Synthesis of prostaglandins by pig blastocysts cultured in medium containing estradiol or catechol estrogen

Two experiments were conducted to determine the effects of 2-hydroxy-estradiol-17β (2---OH---E₂; 0, 50 and 100 μM) and estradiol-17β (E₂; 0, 25 and 50 μM) on prostaglandin (PG) E and PGF₂α synthesis by day-10 pig blastocysts (day 0 is first day of estrus). Blastocysts were incubated in a modified Krebs-Ringer bicarbonate medium, supplemented with bovine serum albumin (4 mg/ml) and the vitamins and amino acids (essential and nonessential) in Minimum Essential Medium (without phenol red or antibiotics). The incubations were conducted at 39°C for three 2-h periods; the second and third periods included an E₂ or catechol estrogen treatment. Release of PGF₂α into the culture medium decreased (p<0.001) linearly with increasing concentrations of 2---H---E₂ in both periods. Release of PGE was not affected by 2---OH---E₂, therefore 2---OH---E₂ increased (p<0.06) the PGE:PGF₂α. When E₂ was added to the medium, release of PGE was decreased (p<0.01) during the second and third periods. Release of PGF₂α also was decreased (p<0.05) by E₂ during period 2, but E₂ did not alter the PGE:PGF₂α. Content of PGs in blastocysts at recovery was less than 10% of the PGs released in vitro. Therefore, these studies demonstrate effects of both the primary and catechol forms of E₂ on the synthesis of PGE and PGF₂α. Catechol estrogens and E₂ may inhibit PG synthesis and modify the PGE:PGF₂α during the establishment of pregnancy in pigs.     

Synthesis of prostaglandins by pig blastocysts cultured in medium containing estradiol or catechol estrogen.

Two experiments were conducted to determine the effects of 2-hydroxy-estradiol-17 beta (2-OH-E2; 0, 50 and 100 microM) and estradiol-17 beta (E2; 0, 25 and 50 microM) on prostaglandin (PG) E and PGF2 alpha synthesis by day-10 pig blastocysts (day 0 is first day of estrus). Blastocysts were incubated in a modified Krebs-Ringer bicarbonate medium, supplemented with bovine serum albumin (4 mg/ml) and the vitamins and amino acids (essential and nonessential) in Minimum Essential Medium (without phenol red or antibiotics). The incubations were conducted at 39 degrees C for three 2-h periods; the second and third periods included an E2 or catechol estrogen treatment. Release of PGF2 alpha into the culture medium decreased (p less than 0.001) linearly with increasing concentrations of 2-OH-E2 in both periods. Release of PGE was not affected by 2-OH-E2, therefore 2-OH-E2 increased (p less than 0.06) the PGE:PGF2 alpha. When E2 was added to the medium, release of PGE was decreased (p less than 0.01) during the second and third periods. Release of PGF2 alpha also was decreased (p less than 0.05) by E2 during period 2, but E2 did not alter the PGE:PGF2 alpha. Content of PGs in blastocysts at recovery was less than 10% of the PGs released in vitro. Therefore, these studies demonstrate effects of both the primary and catechol forms of E2 on the synthesis of PGE and PGF2 alpha. Catechol estrogens and E2 may inhibit PG synthesis and modify the PGE:PGF2 alpha during the establishment of pregnancy in pigs.     

Absence of direct effects of prostaglandins on rabbit blastocysts in vitro

A total of 27 monkeys (M. Fascicularis) whose control cycle lengths ranged from 28 to 32 days were used in this study. All the treatments described below started either on day 17 or 18 of the cycle. Six monkeys received daily injections of 20 μg estradiol-17β (E₂) for 5 consecutive days. Although a drop in blood progesterone (P) did occur due to this treatment, no shortening of the luteal phase of the cycle was recorded. Seven monkeys received daily injections of 15 mg PGF_2α (prostaglandin-F_2α) for 4 or 5 days. These monkeys also showed a drop in blood P levels; moreover 5 of these monkeys had vaginal bleeding for 2–3 days starting either on day 19 or 20 of the cycle. This bleeding did not appear to be a normal physiological menstrual flow, since all of the monkeys commenced menstrual flow at the expected time. Four monkeys received daily injections of 10 mg P for 3 days. These monkeys also had normal cycle lengths in spite of the treatment. Finally 9 monkeys received daily injections of 20 μg E₂ for 3 days, and starting on the third day of E₂ treatment these monkeys also received injections of 15 mg PGF_2α for 4 or 5 days. Shortened cycle lengths were recorded in 8/9 monkeys in this group. Six monkeys had 22-day cycles, 2 monkeys had 24-day cycles and the remaining monkey had a cycle length of 26 days. Thus 8/9 monkeys had shortened luteal phases due to sequential treatment of E₂ and PGF_2α. The cycle lengths in all the treatment groups were normal subsequent to treatments. These results provide potentially useful information for further studies in the human as a method of contraception.     

Production and secretion of C-19 steroids by rat and guinea pig adrenals

The concentrations of C-19 steroids were measured in guinea pig and rat adrenals before and after castration as well as after stimulation with adrenocorticotropin hormone (ACTH). Characterization of adrenal C-19 steroids was also carried out by isolation with high-performance liquid chromatography and gas chromatography/mass spectrometry (GC/MS). From radioimmunoassay (RIA) data, androstenedione (4-DIONE) and 11 beta hydroxyandrostenedione (11 beta-DIONE) were the major C-19 steroids found in guinea pig adrenals, and castration induced a decrease of 4-DIONE levels only while all other C-19 steroids remained unchanged. In rat adrenals, the major C-19 steroids were 4-DIONE and testosterone, and they were also markedly inhibited after castration. With the exception of 11 beta-DIONE, all other C-19 steroids in circulation were eliminated after castration in both animals species. After ACTH administration in the guinea pig, adrenal 4-DIONE and 11 beta-DIONE levels were markedly stimulated, while an increase of only 11 beta-DIONE was observed in plasma. In the rat, ACTH had a small stimulatory effect on adrenal 52-androstane-3 alpha, 17 beta-diol (3 alpha-DIOL) and plasma 11 beta-DIONE levels. Analysis of guinea pig adrenal steroids by GC/MS confirmed the presence of C-19 steroids in adrenals (namely, 4-DIONE and 11 beta-DIONE) while, in the rat, this could not be confirmed. Our data indicate that production of C-19 steroids occurs in guinea pig adrenals, and 11 beta-DIONE is the major C-19 steroid as well as the only C-19 steroid secreted into the circulation. In the rat, the production of C-19 steroids detected by RIA is not supported by GC/MS data.     

Production of carbon dioxide in vitro by blastocysts from intact and ovariectomized mice.

'Normal' and 'delayed implanting' mouse blastocysts were incubated in non-radioactive culture medium for various lengths of time (preincubation), and then placed in culture medium with (14-C)-glucose for 2 hr. The rate of embryonic CO-2 given off by the blastocysts. Normal embryos were unaffected by the length of preincubation. By contrast, 'delayed implantating' embryos had a low level of CO-2 production with short preincubations (i.e. less than 2 hr), but approached the normal range with longer preincubations (i.e. more than 8 hr) and thus, were 'activated ' in vitro. Incubation of the 'delayed implanting' embryos with actinomycin D prevented the expected increase in CO-2 production, indicating that synthesis of new RNA is necessary for their activation. Preincubation of normal and 'delayed implanting' blastocysts with oestradiol-17 beta and/ or progesterone had no effect on the level of CO-2 production, suggesting that the hormones do not directly stimulate or inhibit carbohydrate metabolism in the embryos.     

Accumulation of oestrone by pig blastocysts and its potential physiological significance for blastocyst development in vitro

Oestrone accumulation of Day-5 pig blastocysts and the potential physiological significance of oestrone and oestradiol-17 beta for blastocyst development were investigated in vitro. After 6 h of in-vitro culture in medium supplemented with 10 nM-[3H]oestrone, the accumulation amounted to 550 +/- 49 d.p.m. (s.e.m.) per 10 blastocysts. The accumulation of [3H]oestrone (or its metabolite(s] was reduced (P less than 0.001) in the presence of a 100-fold excess of unlabelled oestrone or oestradiol-17 beta to 135 +/- 14 d.p.m. or 148 +/- 28 d.p.m. per 10 blastocysts, respectively. The accumulation of [3H]oestrone was not affected in the presence of a 100-fold excess of unlabelled progesterone, testosterone or oestrone sulphate. When blastocysts were post-incubated for 30 or 60 min in [3H]oestrone-free medium, blastocysts retained 74.1 +/- 16.8% and 66.0 +/- 10.4%, respectively of their initial radioactivity. In parallel experiments with [3H]progesterone the respective values were 23.8 +/- 3.0% and 21.7 +/- 2.1%. The presence of the antioestrogen nafoxidine (15 micrograms/ml) in basic culture medium impaired (P less than 0.001) the transformation of morulae to blastocysts (21.5 +/- 8.9%) compared to controls (98.3 +/- 1.7%). The inhibitory effects could be overcome (P less than 0.001) by a supplementation with 1 nM- or 100 nM-oestradiol-17 beta (62.5 +/- 12.8% and 80.0 +/- 6.2% development to blastocysts) but not with 1 nM- or 100 nM-oestrone (30.3 +/- 9.6% and 45.2 +/- 10.5%). Blastocyst expansion was also decreased P less than 0.01) to 61.0 +/- 11.4% of control values in the presence of 15 micrograms nafoxidine ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of prostaglandins in development of porcine blastocysts

Rapid elongation of porcine blastocysts between Days 11 to 12 of pregnancy coincides with an increase in uterine luminal content of prostaglandins. The present study evaluated the effect of two prostaglandin synthesis inhibitors (indomethacin and flunixin meglumine) on elongation of porcine blastocysts from spherical to filamentous forms between Day 11 to 12 of pregnancy. Gilts were hemi-hysterectomized on Day 11 of prenancy. The excised uterine horn was flushed with 0.9% saline and diameter of blastocysts recovered were measured. Immediately following surgery, pregnant gilts were assigned to receive either: 1) vehicle every 4 h, 2) flunixin meglumine (banamine) every 4 h, or 3) indomethacin every 12 h. The remaining uterine horn was removed and flushed after the time of blastocyst elongation estimated for each gilt on basis of blastocyst development in the first horn. Uterine flushings were analyzed for total calcium, protein, acid phosphatase activity, estrone, estradiol-17β and prostaglandin F. Pretreatment blastocyst diameter was similar for all groups and ranged from 1 mm to 20 mm. Treatment of gilts with either banamine or indomethacin effectively inhibited (P<0.001) the increase in uterine luminal content of PGF. Total calcium, estrone and estradiol-17β were not influenced by treatment. Total uterine luminal protein and acid phosphatase activity were reduced (P<0.05) in banamine treated gilts compared to those receiving vehicle or indomethacin treatments. Although total PGF recovered in uterine flushings was reduced during the period of blastocyst elongation, treatment with PGF synthetase inhibitors failed to block rapid elongation of blastocysts from the spherical to filamentous forms.     

Role of prostaglandins in development of porcine blastocysts

Rapid elongation of porcine blastocysts between Days 11 to 12 of pregnancy coincides with an increase in uterine luminal content of prostaglandins. The present study evaluated the effect of two prostaglandin synthesis inhibitors (indomethacin and flunixin meglumine) on elongation of porcine blastocysts from spherical to filamentous forms between Day 11 to 12 of pregnancy. Gilts were hemi-hysterectomized on Day 11 of pregnancy. The excised uterine horn was flushed with 0.9% saline and diameter of blastocysts recovered were measured. Immediately following surgery, pregnant gilts were assigned to receive either: 1) vehicle every 4 h, 2) flunixin meglumine (banamine) every 4 h, or 3) indomethacin every 12 h. The remaining uterine horn was removed and flushed after the time of blastocyst elongation estimated for each gilt on basis of blastocyst development in the first horn. Uterine flushings were analyzed for total calcium, protein, acid phosphatase activity, estrone, estradiol-17 beta and prostaglandin F. Pretreatment blastocyst diameter was similar for all groups and ranged from 1 mm to 20 mm. Treatment of gilts with either banamine or indomethacin effectively inhibited (P less than 0.001) the increase in uterine luminal content of PGF. Total calcium, estrone and estradiol-17 beta were not influenced by treatment. Total uterine luminal protein and acid phosphatase activity were reduced (P less than 0.05) in banamine treated gilts compared to those receiving vehicle or indomethacin treatments. Although total PGF recovered in uterine flushings was reduced during the period of blastocyst elongation, treatment with PGF synthetase inhibitors failed to block rapid elongation of blastocysts from the spherical to filamentous forms.     

In vitro production of prostaglandins E and F by the guinea pig ovarian tissue

The ability of guinea pig ovarian tissue to biosynthesize prostaglandins E and F from endogenous precursors has been investigated in vitro. Estimations of prostaglandins were carried out using a sensitive radioimmuno assay during seven days preceding, and up to one day, following oestrous. Prostaglandins E and F were present in the ovarian tissue throughout the period investigated. Prostaglandin concentrations in samples incubated without enzymic inhibition were significantly higher than in samples incubated after enzymic inhibition with ethanol. This indicates that guinea pig ovarian tissue is able to synthesize prostaglandins from endogenous precursors.     

Relationship between release of plasminogen activator and estrogen by blastocysts and secretion of plasmin inhibitor by uterine endometrium in the pregnant pig

Pig blastocysts isolated between Days 10 and 16 of pregnancy release the protease, plasminogen activator (PA), into the medium in a time-dependent manner when cultured in vitro. Production is biphasic. The initial phase (Days 10-12) coincides with the early elongation stages, while release during the second phase (Days 14-16) occurs during a time at which the DNA content of the blastocysts is increasing markedly. Uterine flushings from these pregnant animals contain the zymogen substrate for PA, plasminogen, presumably as a serum transudate. Plasminogen is present in highest amounts at Day 12. The blastocyst, therefore, has the potential ability to generate the broadly specific protease, plasmin, within the uterine lumen. However, during this same period, the endometrium secretes an inhibitor of plasmin into the uterine lumen. In pregnant animals the amount of plasmin inhibitory activity rose 7-fold between Day 10.5, when the blastocysts were spherical, and Day 12, when they had become filamentous. At Day 12 each uterine horn contained about 3 to 4 mg of plasmin inhibitor. A similar release of inhibitor can be initiated in nonpregnant gilts given a single, intramuscular injection of estradiol valerate on Day 11 of the estrous cycle. It is suggested that the initiation of estrogen production by the elongating blastocyst triggers the release of plasmin inhibitor by the maternal endometrium and that the inhibitor serves to prevent a proteolytic cascade of reactions initiated by blastocyst PA, which might otherwise damage the uterine epithelium.     

Development of pig blastocysts in vitro is altered by serum, bovine serum albumin and amino acids and vitamins

We tested the effects of the amino acids and vitamins in minimum essential medium (MEM) and Eagle's medium (BME) on pig blastocyst development and nuclei number. Embryos were recovered either 5 or 6 d after first detected estrus and were cultured for 96 h in U-bottomed wells (0.2 ml). In Experiment 1, addition of MEM amino acids and vitamins to modified Krebs-Ringer bicarbonate (MKRB) medium containing either bovine serum albumin (BSA, 4 mg/ml) or lamb serum (10%, v/v) resulted in fewer (P<0.001) nuclei and smaller (P<0.05) embryo volumes at the end of culture as compared to embryos cultured in MKRB without MEM-supplements. Addition of MEM-amino acids without glutamine (Experiment II) depressed blastocyst volume and rate of hatching, but glutamine (2 mM) had no effect on embryo development. Dialysis (molecular weight > 12,000 retained) of fetal bovine serum (Experiment III) did not affect blastocyst expansion but reduced (P<0.05) the number of nuclei/blastocyst at the end of the culture. Embryos cultured in MKRB with dialyzed serum and the amino acids and vitamins in BME were smaller (P<0.05) and had fewer (P<0.05) nuclei than embryos cultured in MKRB with dialyzed serum but without the BME-supplements. We conclude that, under our culture conditions, MEM and BME amino acids and vitamins are detrimental to the development of early pig blastocysts and that this effect is not due to glutamine. Also, dialysis of fetal bovine serum removes some component(s) that are important for cell division by pig embryos, but it does not affect blastocyst expansion.     

Immunological and non-immunological synthesis and release of prostaglandins and thromboxanes from isolated guinea pig trachea

Specific radioimmunoassays were used to demonstrate the synthesis by the guinea pig trachea of 6-keto PGF_1α, TxB₂, and PGF_2α in addition to PGE₂. The rank order of both spontaneous and stimulated release was PGE₂ > PGF_2α > 6-keto PGF_1α = TxB₂. Ovalbumin-induced prostanoid release from sensitized tissue was antigen-specific. The release was unlikely to be a secondary consequence of tracheal contraction since incubations with calcium ionophore A23187, at a concentration which produces an equivalent magnitude of contraction of sensitized trachea, did not induce a significant PG or Tx production. In contrast, significantly higher prostanoid synthesis was induced by A23187 in unsensitized than sensitized trachea. Thus sensitization altered the profile of arachidonic acid metabolism evoked by the ionophore.     

Production of transgenic porcine blastocysts by nuclear transfer

In this study the in vitro development of porcine nuclear transfer (NT) embryos was investigated. Transgenic fetal fibroblast cells that were frozen after 5 days of serum starvation were injected immediately after thawing into enucleated metaphase II (MII) oocytes. Reconstructed embryos were activated by incubation in 200 microM thimerosal followed by a 30-min treatment of 8 mM DTT. The embryos were subsequently cultured in NCSU23, supplemented with 4 mg/ml BSA for 7 days. The actual cleavage rate (embryos showing > or =2 nuclei) in 6 replicates was 33% (ranging from 15% to 50%). Three blastocysts with cell numbers of 14, 15, and 18 were obtained. The blastocyst rate was significantly lower for NT embryos as opposed to parthenogenetically activated embryos (1% vs. 5%; P<0.05). The neomycin-resistance gene was amplified by PCR in all three NT embryos, indicating their origin from the injected transgenic fibroblasts. Efforts are now being directed in improvements in the nuclear transfer technology, whereby viable fetuses or offspring can be produced from these NT-embryos.     

Regulation of in vitro progesterone release from caprine luteal tissues by prostaglandins E2 and F2a

Luteal slices obtained from Day-10 cyclic, sexually mature, mixed-breed, superovulated goats were used to study the effects of prostaglandins E(2) and F(2)a (PGE(2) and PGF(2)a) on the release of progesterone. The goats were synchronized for estrus using a single intramuscular injection of 5 mg PGF(2)a given during the mid-luteal phase of the estrous cycle. Multiple follicular growth and superovulation were induced using a treatment regiment of follicle stimulating hormone (FSH) and luteinizing hormone releasing hormone (LHRH) previously standardized in our laboratory (1). The luteal slices were treated with PGE(2) or PGF(2)a at concentrations of 1 and 10 ng/ml each. Untreated luteal slices continued to release significant amounts of progesterone over the entire period of incubation (30 to 360 minutes). There was a progressive increase in progesterone accumulation following treatment with PGE(2) at both concentrations. The mean progesterone values were significantly higher in the PGE(2)-treated groups at all incubation periods than in the controls. Progesterone values at 10 ng/ml were higher (P<0.05) than at 1 ng/ml. Treatment with PGF(2)a decreased (P<0.05) progesterone release at 60 to 360 minutes of incubation compared with that of the corresponding controls for each incubation period. However, there appeared to be no differences (P>0.05) in mean progesterone values between the two concentrations of PGF(2)a. The results of this study showed that PGE(2) enhanced the release of progesterone by caprine luteal tissues, whereas PGF(2)a inhibited its release.     

Development of pig blastocysts in a uterine environment advanced by exogenous oestrogen

Routine embryo transfer techniques were used to establish recipient groups in which blastocysts were either asynchronous (blastocysts 24 h behind recipient uterus) or synchronous with their uterine environment. Oestradiol valerate (5 mg) was administered on Day 11 of the recipient's cycle to stimulate release of uterine secretion in the synchronous gilts (Group SE) and one group (AE) of asynchronous gilts. The gilts in the other asynchronous group (Group AC) were injected with vehicle (sesame oil). Embryos recovered on Day 14 by hysterectomy and flushing were evaluated for morphological development. Oestradiol treatment resulted in a failure of blastocyst development in Group AE gilts only. Recoverable oestradiol in the uterine flushings was increased in gilts in Groups AC and SE which contained elongated blastocysts. Plasmin inhibitor levels were lower in Groups AC and SE while PGF tended to be increased. Acid phosphatase activity was higher and recoverable Ca2+ was lower in Groups AE and SE. Failure of blastocyst development in Group AE is believed to have resulted from a failure to undergo trophoblastic elongation due to premature alteration of the uterine environment at a critical period of blastocyst development or from the presence of an unfavourable uterine environment for blastocyst attachment and development shortly after Day 12.     

Prostaglandin synthesis by day-6 rabbit blastocysts in vitro

Day-6 rabbit blastocysts were recovered from superovulated donor animals, washed in ice-cold Krebs-Ringer-bicarbonate (KRB) buffer, pooled and randomly allocated to polypropylene incubation tubes, usually 10 blastocysts in 1 ml KRB. The blastocysts were ruptured with a dissecting needle and incubated at 37 degrees C for periods of 1-3 h with 10 microCi [3H]arachidonic acid/tube. A control tube without blastocysts was run in each experiment. At the end of the incubation, the samples were acidified, extracted with ethyl acetate, dried down and resuspended in h.p.l.c., using a solvent system for prostaglandins (PGs), was subtracted from each experimental run in the same experiment. The remaining radioactivity constituted 0.14% of the original [3H]arachidonic acid added to each incubation tube. This was considered to have been the result of conversion of the radiolabelled arachidonic acid to prostanoids. In the absence of 10 mM-EDTA no conversion occurred, whereas in its presence peaks of radioactivity co-eluting with [3H]PGF-2 alpha and [3H]PGE-2 were seen. A third peak that eluted was either 15-keto metabolites of these PGs or PGD-2. These 3 peaks were always significantly above background, and usually did not differ from each other. No differences in amount of conversion could be related to incubation time. Addition of indomethacin (100 micrograms/ml) or radioinert arachidonic acid (10 micrograms/ml) inhibited production of [3H]PG, even in the presence of EDTA. Removal of calcium from the incubation medium was per se without effect. Addition of atropine (0.15 mM) or carbachol (0.15 mM) in the presence or absence of EDTA did not change the pattern of conversion of [3H]arachidonic acid to [3H]PGs. These experiments demonstrate that rabbit blastocysts have the capacity for de-novo synthesis of PGs from exogenous substrate, when utilization of endogenous substrate is inhibited. The extent of conversion observed may not be a true reflection of the capacity for conversion of endogenous substrate.