Corpora lutea in submerged organ cultures at low incubation temperatures

Summary Corpora lutea in submerged organ cultures of mouse ovary retained viability when incubated at 24°C. At harvest, whole corpora lutea appeared uniformly viable, and mitotic figures were occasionally seen among the luteal cells. At 24°C, the observed excellent cytological condition of the corpora lutea was in contrast to the necrosis seen when the explants were incubated at 34°C.     

Rapid block of gonadotropin uptake by corpora lutea in vivo induced by prostaglandin F2α

Intravenous administration of ¹²⁵I-hCG to 7–8 day pseudopregnant rats resulted in maximum uptake of radioactivity to corpora lutea 2 hours after treatment. At this time tissue/plasma radioactivity ratios on an equal weight basis were: corpora lutea, 70.2 ± 12.8; ovarian interstitium, 4.6 ± 0.2; kidney, 2.2 ± 0.1. No appreciable uptake was seen by adrenals or liver. Radioactivity in corpora lutea was associated primarily with membranes which sedimented at 2000g and when released by heat it was more than 63% bound to luteal LH receptor preparation in vitro. Radioactivity in renal tissue was associated primarily with the 100,000g supernatant fraction and was bound less than 1% to luteal LH receptors in vitro.PGF₂α significantly reduced uptake (p<.001) of ¹²⁵I-hCG by corpora lutea within 30 minutes (?63%) as well as at 1 (?64%), 2 (?75%), 4 (?68%) and 24 hours (?85%). No clear effect of PGF₂α on uptake of ¹²⁵I-hCG by ovarian interstitial tissue was seen. Plasma progesterone was significantly decreased (p<.001) within 30 minutes (?47%; p<.01) after PGF₂α treatment and also at 1 (?65%), 2 (?82%), 4 (?68%) and 24 hours (?92%). Two hours after PGF₂α treatment the content of progesterone in corpora lutea was depressed (?46%; p<.001). It is suggested that the rapid inhibition of luteal progesterone production induced by PGF₂α in vivo occurs through a block in gonadotropin uptake by corpora lutea.     

Contrasting effects of oestradiol-17 beta and human chorionic gonadotrophin on steroidogenesis in the rabbit corpus luteum

On Day 10 of pseudopregnancy, rabbits were given an i.v. injection of hCG (10-20 i.u.) that was sufficient to cause new ovulations and the loss of follicular oestradiol secretion. There was an immediate 3-4-fold rise in serum progesterone which returned to near prestimulation values (approximately 27 ng/ml) within 12 h in the presence of an implant containing oestradiol-17 beta. In the absence of oestradiol, serum progesterone continued to decline to reach low values (approximately 4 ng/ml) within 24 h and the original corpora lutea subsequently regressed. The administration of oestradiol 24 h after injection of hCG, when progesterone secretion was low, arrested any further decline in progesterone and then restored serum progesterone to normal values. This steroidogenic effect of oestradiol in vivo was a function of enhanced luteal steroidogenesis; corpora lutea removed and incubated for 12 h produced progesterone at high, linear rates, whereas the corpora lutea from animals that did not receive oestradiol produced low or insignificant quantities of progesterone in vitro. We conclude that hCG at these doses is compatible with continued responsiveness of the corpora lutea to oestrogen and that hCG produces its luteolytic effect primarily by ovulating follicles, thus stopping the secretion of the luteotrophic hormone, oestradiol.     

Effects of incubation temperatures on sexual differentiation in the turtle,Chelydra serpentina

Eggs of the common snapping turtle, Chelydra serpentina, were incubated at constant temperatures ranging from 20°C to 30°C, At hatching, the oviducts were absent or incomplete in males; the testes were differentiated. In females at hatching, the oviduct was intact hut in some cases the gonad retained bisexual characteristics. Three months after hatching, the ovary was differentiated and contained follicles. Eggs incubated at 20°C and at 30°C developed into females in 100% of the cases. At 26°C, 99% of the individuals were males; at 24°C, 100% were males. More males than females developed at incubation temperatures of 22°C and 28°C.     

Inhibition of induction of 20 alpha-hydroxysteroid dehydrogenase activity in rat corpora lutea in vitro by the progesterone antagonist RU486.

To determine if the antiprogestagen RU486 has a direct effect on luteal progesterone secretion, whole corpora lutea or dispersed luteal cells were incubated in the presence of RU486. Whole corpora lutea, isolated from rats at day 5 of pseudopregnancy, were incubated individually in hormone-free medium. The concentrations of progesterone and 20 alpha-dihydroprogesterone in the medium plus corpus luteum was measured before and after 24 h of incubation. In the absence of RU486 the concentration of 20 alpha-dihydro-progesterone increased, while that of progesterone remained unchanged. In the presence of RU486 (230 microM) the concentration of both progesterone and 20 alpha-dihydro-progesterone was increased. Dispersed luteal cells were incubated for 24 h in the presence of various amounts of RU486. In the absence and in the presence of 0.2 and 2.3 microM RU486 a high ratio between 20 alpha-dihydro-progesterone and progesterone was found, while in the presence of 23 microM RU486 the concentrations of progesterone and 20 alpha-dihydro-progesterone were equal. 20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSD) activity measured in luteal homogenates started to increase between 6 and 12 h of incubation. This increase could be prevented after incubation of the corpora lutea in the presence of 23 or 230 microM RU486 for 24 hrs. It is concluded that the progesterone antagonist RU486 can have a direct effect on luteal progesterone production. RU486 prevents the increase in 20 alpha-HSD activity that normally occurs during in vitro incubation. However, since these effects in vitro can only be obtained with high concentrations of RU486, it is unlikely that this antiluteolytic effect plays a role after injection of RU486 in vivo.     

Cyclic AMP formation of isolated human corpora lutea in response to HCG - interference by PGF2 alpha.

Human corpora lutea of defined ages were excised at operation, cut into pieces and incubated in the presence of HCG, PGF2 alpha and PGE2 alone or in combination. Following incubation cAMP formation in tissue and medium was determined. HCG-stimulated tissue cAMP content was most pronounced at a corpus luteum age of 7-10 days after ovulation. This stimulation was antagonized by PGF2 alpha in corpora lutea older than 6 days. PGE2 stimulated cAMP formation per se and this effect was more pronounced when HCG and PGE2 were combined. A possible role for PGF2 alpha as a luteolytic substance in the human is suggested.     

Invitro responsiveness of hamster corpora lutea undergoing luteolysis to luteinizing hormone

Corpora lutea removed from pregnant hamster deprived of endogenous luteinizing hormone for varying periods were compared for their responsiveness to externally added luteinizing hormone. The corpora lutea removed on the 8th day of pregnancy exhibited a dose-dependent increase in progesterone production in response to added luteinizing hormone upto a concentration of 2.5 Μg/ml. The total progesterone synthesised by the corpora lutea decreased with increase in the duration ofin vivo luteinizing hormone deprivation. However, the hormone deprivation had to be for a minimum period of 24 h before a marked reduction in thein vitro responsiveness could be seen. Neutralisation of endogenous luteinizing hormone increased the luteal cholesterol ester concentration, whilein vitro incubation of such tissue with luteinizing hormone resulted in a marked reduction in cholesterol ester levels. Corpora lutea removed from hamsters on day 8, 15 and 16 of pregnancy when compared for their responsivenessin vitro to added luteinizing hormone showed that the luteal tissue of day 8 produced more progesterone relative to those of day 15/16. In contrast, depletion of free and esterified cholesterol increased with the increase in age of corpora lutea (from 15% on day 8 to 67% on day 16).     

Regulation of the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the bovine ovary in vivo and in vitro

In order to investigate the pattern of ovarian cholesterol biosynthesis during the bovine estrous cycle, tissue concentrations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the synthesis of cholesterol, were determined by immunoblot techniques. Medium-sized (9-11 mm) and large (14-18 mm) follicles, after removal of follicular fluid by centrifugation, and corpora lutea from the early, early-mid, late-mid, and late stages of the luteal phase were used (n = 5 per group). The specific content (per microgram of tissue homogenate protein) and total content of HMG-CoA reductase in medium-sized and large follicles were substantially lower than those of corpora lutea of the early-mid and late-mid luteal phase. The specific content was elevated in a number of the corpora lutea from the early luteal phase and was low in regressing corpora lutea. Thus during the midluteal phase, when steroid hormone production is elevated, the total and specific contents of HMG-CoA reductase are also elevated. To investigate the mechanisms whereby the levels of HMG-CoA reductase are regulated, primary monolayer cultures of bovine luteal cells (early-mid and late-mid luteal phase) were used. Cells were cultured for 24 h in Dulbecco's modified Eagle's medium containing lipoprotein-poor fetal calf serum (2% vol/vol). At this concentration there was no stimulation of the production of progesterone above that seen with no addition of serum. Under these conditions the total and specific contents, and the synthesis, of HMG-CoA reductase were stimulated by treatment with (Bu)2cAMP (1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in porcine,ovine, bovine and equine blood progesterone concentrations between collection and centrifugation

The aim of this study was to determine if different methods of handling porcine, ovine, bovine and equine blood between collection and centrifugation influence measurable progesterone levels. A 2 × 2 × 5 factorial experiment was conducted for each species with heparin (with or without), temperature of incubation (4 and 22°C) and time of incubation (0, 6, 12, 24 and 48 h) as the main effects. Following centrifugation, plasma and serum samples were stored at ?20°C until progesterone concentrations were determined by radioimmunoassay. Method of handling porcine and equine blood between collection and centrifugation did not affect the levels of progesterone. However, heparinized blood held at 4°C resulted in the most consistent levels of progesterone over time. Progesterone levels were fairly consistent across time in the ovine blood by all methods of handling except heparinized blood incubated at 22°C. By 24 h after collection, plasma progesterone concentrations decreased by 50% for the ovine blood incubated at 22°C with heparin. Decreases were detected by all the methods of handling the bovine blood between collection and centrifugation. The rate of decline, however, was considerably faster for blood held at 22°C than blood held at 4°C. At 12–48 h after collection, the concentrations of progesterone averaged only 5% of the time 0 sample for blood incubated at 22°C. In contrast, at least 30% of the progesterone values in the time 0 sample were detected between 12 and 48 h of incubation for the blood held at 4°C.     

Temperature linked degenerative tissue rots in Capsicum annuum (L.) varieties diseased with Phytophthora capsici

Five-week-old seedlings of Capsicum annuum variety SAMPEP 4, Californian Wonder and Ex Dandamasa drenched with 15,000 infectious units per ml of Phytophthora capsici were incubated at 5°C, 20°C, 30°C and 35°C in alternating light–dark cool cycle Gallenkamp incubators and monitored for root rot development. Each host–pathogen system was replicated five times. Successful disease development was contingent on been incubated at ambient temperature for not less than 3.5 ± 0.5 h. Depending on variety, degenerate tissue rots were aggravated ≤2–3 days after a preconditioning temperature treatment for 24 h possibly linked to cell wall constitution, composition and permeability. Lesion development on stem heightened (27.8%) when incubated at temperatures above 20°C. Ten days after treatment, plant mortality and disease severity were not affected significantly by post-inoculation temperature.     

The development of Puccinia hordei on barley cv. Zephyr

Germination of uredospores of Puccinia hordei was similar on cover-slips and on the first leaves of barley seedlings (cv. Zephyr) at 100 % r.h. over the range 5–25 °C, being greatest at 20 °C. At 15, 20 and 25 °C maximum germination was attained in 6 h. No uredospores germinated on coverslips in humidities below saturation. The numbers of pustules which subsequently developed on plants incubated at 5, 10, 15 or 18 °C and 100 % r.h. for varying periods up to 24 h, were directly related to rise in temperature and length of incubation. The time from inoculation to eruption of pustules (generation time) was 6 days at 25 °C, 8 days at 20 °C, 10 days at 15 °C, 15 days at 10 °C and 60 days at 5 °C. Pustule production on inoculated plants which had been kept at 5 °C was rapidly accelerated when they were transferred to 20 °C. Data obtained at constant temperatures were used to predict generation times of the fungus in the field. The productivity of pustules, determined as weight of uredospores, was examined at 10, 15 and 20 °C. Significantly more spores were produced at 15 than at 10 °C and most were produced at 20 °C. The results are discussed in relation to those obtained by other workers and to the development of brown rust in the field.     

Effect of prostaglandin F2 alpha on release of progesterone and leukotriene B-4 by cells from corpora lutea of mares.

Corpora lutea were recovered from mares either 4 to 5 days or 12 to 13 days after ovulation. Mixed populations of luteal cells were prepared by collagenase digestion and were incubated for 24 h in the presence or absence of prostaglandin (PG) F-2 alpha (250 ng/ml). PGF-2 alpha significantly (P = 0.03) reduced progesterone secretion by cells from late diestrous corpora lutea and tended (P = 0.06) to reduce secretion by early diestrous cells. PGF-2 alpha had no significant effect on leukotriene B-4 (LTB-4) production by cells from early diestrous corpora lutea, but significantly (P = 0.03) increased LTB-4 production by late diestrous luteal cells. It seems possible that LTB-4 could play a role as an intermediary in the action of PGF-2 alpha in luteolysis in the mare.     

The effect of the weight of the seedling-derived tuber on subsequent clonal generations in a potato breeding programme

Cultures of Polymyxa graminis were maintained in roots of barley plants grown in sand at different temperatures using Wisconsin soil temperature tanks. At 17 – 20°C, the minimum time from inoculation with cystosori to the production of zoospores from the inoculated roots was 2 – 3 wk. At 11 – 20°C many zoospores were produced but the incubation period was longer at the lower temperatures. Above 20°C little fungal development occurred. The duration of motility of zoospores ranged from c. 1 h to > 24 h. Bovine serum albumen (BSA) prolonged motility but glycine and glucose had no effect or, at higher concentrations, were toxic. Zoospores were rapidly immobilised by zinc ions in solution at or above 10μg/ml. In some experiments BSA added to the zoospore suspension greatly increased transmission of barley yellow mosaic virus (BaYMV) while glucose, glycine and ovalbumen decreased it. When seedlings were incubated with zoospore suspensions for 24 h at different temperatures, BaYMV transmission was high (> 60%) at 10, 15 and 20°C but there was little at 5 or 25°C. In experiments to determine the time taken for zoospore penetration, seedlings were incubated in suspension for different periods of time and then rinsed in zinc sulphate solution to kill free zoospores. Between 3 and 3·5 h was needed for zoospores to establish infection. Transmission occurred equally to plants of various ages between 3 days and 7·5 wk.     

Temperature and genotype affect asparagus somatic embryogenesis

Summary Calluses from five asparagus genotypes G14, G32, G171, G203, and G447 and hybrid Jersey Giant (JG) were incubated at three temperature regimes (24, 27, and 30°C) on embryo induction medium to assess somatic embryo development and conversion to plantlets. The calluses from three genotypes (G14, G32, and G171) were not responsive, failing to produce somatic embryos at any temperature regime. For three responsive genotypes (G203, G447, and JG), both incubation temperature and genotype significantly affected the numbers of somatic embryos produced. The calluses produced the most and the least numbers of total, bipolar, and globular embryos when incubated at 27°C and 24°C, respectively. When incubated at 27°C, G203 produced the highest numbers of total and globular embryos, 178 g⁻¹ callus and 142 g⁻¹ callus, respectively while G447 produced the highest number of bipolar embryos, 77 g⁻¹ callus. Incubation temperature but not genotype significantly affected the conversion of somatic embryos to plantlets. The somatic embryos recovered from the three responsive genotypes incubated at 27°C also converted to plantlets at the highest frequencies, 60–63% of the bipolar embryos and 42–43% of the globular embryos converted to plantlets, while the somatic embryos recovered from the calluses incubated at 24°C converted to plantlets at the lowest frequencies.     

An autoradiographic study of rabbit ovarian surface epithelium before and after ovulation

Morphologic studies suggest that the proliferative activity of the ovarian surface epithelium (OSE) may vary during the reproductive life cycle. To further investigate this phenomenon, rabbit ovaries obtained before and after induction of ovulation with human chorionic gonadotropin (hCG) were incubated in medium containing 3H-methylthymidine and processed for autoradiography. Before ovulation, the labeling index (LI) of OSE cells varied from 0.04% to 0.22%. Twelve hours after hCG, the maximal LI (9.02 +/- 0.38%) was seen in OSE cells adjacent to the ovulatory stigma. The LI remained elevated at Days 1 and 5 post-hCG in OSE cells overlying corpora lutea. At Day 12, numerous papillary processes were observed at the apex of each corpus luteum. The maximal LI (16.44 +/- 1.31%) had now shifted to the OSE cells covering these processes. Eighteen days after hCG stimulation, the LI of OSE cells near the corpora lutea had returned to preovulatory levels. A slight increase in the LI of OSE cells not associated with ovulatory sites was also observed after ovulation. This study shows that a significant fraction of OSE cells undergoes DNA synthesis throughout most of the postovulatory period.     

Cyclic AMP formation of isolated human corpora lutea in response to HCG — Interference by PGF2α

Human corpora lutea of defined ages were excise at operation cut into pieces and incubated in the presence of HCG, PGF_2α and PGE₂ alone or in combination. Following incubation cAMP formation in tissue and medium was determined. HCG-stimulated tissue cAMP content was most pronounced at a corpus luteum age of 7–10 days after ovulation. This stimulation was antagonized by PGF_2α in corpora lutea older than 6 days. PGE₂ stimulated cAMP formation $\text{per se}$ and this effect was more pronounced when HCG and PGE₂ were combined. A possible role for PGF_2α as a luteolytic substance in the human is suggested.     

Effect of high temperature on the development of nuclear polyhedrosis virus in the silkworm,Bombyx mori

Effect of a high temperature on the development of nuclear polyhedrosis and nuclear polyhedrosis virus (NPV) was studied employing pupae and isolated pupal abdomens of the silkworm, Bombyx mori. It was shown that pupae inoculated with an NPV and incubated at 35°C survived longer than those incubated at 25°C. At lower dosages of virus, pupae at 35°C escaped death from NPV. When inoculated pupae were incubated at 35°C for varying periods and then transferred to 25°C, the longer the pupae had been kept at 35°C the longer they survived. In contrast, when inoculated pupae were transferred from 25° to 35°C, the longer the pupae had been kept at 25°C the sooner after inoculation they died. Essentially the same results were obtained in isolated abdomens which were in an arrested state of development, excluding the possibility that observed thermal inhibition of viral diseases is dependent upon the altered developmental processes at high temperatures. Virus titration experiments showed that, under experimental conditions utilized, no detectable accumulation of infectious NPV was present in abdomens inoculated with an NPV and incubated at 35°C. When inoculated abdomens were shifted up from 25° to 35°C at 3 days postinoculation, NPV accumulation was inhibited almost immediately, and when inoculated abdomens were shifted down from 35° to 25°C, infectious NPV started to accumulate as early as 1 day after the shift. It was also shown that the pattern of infectious NPV accumulation and that of nucleic acid increase in infected abdomens gave a rough correlation. These results indicate that the thermal inhibition of viral diseases is attributed, at least in part, to the restricted accumulation of infectious progeny and suggest that the virus replication mechanism itself is more sensitive to high temperatures than that related to other events necessary for viral replication to be initiated.     

Reversal of embryonic diapause in the Australian plague locust, Chortoicetes terminifera (Walker), by temperatures above the development threshold

Diapause was induced in embryos of Chortoicetes terminifera (Walker) by transferring adults from an L:D 15:9 regime to an L:D 12.5:11.5 regime. When incubated at 20°C all eggs in all pods entered and remained in diapause but when incubated at 26, 32 and 38°C a proportion of eggs in some pods did not. Pods incubated at 32°C for up to 6 days, when diapause intervenes and then transferred to 20°C gave the same result as pods incubated at 20°C throughout development. All eggs entered and remained in diapause. If the period at 32°C was extended to 8 days, the proportion remaining in diapause was not significantly different from that found when pods were incubated at 32°C throughout development. In eggs which broke diapause at 32°C there was a pause or slowing down of development for about 2 or 3 days around the stage at which diapause intervenes.     

Glutamine synthetase in cultured whole retinas from the embryonic chick: Enhancement of basal and induced activity by 4 °C storage

Storage of whole retinas from the embryonic chick for 24 h at 4 °C resulted in increased basal levels of glutamine synthetase (GS) during subsequent incubation at 37 °C in the absence of cortisol. GS levels in these retinas maintained initially at 4 °C (CS), in many cases, exceeded GS levels in cortisol-induced whole retinas incubated solely at 37 °C. The increase in basal GS activity is seen within 48 h of the transfer of the retinas from 4 to 37 °C. If cortisol (0.001 μg/ml = 2.8 nm or 0.01 μg/ml = 28 nm) is added during the last 24 h of culture to CS retinas subsequently transferred to 37 °C, levels of GS are attained that are higher than those in the corresponding retinas cultured continually at 37 °C. However, the activity ratios (GS specific activity in cortisol-treated retinas/GS specific activity in retinas not exposed to cortisol) are similar for CS retinas and those maintained at 37 °C throughout. Monolayers of retinal cells display similar basal and cortisol-induced levels of GS independent of treatment. Retinal monolayers maintained at 4 °C for 24 h and subsequently incubated at 37 °C do not exhibit increases in either basal or cortisol-induced levels of GS over those in monolayers maintained at 37 °C throughout. The CS-promoted increase in the basal and cortisol-induced GS activity of whole retinas is eliminated by enzymatic dispersion of the retina just prior to 37 °C culture of the cells as monolayers. Both basal and cortisol-induced GS levels in the latter monolayers resemble those in retinal cells kept as monolayers throughout.     

Effect of temperature on recombinant protein expression in Semliki Forest virus infected mammalian cell lines growing in serum- free suspension cultures

The firefly luciferase gene was introduced into the Semliki Forest virus (SFV) vector and high titer recombinant SFV particles generated. The broad host range of SFV allowed efficient infection and high level expression of four mammalian cell lines growing in serum-free suspension cultures. The incubation temperature had dramatic effects on the level and duration of recombinant protein expression. For example, the luciferase activity was significantly higher in the rodent BHK and CHO cell lines incubated at 33 °C compared to 37 °C when harvested 19 h post-infection. At 33 °C the specific expression levels increased 10–20 fold during prolongation of the post-infection time up to 50 h. In contrast, a significant decrease in luciferase activity was observed from 26 h post-infection for cell cultures incubated at 37 °C. Only a slight temperature effect on luciferase expression was seen in the human cell line HEK293 and no effect was observed for the subclone293(EBNA). This revised version was published online in August 2006 with corrections to the Cover Date.