Blastocyst Size Research Articles

Stimulation of protein synthesis and expansion of pig blastocysts by insulin in vitro.

Present evidence indicates that insulin may act as a growth factor during preimplantation development. This hypothesis has been tested on pig blastocysts by determining the effect of insulin on protein synthesis and blastocyst expansion over 24 h. Blastocysts were collected from superovulated gilts or sows on Day 5 or 6 and incubated overnight in a modified BMOC2 medium. Those that were cultured with 1.7 nM insulin had 14% larger radii, and were 36% more active in their incorporation of [3H]leucine (protein synthesis) than those that had been cultured in non-supplemented medium. There was a significant linear correlation between the rate of protein synthesis and the radius of blastocysts when all blastocysts and only those cultured with insulin were examined, but the correlation for the blastocysts in non-supplemented medium was just outside statistical significance. The regression coefficient for the insulin-treated blastocysts was 132% of that for blastocysts cultured in unsupplemented medium; this suggests that insulin increased the size of blastocysts and the rate of protein synthesis per unit size. The results indicate that pig blastocysts respond to physiological levels of insulin in similar fashion to those of mice and cattle, supporting the hypothesis that insulin may act as a general embryonic growth factor. Because of the cross reaction between the insulin receptor and the ligands, insulin and insulin-like growth factor 1 (IGF-1), the results also suggest that IGF-1, reported to be present in pig uterine fluid, could be involved in this stimulation in utero.

Patterns of plasma progesterone, androgen and oestrogen concentrations and in-vitro ovarian steroidogenesis during embryonic diapause and implantation in the mink (Mustela vison).

Peripheral plasma progesterone concentrations exhibited an increase 10 days before implantation, coinciding with the resumption of blastocyst growth and with a decrease in plasma androgen values (DHA, androstenedione, testosterone). No definite pattern of oestrone was observed and oestradiol concentrations remained undetectable. The production of steroids by dispersed luteal cells showed that the growth of the corpora lutea paralleled that of blastocysts and resulted in hypertrophy followed by hyperplasia of the luteal cell. The production of progesterone in the medium increased with blastocyst size up to implantation; it was enhanced by mink charcoal-treated serum, but prolactin, LH, FSH or a combination of these hormones did not affect the progesterone production, whatever the stage of diapause. DHA and androstenedione secretion increased in the two last stages of blastocyst growth and was enhanced by LH. The conversion of androstenedione and testosterone into oestrone and oestradiol was observed at all stages of embryonic diapause, indicating that corpora lutea contain aromatase activity even at an early stage. The secretion of oestrone was higher than that of oestradiol. The non-luteal tissue contributed up to 50% of the steroid production; while progesterone and androgen production remained constant, that of oestradiol decreased at the end of the delay period. These results indicated a change in the size and the secretory capacity of the luteal cell related to blastocyst development and implantation. Although progesterone was the main product of the corpora lutea, androgens and oestrogens were also secreted.

Loss of polar trophoblast during differentiation of the blastocyst of the horse.

Twelve blastocysts, collected 7-12 days after ovulation (Day 0), were examined by light and electron microscopy to investigate the nature of the relationship of the polar trophoblast (Rauber's layer) to the inner cell mass. On Day 7, the polar trophoblast was intact and formed a flattened layer overlying the epiblast cells of the inner cell mass. As blastocysts enlarged to greater than 1 mm in diameter, small discontinuities appeared in the polar trophoblast, where epiblast cells intruded onto the surface. At this time, trophoblast cells adhered closely to adjacent and underlying epiblast cells, forming an irregular layer of cells capping the epiblast. With continued increase in blastocyst size, polar trophoblast cells became isolated but maintained their characteristic apical endocytic structures. By Days 10-12, the scattered trophoblast cells showed evidence of deterioration, and vacuoles containing cell debris were common within the epiblast. It is suggested that polar trophoblast cells become scattered, rather than withdrawing as a unit, because they become more adherent to subjacent epiblast cells than to adjacent trophoblast cells. It is further suggested that most of the isolated cells are eventually phagocytosed by epiblast cells.

Establishment of Pregnancy in the Pig: I. Interrelationships Between Preimplantation Development of the Pig Blastocyst and Uterine Endometrial Secretions12

Pregnancy recognition in pigs occurs between Days 11 and 12 when blastocysts undergo transformation from the spherical to filainentous form. This study evaluated the relationship between blastocyst development and total calcium (Ca), estrone (E1), estradiol (E2), estriol (E3), E1 sulfate (E1S), E2S, E3S, prostaglandin F (PGF), PGE2, protein (Pr) and acid phosphatase (AP) activity in uterine flushings obtained from pregnant gilts between Days 10 and 14. Data from pregnant gilts were compared to those obtained from uterine flushings collected between Days 10 and 14 of the estrous cycle. Surface and ultrastructural changes in the endometrium associated with blastocyst development were evaluated by scanning (SEM) and transmission (TEM) electron microscopy. Pregnant uterine flushings were analyzed relative to average size of blastocysts recovered: 5 mm spherical, 5–8 mm spherical, 9–50 mm tubular, and >50 mm filamentous Day 12 and Day 14. Nonpregnant gilt uterine flushings were analyzed by Day of the estrous cycle (10.5, 11, 11.5, 12 and 14). Total E2 content increased almost 4-fold in pregnant uterine flushings containing tubular blastocysts (2.3 ng) as compared with flushings with spherical blastocysts (0.6 ng) and continued to increase as the blastocysts became filamentous (4.4 ng) but had declined by Day 14 (0.4 ng). The pattern of change for total recoverable E1 and E2 was similar to that for E2 and the highest values were obtained in uterine flushings with filamentous blastocysts. A concomitant increase in E1S and E2S content also was detected in flushings with tubular and Day 12 filamentous blastocysts. Total Ca, Pr, AP, PGF, and PGE2 content increased in association with increased E2 in flushings. The increase in Pr, AP, PGF and PGE2 content in pregnant flushings continued to Day 14. However, Ca content had declined by Day 14 (0.1 mg) after a transient increase to 1.5 mg in flushings that contained tubular blastocysts (Days 11 and 12). Comparable changes in estrogens, proteins, Ca, PGF and PGE2 content in nonpregnant uterine flushings collected between Days 10.5 and 14 were not detected. Electrophoresis of protein in pregnant uterine flushings indicated that the appearance of three basic uterine proteins (Mr 32K to 60K) were associated with the increase in estrogen. These proteins were detected in flushings with tubular and filamentous blastocysts (Day 12), but were not found until Day 14 in nonpregnant gilts. A synchronized release of secretory vesicles from the glandular epithelium was observed by TEM which indicated a close association between formation of tubular blastocysts, onset of blastocyst estrogen production and increased protein in uterine flushings. Although secretion was detected in nonpregnant glandular epithelium, no synchronized release was observed. Results suggest that estrogen production by tubular and early filamentous blastocysts (Day 11.5–12.0) stimulates secretion of protein from the endometrium which may be mediated through an effect of free Ca on the uterine glandular epithelium. Increases in PGF and PGE2 were associated closely with estrogen production and blastocyst elongation.

Copper IUD-induced loss of endometrial arylamidase activity in the rabbit.

Copper IUDs of blastocyst size cause, in the rabbit uterine epithelium, local reduction of activity of amino acid arylamidase, an aminopeptidase type enzyme found in the endometrium and in uterine secretion during preimplantation phases. By this means (although evidently by a different mechanism, namely direct enzyme inhibition) copper IUDs mimic an effect exerted by the late preimplantation and early implantation stage blastocyst which locally stimulate discharge of this enzyme from the uterine epithelium into the uterine secretion. The effect has not been seen in experiments with copper-free IUDs and hormone-containing IUDs.

Effects of low temperature upon subsequent nucleic acid and protein synthesis of rabbit embryos

Two-cell rabbit embryos, which were maintained without cleavage for 24 h at 10 °C, and then allowed to develop into blastocysts in vivo or in a completely defined medium at 37 °C, incorporated 3H-uridine into RNA and 14C-leucine into protein at rates comparable to blastocysts not previously exposed to 10 °C temperature. Thus, temporary suppression of development did not impair nucleic acid and protein synthesis by embryos developing into blastocysts. Blastocysts which developed in vivo incorporated more 3H-thymidine into DNA, 3H-uridine into RNA and 14C-leucine into protein than did blastocysts which developed in vitro. These differences probably reflect not only differences in blastocyst size and number of cells, but also differences in endogenous precursor pools.

Effects of uterine fluids, and immunoglobulins from semen-immunized rabbits on rabbit embryos cultured in vitro.

SummaryRabbit embryos in the morula stage were cultured in vitro in Ham's F-10 medium containing different serum samples and uterine fluid samples from control and semen-immunized rabbits. The percentage of embryos that blastulated and developed and the average blastocyst size did not differ between control and immune serum samples, whereas immune uterine fluid significantly depressed these parameters in comparison to control uterine fluid. Control and immune uterine fluids were fractionated by gel filtration and ion exchange column chromatography and the two predominant immunoglobulin classes, SIgA and IgG, separated. Of the two classes of immunoglobulins and two other fractions obtained from the gel filtration column, only immune SIgA proved to be embryotoxic. This anti-embryo effect of the immune SIgA was removed by absorption with spermatozoa and goat anti SIgA serum. Use of immunofluorescence also indicated that immune SIgA, but neither uterine nor serum IgG, cross-reacted with embryonic tissue. Thus...

Uteroglobin and serum proteins of rabbit blastocysts as affected by maternal factors

Proteins in the blastocoelic fluid of the rabbit were studied electrophoretically at 12 hr. intervals between 5 and 10 days post coitus (p.c.). Uteroglobin appeared in the fluid at 5.5 days p.c., occurred in large and maximal amounts between 6.5 and 7 days p.c., and disappeared by 8 days p.c. Albumin appeared at 5 days p.c.; prealbumin and γ-globulin appeared at 6 days p.c. The postalbumin, transferrin, and posttransferrin bands at 7 days p.c. resembled those found in maternal serum or peritoneal fluid. These 6 protein fractions increased in intensity and persisted after implantation. The distribution and intensity of protein bands in blastocoelic fluid was the same as those in the maternal serum at 8.5 days p.c. The total protein content of the fluid increased gradually until 6.5 days p.c. and then increased rapidly, at 8 days p.c., to the levels of peritoneal fluid (40 mg. per milliliter). The effects of maternal environment on uteroglobin content of blastocoelic fluid was studied at 7 days p.c. The intensity of uteroglobin bands was less with exogenous progesterone than with exogenous progesterone, probably as a result of promoted blastocyst growth with progesterone. The intensity of uteroglobin bands was higher in the blastocoelic fluid from ovariectomized animals and overcrowded uteri than in controls. The intensity of uteroglobin was higher in lactating rabbits than in postpartum rabbits. The strong staining of uteroglobin seems to be associated with the maternal factors which result in the reduction in blastocyst size.

Observations on pregnancy in intact rabbits and in ovariectomized rabbits treated with 17 alpha-hydroxyprogesterone caproate.

Rabbits ovariectomized 24 hr post coitum (p.c.) were injected i.m. with 2 mg or 4 mg 17\g=a\-hydroxyprogesteronecaproate daily. Pre\x=req- natal development was compared with that of control litters at 9 and 29 days, 5 days and 7 days p.c. The 2 mg treatment supported 26\m=.\4% pre-natal survival to 29 days p.c., most mortality occurring between 7 and 9 days. The 4 mg dose rate permitted 71 \m=.\0%survival to 29 days p.c. which was not significantly different from controls (81\m=.\5%).Blastocyst size at 5 days p.c. was not significantly affected by treatment but both ovariectomized groups showed slight retardation at 7 days when stage ofattachment was assessed by observation and by analysis ofblastocyst fluid for lactic acid. Attachment was least advanced in the group receiving the lower dose rate. In 29-day foetuses the degree ofossification of the femur was positively related to foetus weight. In control litters deposition ofcalcium and total ash components and relative decrease in water content were also further advanced in the larger foetuses, suggesting a general association of size and maturity.

GROWTH AND SURVIVAL OF BLASTOCYSTS IN THE DOMESTIC RABBIT

Summary. A total of 180 nulliparous rabbits was bred, bilaterally ovariectomized 2 or 4 days post coitum, and injected daily with 0·5, 0·75, 1, 4 or 40 mg of progesterone alone or in combination with oestradiol at a ratio of 1 : 400 or 1 : 4000. Uninjected, unilaterally and subtotally ovariectomized rabbits and progesterone-treated intact animals served as controls. The animals were autopsied at 6 days post coitum and 1111 blastocysts were recovered, examined for their normality and their diameters measured. The length and weight of uterus were affected neither by ovariectomy nor by the dose of injected progesterone. Intact does had an average of 6·6 to 9·0 viable blastocysts and 0·3 to 0·6 degenerating blastocysts. Ovariectomized does had an average of 4·1 to 10·2 viable blastocysts and 0 to 2·2 degenerating blastocysts. In ovariectomized does the percentage of viable blastocysts, the size of blastocysts and diameter of embryonic disc were decreased when the daily dose of progesterone was less than 1 mg and when oestradiol was combined with progesterone at a ratio of 1 : 400. There was no quantitative relationship between dosage of progesterone on one hand and percentage of viable blastocysts, size of blastocysts and diameter of embryonic disc on the other hand. Salpingectomy, unilateral ovariectomy, subtotal ovariectomy or administration of progesterone in intact rabbits had no significant effect on viability and size of blastocyst or diameter of embryonic disc.

Variation in the number of corpora lutea and in the number and size of 6-day blastocysts in rabbits subjected to superovulation treatment.

SUMMARY The numbers of corpora lutea and the numbers and sizes of 6-day blastocysts have been studied in rabbits, widely varying in ages and weights, that had been subjected to a superovulation treatment. For all does, the average value of each characteristic on the left sides of the animals was similar to that on the right, the average left-to-right difference being not significant. An average of thirty-one corpora lutea and thirteen blastocysts per doe was obtained. Maximum numbers per doe were one hundred and fifty-three corpora lutea and seventy-three blastocysts. Average blastocyst diameter was 2·203 mm, the average volume being 6·780 mm3. There was a fairly large variation from one doe to another in the relative numbers of corpora lutea (or in the relative numbers of blastocysts) on the two sides of the animal. The number of blastocysts per doe was positively related to the number of corpora lutea. The number of corpora lutea per doe was inversely related to the age of the doe. An inverse relation between the number of blastocysts and the age of the doe was presumably indirect, being mediated by the number of corpora lutea. All these relationships were highly significant. There was no demonstrable relationship between the number of corpora lutea and the weight of the doe. No firm evidence was found for a direct relationship (independent of weight and number of corpora lutea) between the number of blastocysts and age, or for any relationship with weight. Diameter of blastocysts seemed more appropriate than volume as a measure of size suitable for analysis. Transformations tending to give linear relationships between variables have been studied. Size differences between blastocysts within the same uterine horn formed a large source of variation, probably not attributable to the superovulation procedure. Differences in blastocyst size between uterine horns within does were significant but introduced little extra variation. Mean blastocyst size varied greatly from one doe to another, but none of this variation was related significantly to known factors such as the weight of the dam or sire, or the age of the dam. The experiment appeared to be fairly sensitive in detecting these relationships. The relationships found between the number of corpora lutea and the age or weight of the doe can be reconciled with the previous literature. The lack of demonstrable connexion between blastocyst size and the weight of dam or sire is in conflict with the results of other authors, and no specific reconciliation can be made. It is suggested that uniformity in age is more important than uniformity in weight when test animals are selected for assay of follicle stimulating hormone under conditions comparable with those of the present work.

THE COMPARATIVE EFFECTS OF PROGESTERONE, ESTRADIOL, DIETHYLSTILBESTROL ANDITS MONOMETHYL ETHER ON THE EARLY BLASTOCYST OF THE RABBIT1

THE SIZE of the 5 day rabbit blastocyst has been shown to be function of theamount of progesterone administered (Pincus & Werthessen, 1937). It was further demonstrated that the simultaneous injection of natural or synthetic estrogens profoundly altered the degree of response to a fixed dose of progesterone (Pincus & Wer-thessen, 1938). Thus, estradiol and estriol appeared to stimulate the growth of the blastocyst further, while estrone definitely inhibited it. The response of the endometrium differed from that of the blastocyst, in that estriol, for example, produced in the same rabbit a significant increment in blastocyst size and a notable diminution in endometrial responseto the standard dose of progesterone, estradiol produced significant increments in both blastocyst size and endometrial growth within the lower concentrations of the dosage employed, while the higher concentrations resulted only in significant increases in blasto-cyst size; the endometrial response was essentially that of the controls.