Development and survival of pig blastocysts after oestrogen administration on day 9 or days 9 and 10 of pregnancy

In Exp. 1, administration of 5 mg oestradiol valerate i.m. to pregnant gilts on Days 9 or 9 and 10 advanced the uterine secretion of calcium, protein, and acid phosphatase as demonstrated by levels recovered in the uterine flushings of females unilaterally hysterectomized on Day 11. Upon removal of the remaining uterine horn on Day 12, protein and acid phosphatase increased while Ca2+ decreased in oestradiol-treated gilts as did PGF. In contrast, a 4-fold increase in recoverable Ca2+ occurred from Days 11 to 12 in control gilts. Recoverable oestradiol-17 beta was increased in all 3 groups on Day 12 and plasmin inhibitor concentration increased in oestradiol-treated gilts. Two-dimensional PAGE demonstrated the appearance of a group of very acidic polypeptides in oestradiol-treated gilts. Blastocysts recovered from the second uterine horn had undergone elongation to the filamentous morphology in all 3 groups. In Exp. 2, oestradiol valerate was administered to pregnant gilts on Day 9 or Days 9 and 10 followed by total hysterectomy on Day 16. No differences in recoverable Ca2+ or protein were found, but acid phosphatase was decreased by 75% after oestradiol treatment. Recoverable oestradiol was decreased in oestradiol-treated gilts while PGF and plasmin inhibitor concentrations were unaffected. Compared with the control gilts, blastocysts recovered from oestradiol-treated gilts were fragmented and degenerating on Day 16. PAGE demonstrated greatly intensified staining of the group of acidic polypeptides in oestradiol-treated gilts. These results indicate that oestradiol treatment on Day 9 of pregnancy advances uterine secretory response, but that blastocyst elongation can occur in this uterine environment and in the presence of declining intraluminal Ca2+ levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Length of pseudopregnancy and pattern of uterine protein release as influenced by time and duration of oestrogen administration in the pig

Treatment of gilts with 5 mg oestradiol benzoate on Day 9.5, 11, 12.5, 14, 15.5 or Days 14-16 resulted in an interoestrous interval of about 30 days. Administration of oestradiol benzoate daily from Days 11 to 15 or two periods of treatment on Days 11 and 14 to 16 resulted in prolonging CL function beyond 60 days from the pre-treatment oestrus. Endometrial secretory response to oestrogen stimulation, based on the ability of oestrogen to release calcium and uterine protein into the lumen appears to occur after Day 10 of the oestrous cycle. The results suggest that maintenance of prolonged CL function appears to require two periods of oestrogen stimulation. The first period occurs on Day 11 when the endometrium has become responsive to oestrogen stimulation followed by a second prolonged increase in oestrogen stimulation after Day 14. These findings accord with the normal patterns of oestrogen released by pig blastocysts during early pregnancy.     

Development of OPS vitrified pig blastocysts: effects of size of the collected blastocysts, cryoprotectant concentration used for vitrification and number of blastocysts transferred

Unhatched blastocysts from Large White hyperprolific gilts (n=103) were identified, measured and vitrified using the Open Pulled Straw (OPS) technique to evaluate the effects of the collected blastocyst size and cryoprotectant concentrations used for vitrification, and the number of embryos transferred per recipient. Vitrified/warmed blastocyst viability was estimated in vitro, as the percentage of embryos developing after 72h, and in vivo, on pregnancy Day 30. In the in vitro study, we compared the use of three cryoprotectant concentrations (16.5, 18, or 20% DMSO+16.5, 18, or 20% EG+0.4M sucrose). Survival rates differed significantly between the control (98.3%) and the three cryoprotectant concentrations (67, 62.3, and 57%, respectively). Blastocyst size at vitrification determined the further in vitro development of embryos (26% survival for blastocysts 126-144microm versus 100% for blastocysts >199microm). For the in vivo study, blastocysts were vitrified using cryoprotectant concentrations of 16.5 or 18% DMSO+EG and transferred surgically in groups of 20 or 30 per recipient (n=40). Recipients were slaughtered on pregnancy D30. No significant differences were detected in gestation rates (50-70%) and embryo survival rates (14.7-25%), although survival was higher (P=0.0003) when 20 blastocysts were transferred compared to 30 (24.7% versus 15.5%). Our findings indicate that best results, in terms of subsequent in vivo embryo survival, were achieved after transferring 20 embryos at the blastocyst or expanded blastocyst stage, previously vitrified using cryoprotectant concentrations of 16.5 or 18%.     

Aromatase activity in individual day-11 pig blastocysts

Blastocysts were flushed from both uterine horns of 10 gilts on Day 11 of pregnancy. In these gilts 15.1 +/- 2.3 (mean +/- s.d.) corpora lutea were present and 12.7 +/- 3.1 spherical blastocysts were recovered. In all the gilts variation in blastocyst diameter was observed. Aromatase activity was measurable in 118 of 121 examined blastocysts and varied from 0.005 to 19.330 pmol [1 beta-3H]androstenedione converted into 3H2O in 20 min (mean 1.31). This variation in aromatase activity reflected a difference between and within gilts. Of the total variation between all blastocysts, 67% was due to differences between gilts. A positive exponential relationship existed between blastocyst diameter and aromatase activity, but this relationship was different between gilts (P less than 0.0001). The observed variation in aromatase activity may be caused by differences in germ layer differentiation of the blastocysts.     

Production of steroids and release of prostaglandins by spherical pig blastocysts in vitro

Levels of pregnenolone and progesterone in spherical pig blastocysts (near 4 and 15 microM respectively) exceeded respective levels in histotroph by about 400-fold. When blastocysts were cultured for 5 days in a synthetic medium containing pregnenolone sulfate (1 microM), daily rates of release of pregnenolone, progesterone, androstenedione, testosterone, oestrone and oestradiol were determined to be near 320, 45, 26, 27, 0.8 and 9.2 fmol per blastocyst respectively. Daily outputs of progesterone and testosterone (fmol per blastocyst) diminished (P less than 0.05) to 1.3 and undetectable levels (less than 2) respectively in the presence of Trilostane (94 microM). Increasing the content of pregnenolone sulfate in the culture medium (to 4.5 microM) resulted in higher daily rates of release of pregnenolone and progesterone (to near 1740 and 380 fmol per blastocyst respectively), verifying activity of 3 beta-hydroxy-delta 5-steroid dehydrogenase, and of arylsulfatase, in tissues of intact spherical pig blastocysts. Prostaglandin E2 was the predominant prostaglandin (PG) released by cultured blastocysts (about 1 fmol per blastocyst per hour), hourly rates of release of PGH2 (derived) and PGF2 alpha being near 0.1 and less than 0.06 fmol per blastocyst respectively. The data establish a capacity for spherical pig blastocysts to release a range of steroids and PGs of possible significance to embryonic growth and development in vivo.     

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.     

3H]prostaglandin uptake in vivo by rabbit uterine tissues and blastocysts

Day-6 pregnant rabbits were anesthetized and subjected to a mid-ventral laparotomy. [3H] Prostaglandin F2alpha) (PGF2alpha) [3H]PGE2, [14C]Urea or [14C]Sucrose were instilled into the uterine lumen via the uterotubal junction. The amounts instilled/uterine horn were respectively 3.7 +/- 0.3, 3.5 +/- 0.3, 5.7 +/- 1.3 and 2.7 +/- 1.6 muCi in 20mul of buffer. Animals were killed at 1, 2, 9, 19 or 21 h after radioactive instillation, and the amounts of radioactivity in blastocysts, uterine tissue, peritoneal cavity washings and urine evaluated by liquid scintillation spectrometry. A gradient of radioactivity was observed from the uterotubal junction to the cervical end of the uterus. Large amounts of [3H]PG were found in the injected horn and associated blastocysts with a considerable crossover to the non-injected horn, but little in the associated blastocysts. Much of the blastocysts associated- [3H]PG remained unmetabolized. Large amounts of metabolized [3 H] were found in urine. [14C]Urea was taken up by uterine tissue in the injected horn, but there was little cross over to the non-injected horn. Urea was also found in urine. Much of the [14C]Sucrose remained in the injected horn, and little was recovered from the urine. It was found that at 9 h, but not at 19 h, after [3 H]PG instillation, the PG was localized at the site of the blastocysts in the injected but not in the contralateral horn. Significantly more [3H]PGF2alpha than [3H]PGE2 was localized in this situation. [14C]Urea was not localized at the site of the blastocysts in urea injected horns. (ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Implantation and invasiveness of mouse blastocysts on uterine monolayers

We have utilized monolayers of mouse uterine cells as a substratum for blastocyst implantation in vitro. In this system, blastocysts attach, and trophoblast cells initially grow out in an invasive manner, displacing the cells of the monolayer. A similar phenomenon is observed when any of a number of established cell types constitute the monolayer. After 5 to 6 days in culture, trophoblast cells lose their invasive capacity, although viability is unaffected. These events appear to be analogous to those taking place during implantation in utero or in ectopic sites. After treatment with dextran-norit, progesterone and estrogen can no longer be detected in fetal calf serum by a sensitive radioimmunoassay. Nevertheless, blastocysts can implant normally in medium supplemented with treated serum, indicating the likelihood that, unlike implantation in vivo, progesterone and estrogen are not required for the analogous event in vitro.     

Affinity of uterine luminal proteins for rat blastocysts.

The binding of 125I-labelled rat uterine luminal proteins from Day-5 pregnant rats showed higher binding affinity to blastocysts than did the binding of proteins in uterine fluid from pro-oestrous rats (Day 0), rat serum albumin (RSA) or bovine serum albumin (BSA). Apparently little uptake of proteins into cells by phagocytosis or entry into the blastocoelic cavity occurred since similar results were obtained in the presence of sodium azide or cytochalasin B. Autoradiographic studies showed that the proteins were localized on the outer surface of the blastocyst. The binding was Ca2+-dependent. Denaturation of Day-5 uterine proteins at 80 degrees C reduced the counts to the values obtained with undenatured RSA and Day-0 fluids; this residual binding was considered as non-specific. The binding of labelled Day-5 uterine proteins was substantially reduced in the presence of unlabelled Day-5 proteins but to a lesser extent in the presence of RSA or rat serum. The dissociation of the bound labelled Day-5 uterine proteins occurred most rapidly in the presence of unlabelled Day-5 proteins. However, dissociation occurred within 2 h in the presence of other macromolecules, suggesting that the binding was not strong.     

Estradiol-15 alpha-hydroxylation: a new avenue of estrogen metabolism in peri-implantation pig blastocysts

Pig blastocysts have the capacity to convert estradiol into catechol estrogens. Our present study shows that they also have the capacity to hydroxylate estradiol in cycloaliphatic C-atom 15, and this aliphatic hydroxylation reaction is more predominate than the aromatic hydroxylations. The conversion of [4-14C]estradiol to [4-14C]15 alpha-hydroxyestradiol by mitochondrial-rich/microsomal fractions was examined by isolation of this product using reversed phase high-performance liquid chromatography (HPLC) attached to a radiometric flow detector, and its identification by gas chromatography-mass spectrometry. The enzyme kinetics for estrogen 15 alpha-hydroxylase were performed in the pig blastocyst obtained on Day 13 of pregnancy (Day 0 = first acceptance of the male). The enzyme follows classical Michaelis-Menten kinetics. The apparent Kms for estradiol were 2.47 and 1.85 microM, and the apparent Vmaxs were 0.25 and 0.197 nmol/mg/min in the mitochondrial-rich and microsomal fractions, respectively. The enzyme activity was inhibited by different steroidal compounds and non-steroidal estrogens, as well as by CO, SKF-525A, piperonyl butoxide and antibody to cytochrome P450 reductase. Ontogenesis of the blastocyst's estrogen 15 alpha-hydroxylase follows a similar pattern to that of estrogen-2/4-hydroxylase. Thus, highest activity was observed on Days 12 and 13 and lowest was on Day 15 of pregnancy. Furthermore, the enzyme is abundant primarily in the extraembryonic tissues rather than in the embryo proper. The abundance of the enzyme in the extraembryonic tissues, and its surge at a critical time of pregnancy recognition and just prior to implantation suggest that 15 alpha-hydroxylated estradiol could be involved in these processes.     

Aspartate and glutamate transport in unfertilized pig oocytes and blastocysts

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. Mediated Na⁺-independent transport of L-glutamate can be easily detected in mouse oocytes, but it is nearly undetectable in blastocyst-stage embryos. In contrast, the Na⁺-dependent transport of L-aspartate is not detectable in oocytes, but it is detectable in eight-cell embryos and reaches relatively high levels by the blastocyst stage. It is believed that the amino acid transporters responsible are systems x^?_c and X^?_AG, respectively. Here we report the detection of Na⁺-dependent L-aspartate transport, which increased as pig blastocysts developed, although Na⁺-dependent aspartate transport was not detected in pig oocytes. Mediated Na⁺-independent L-glutamate transport was not detected in pig oocytes, in contrast to the mouse, nor in early or hatched pig blastocysts. Thus, while the developmental regulation of system X^?_AG is similar in both the pig and the mouse, system x^?_c was not detectable in pig oocytes or blastocysts. Elucidation of the molecular mechanisms controlling amino acid transport and other gene expression in early embryos should contribute to an understanding of whether and even why some aspects of developmental regulation of gene expression may need to differ among species. © 1993 Wiley-Liss, Inc.     

Role of the fetal pituitary in cryptorchidism induced by exogenous maternal oestrogen during pregnancy in mice

A single subcutaneous injection of 5 or 1 mg oestradiol given to pregnant female mice on Day 14 of pregnancy resulted in all male offspring being cryptorchid. Pituitary LH content, testicular weights and structure, seminal vesicle weights and the structure of the reproductive tract as a whole were monitored on the day of birth and at 2, 4, 8 and 14 weeks of age. Apart from an initial significant reduction in pituitary LH at the time of birth, no other marked differences were seen between control and treated animals except that all oestrogen-treated males lacked a gubernaculum and the testes were freely mobile within the abdomen. Hypogonadal (hpg) male mice lacking GnRH are cryptorchid but have a normal gubernaculum and their testes develop and descend normally if treated with gonadotrophins. When the mothers of hpg mice were treated with oestradiol the male offspring lacked a gubernaculum. These results indicate that perturbations of the fetal hypothalamic/pituitary axis play no significant part in oestrogen-induced cryptorchidism in mice.