioners or to exclude competent candidates from practicing. This paper begins by examining the intended outcomes of licensure and certification programs and by outlining the interpretive argument that is typically used for written credentialing examinations. Criteria are then developed for evaluating standard-setting methods in terms of how well they serve the goals of protecting the public, maintaining an adequate supply of practitioners, and protecting the rights of candidates. Finally, the criteria are used to evaluate the Angoff method. This analysis identifies two potential sources of bias in the Angoff method, and suggests ways to control these weaknesses.

Peptide growth factors and preimplantation development.

This paper examines some of the problems and questions that must be considered in relation to research on the role of growth factors in preimplantation embryos. It reviews and summarizes the large body of work on gene expression of growth factor receptors and ligands in preimplantation embryos and in oviduct and uterine tissue. It also reviews the literature on the effects of gene knockout in preimplantation embryos and concludes with a review of work on the effects of growth factors on cultured embryos.

Partial purification from bovine follicular fluid of a factor of low molecular mass with inhibitory effects on the proliferation of bovine granulosa cells in vitro and on rat follicular development in vivo.

Bovine follicular fluid was aspirated from follicles of 2-20 mm in diameter, charcoal-treated to remove steroids and then separated into low and high molecular mass fractions. The low molecular mass (< 10 kDa) fraction was purified on a Sephadex G-25 chromatography column with formic acid as the eluent. Seven peaks were isolated and assayed for biological activity in cultures of bovine granulosa cells at concentrations of 10, 100 and 1000 ng ml-1. One peak (peak 4) inhibited (P < 0.001) the proliferation of granulosa cells when measured by cell counting and by [3H]thymidine incorporation (33-37% inhibition). This peak inhibited proliferation of granulosa cells from both small (< 2 mm) and medium (2-10 mm) follicles, but not large (> 10 mm) follicles. The inhibitory effect of peak 4 was not due to a toxic effect on cells. Administration of peak 4 to rats did not affect liver or kidney masses but did decrease uterine (25%, P < 0.01) and ovarian (35%, P < 0.01) masses. Peak 4 also caused a reduction in the number of large follicles (65%, P < 0.01) but increased the number of small follicles (55%, P < 0.01). We have named the inhibitory factor associated with peak 4, granulosa cell-inhibitory factor (GCIF). The results presented suggest that GCIF may be a factor secreted by dominant follicles that inhibits the development of subordinate follicles.