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.

Mechanism of luminal ATP activated chloride secretion in a polarized epithelium.

There are both secretory and absorptive pathways working in tandem to support ionic movement driving fluid secretion across epithelia. The mechanisms exerting control of fluid secretion in the oviduct is yet to be fully determined. This study explored the role of apical or luminal extracellular ATP (ATPe)-stimulated ion transport in an oviduct epithelium model, using the Ussing chamber short-circuit current (Isc) technique. Basal Isc in oviduct epithelium in response to apical ATPe comprises both chloride secretion and sodium absorption and has distinct temporal phases. A rapid transient peak followed by a sustained small increase above baseline. Both phases of the apical ATPe Isc response are sensitive to anion (HCO3-, Cl-) and cation (Na+) replacement. Additionally, the role of apical chloride channels, basolateral potassium channels and intracellular calcium in supporting the peak Isc current was confirmed. The role of ATP breakdown to adenosine resulting in the activation of P2 receptors was supported by examining the effects of non-hydrolyzable forms of ATP. A P2YR2 potency profile of ATP = UTP > ADP was generated for the apical membrane, suggesting the involvement of the P2YR2 subtype of purinoceptor. A P2X potency profile of ATP = 2MeSATP > alpha,beta-meATP > BzATP was also generated for the apical membrane. In conclusion, these results provide strong evidence that purinergic activation of apical P2YR2 promotes chloride secretion and is thus an important factor in fluid formation by the oviduct.

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.

Effect of level of eicosapentaenoic acid on the transcriptional regulation of Δ-9 desaturase using a novel in vitro bovine intramuscular adipocyte cell culture model.

Ruminant fat is often perceived as having a negative impact on human health; however, the composition of the fat is under complex biochemical control and can be improved through strategic manipulation of the animal's diet. There were two major objectives of this study, namely (i) to develop and validate a primary bovine intramuscular adipocyte cell line and (ii) to examine the effect of eicosapentaenoic acid (EPA) on the transcriptional regulation of Δ-9 desaturase in vitro using the novel cell line. Intramuscular adipose tissue was obtained from the Musculus longissimus thoracis of a beef heifer. Mature adipocytes were isolated and cultured, and subsequently harvested and evaluated for lipid accumulation and the expression of genes regulating key functional adipocyte protein markers at passages 10, 20 and 30. Isolated cells were shown to accumulate lipid in culture over time. Fatty acid analysis by gas chromatography was carried out at passage 30. Thirteen fatty acids ranging from tetradecanoic acid (C14:0) to the polyunsaturated fatty acid, docosahexaenoic acid (C22:6), were easily detected and measured. High-quality total RNA was isolated from adipocytes and the expression of peroxisome proliferator-activated receptor-γ, fatty acid synthase, fatty acid-binding protein-4, adipocyte lipid-binding protein, CD36, Δ-9 desaturase, sterol regulatory element-binding protein (SREBP), microsomal triglyceride transfer protein and leptin genes were identified by reverse transcriptase-PCR and sequence analysis. Expression of the negative control, liver-specific hepatocyte nuclear factor-1alpha, was not detected. Adipocytes were subsequently incubated in medium containing 0, 50 or 100 µM EPA for 24 h. Increasing the EPA concentration of the culture media led to a linear increase in adipocyte EPA concentration (P < 0.01). Expression of Δ-9 desaturase mRNA was decreased five- and seven-fold, respectively, following 50 and 100 µM EPA incubation compared to the control. Gene expression of SREBP-1c was decreased by 6- and 18-fold in cells supplemented with 50 and 100 µM EPA, respectively, compared to the control. Regression analysis showed a negative linear relationship between EPA concentration and the gene expression of both Δ-9 desaturase (P < 0.001) and SREBP-1c (P < 0.001), while a significant positive relationship was observed between Δ-9 desaturase and SREBP-1c gene expression (P < 0.001). This is the first report demonstrating that EPA treatment of bovine intramuscular adipocyte cells decreased gene expression of both Δ-9 desaturase and SREBP-1c in vitro. The bovine adipocyte cell line developed here is an important resource for future studies facilitating less-expensive, rapid screening of research hypotheses and circumventing the limitations associated with the use of experimental animals including cost, inter-animal variation, pre-experimental management and ethics.

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.

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.

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.

Modulation of the effects of FSH, androstenedione, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on bovine granulosa cells by GCIF, a growth-inhibitory factor of low molecular mass from bovine follicular fluid.

A granulosa cell-inhibitory factor (GCIF) of low molecular mass from bovine follicular fluid inhibits the proliferation of bovine granulosa cells in vitro and the growth of large follicles in rats in vivo. The present study examined the ability of GCIF to modulate the effects of FSH, epidermal growth factor (EGF), insulin-like growth factor I (IGF-I) and androstenedione on the proliferation of bovine granulosa cells and on aromatase activity in vitro. Granulosa cell proliferation was assayed by counting haemocytometric cells and by measuring the incorporation of [3H]thymidine into acid-precipitable material. Assay of aromatase activity was based on the conversion of [3H]androstenedione to [3H]H2O. FSH, androstenedione, EGF and IGF-I all stimulated (P < 0.01) granulosa cell proliferation; however, the addition of GCIF reduced (P < 0.01) cell proliferation in their presence. In the case of EGF, the addition of GCIF almost abolished the stimulatory response. FSH and IGF-I, but not EGF, stimulated (P < 0.01) aromatase activity of granulosa cells. The stimulatory effect of IGF-I was decreased by GCIF. The inhibitory effects of GCIF indicate that it may play a significant role in regulating the effects of intraovarian growth factors on granulosa cells and the growth of follicles.

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.