Protein synthesis and degradation in non-cultured and in-vitro cultured rabbit blastocysts

In 'pulse-chase' experiments synthesis and half-lives of leucine-labelled proteins were determined in rabbit blastocysts. Embryos were either non-cultured controls or were cultured for 24 h or 48 h in Ham's F-10 medium supplemented with homologous serum or uterine flushings. In control blastocysts protein synthesis increased by a factor of 10 between Day 4 and Day 5. Half-lives of newly synthesized proteins were 32 h in Day-4 and 99 h in Day-5 control blastocysts. In-vitro culture of Day-4 blastocysts led to dramatically shortened half-lives, amounting to 6-10 h. Blastocysts developing in uterine flushing-supplemented media differed significantly from those cultured in serum-supplemented media. Protein synthesis was enhanced and protein degradation was normal for culture times up to 24 h. These results demonstrate (1) that half-lives of proteins in rabbit blastocysts increase with advancing embryonic age, and (2) that a characteristic feature of the altered metabolism of cultured blastocysts is a dramatically accelerated protein degradation, which (3) can be prevented for some time by supplementation of the culture medium with uterine secretions.     

Influence of uterine flushings from superovulated cows on in vitro bovine morulae development

To evaluate early embryo development, 248 good to excellent bovine morulae were cultured in Ham's F-10 medium, supplemented with 10% steer serum, uterine flushings from Days 6, 10 or 15 following estrus (0.01, 0.1, 1.0 and 10% protein; 64 mg protein/ml), and 1.0% uterine flushings and 10% steer serum. Final development scores for embryos in steer serum were significantly higher (range across experiments was: 4.06 to 4.37) than for embryos cultured in uterine flushings alone (-0.23 to 0.52). Treatment means were not different (P >0.05) when 10% steer serum was added to 1.0% uterine flushings. A higher percentage of embryos in 10% steer serum (92%) than in 10% steer serum plus 1.0% uterine flushing from Day 6 (33%), Day 10 (45%) and Day 15 (50%) developed to hatched blastocysts. Embryos cultured in 1.0% Day 6 uterine flushings plus 10% steer serum required more time to attain the early blastocyst and blastocyst stages, while embryos in 1.0% Day 15 uterine flushings and 10% steer serum developed at the same rate as controls to the expanded blastocyst stage, but hatched sooner (72.8 vs 96.5 h). These results suggest substance(s) in uterine secretions can have inhibitory and stimulatory influences on early bovine embryo development.     

Development retardation in cultured preimplantation rabbit embryos

Day 3 to Day 5 preimplantation rabbit embryos were cultured for 24 h in chemically defined media which are widely used in early embryo culture (BSM II and Ham's F-10) supplemented with BSA or homologous serum. For the next 24 h, the embryos were left in the same culture medium, placed in freshly made medium, or cultured in medium which was supplemented with uterine flushings. In addition, 24-h cultured embryos were transferred into uteri of synchronous recipients for 1 day. After culture or transfer, development was assessed by cell proliferation evaluated by incorporation of tritiated thymidine. In comparison to non-cultured controls, thymidine incorporation demonstrated a considerably impaired cell proliferation after culture in defined media irrespective of medium, supplement, or replenishment with fresh medium. For Day 3 embryos, there was a developmental retardation amounting to about 1 day after 2 days in culture. Compared to Day 3 embryos, delay was clearly more pronounced in Day 4 and Day 5 blastocysts, i.e. in stages which had been retrieved from the uterus before culture. Supplementation with uterine flushings markedly promoted blastocyst cell proliferation (P less than 0.001). Incorporation data examined after transfer showed that impairment of cell proliferation caused by 1 day in culture had been compensated for to a large extent within 1 day in utero.     

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.     

Effect of human uterine flushings collected at various stages of the menstrual cycle on mouse blastocysts in vitro

Mouse blastocysts were cultured in vitro in a defined medium supplemented with uterine flushings (containing 500 microgram protein/ml) obtained from normal women at various stages of the menstrual cycle. With one exception (uterine flushing collected on the last day of a menstrual period) blastocyst hatching and attachment were not impaired by flushings collected before or after ovulation.     

The ovine uterus as a host for in vitro-produced bovine embryos

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.     

Analysis of proteins secreted by mouse embryos developing in vivo and in vitro

Proteins secreted by mouse blastocysts developing in vitro were compared to these from blastocysts developing in utero to determine if a simple medium supporting blastocyst development also supports secreted protein expression. In-vivo embryos were collected on days 3, 4, or 5 of pregnancy and incubated in 35S-methionine to produce conditioned medium containing released, labeled proteins. Embryos for culture were collected on day 3 and after 48 or 72 h labeled conditioned medium was produced. Labeled proteins were separated by two-dimensional electrophoresis and compared using a digital image analysis system. Day 3 embryos did not release proteins in detectable amounts, although synthesis of intracellular proteins was substantial. Day-4 and -5 blastocysts released proteins in increasing amount and complexity, consistent with previous results. When day-3 embryos were cultured in medium containing 4 mg/ml BSA for 48 h, secreted protein patterns were similar but not identical to those of day-5 uterine blastocysts. Although most of the proteins produced by uterine blastocysts were secreted by cultured embryos, differences were found in the relative quantities of certain proteins. Neither crystallized BSA nor polyvinyl alcohol at 4 mg/ml supported development of protein secretion as well as the crude fraction-V BSA. Blastocysts restricted to the oviduct also exhibited quantitative differences in protein secretion patterns compared to uterine blastocysts. Thus, although blastocyst development and the expression of many secreted proteins are supported outside the uterus, the full pattern of secretion characteristic of the peri-implantation embryo may be dependent on specific uterine influences.     

Embryonic mortality and the uterine environment in first-service lactating dairy cows

Embryos were collected non-surgically from the tip of one uterine horn of 23 lactating dairy cows on Day 7 of pregnancy. Embryos were classified on the basis of morphological criteria as normal (n = 12) or abnormal (n = 13). Abnormal embryos were further classified as cleavage stage (n = 9) or morula/blastocyst (n = 4). Cows producing an abnormal embryo did not differ in days post partum at oestrus, age or parity from cows producing a normal embryo. Cows with an abnormal morula/blastocyst also did not differ with respect to days post partum at oestrus from cows with abnormal cleavage-stage embryos but cows with an abnormal morula/blastocyst were significantly older and of greater parity than cows with an abnormal cleavage-stage embryo. Hepes-saline-PVP solution (30 ml) was initially infused into the uterine tip, mixed and then withdrawn with a syringe. Analysis of this fluid revealed that the concentrations of glucose, total protein, calcium, magnesium and potassium were significantly higher in the flushings from the uterus of cows with abnormal embryos than from cows with normal embryos and zinc and phosphorus tended to be higher in the uterine flushings of cows with abnormal embryos. Phosphorus, total protein, calcium and magnesium tended to be higher in the flushings from cows with abnormal morulae/blastocysts than from cows with abnormal cleavage-stage embryos. Plasma progesterone did not differ between cows with normal or abnormal embryos or in cows with abnormal morulae/blastocysts or abnormal cleavage-stage embryos. Most embryonic mortality therefore occurred before Day 5 (during cleavage) in these cows.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of preimplantation rabbit embryos in uterine flushing-supplemented culture media

The development of cultured rabbit preimplantation embryos grown in standard media (Ham's F-10 or BSM II supplemented with bovine serum albumin (BSA) or homologous serum) or in Ham's medium supplemented with uterine flushings was compared. The uterine flushings derived from donors of 0.5-6 years of age. Uterine flushing supplemented media were used natively or after treatments like sterilization by filtration, lyophilization, three times freezing/thawing, heat denaturation, dialysis, or ultrafiltration. Compared with in vivo controls, embryonic growth was substantially reduced during in vitro culture, demonstrably by smaller diameters and impaired cell proliferation (measured by thymidine incorporation). The growth retardation was more pronounced in blastocysts (recovered at day 4 post coitum [p.c.]) than in morulae (recovered at day 3 p.c.). Development in uterine flushing media was notably better than in standard media but did not comply with in vivo development. Highest thymidine incorporation was observed in media with increased concentrations of uterine secretions and after sequential supplementation of flushings from subsequent progestational stages. Advanced donor ages, heating up to 80 degrees C, freezing, and lyophilizing did not affect incorporation data statistically significantly, whereas sterilization by filtration, ultrafiltration, and dialysis led to a significantly reduced thymidine incorporation in the cultured embryos. The positive effects of uterine flushing supplementation are attributed to the supply of components more adjusted to the needs of the cultured embryos and/or to a reduction of pathological effects in vitro like washing out of nutritive and regulatory components from the embryo into the surrounding culture medium.     

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.     

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.     

Ultrastructural and Autoradiographic study of Preimplantation rabbit embryos grown in conventional or uterine flushing-supplemented culture media

Rabbit morulae and blastocysts were cultured in conventional culture media [Ham’s F10 or BSM II supplemented with bovine serum albumin (BSA) or serum] or in Ham’s medium supplemented with synchronous or asynchronous uterine flushings, mostly for 2 days, and afterwards investigated by light and electron microscopy and by autoradiography. Ultrastructure and cell proliferation differed considerably between cultured embryos and noncultured controls. Cultured embryos displayed more dead cells. They were developmentally retarded (predominance of smooth endoplasmic reticulum rather than the age-specific rough endoplasmic reticulum, mitochondria still round to ovoid shaped) and showed nonspecific signs of cells damage (swollen mitochondria and Golgi complex vesicles, increased number of lysosomes). All these features were also present in embryos grown in uterine flushing-supplemented media, but were less pronounced. Cell damage and impaired cell proliferation had affected trophoblast cells more than embryoblast cells. Endoderm could be differentiated only if culture had been started with blastocysts—not with morulae—and seems to require uterine secretions. No significant ultrastructural differences were observed between embryos cultured in synchronous or in asynchronous uterine flushings. Present results indicate that cultured preimplantation rabbit embryos deviate clearly from those grown in vivo and maintain, for some time, a better cellular structure—and probably function —in the presence of uterine flushings than in conventional culture media. Specific abnormal morphologic features related to a particular medium could not be identified.     

Effects of asynchrony on rabbit blastocyst development

The development of synchronously and asynchronously transferred rabbit morulae (recovered at Day 3 p.c.) and blastocysts (recovered at Day 4 p.c.) was investigated before the anticipated time of implantation. The results obtained with various techniques (evaluation of gross morphology, measurement of diameter, thymidine incorporation, light and electron microscopy) led basically to the same conclusions. Embryos being asynchronously transferred to the uterus of recipient rabbits survived, at least in terms of certain cellular functions like cell proliferation, for more than 2 days. Development, however, was clearly retarded and ultrastructural examination revealed substantial cell damage. Some blastocysts showed, even after 3 days, normal growth and cell proliferation indicating considerable differences between individuals in the ability to compensate for suboptimal developmental conditions before implantation. In general, this ability was greater in the transferred Day-3 morulae than in the Day-4 blastocysts. Embryonic growth and the ability to dissolve the zona pellucida, to synthesize crystalloid bodies and to differentiate extraembryonic endoderm indicated the maintenance of some developmental functions under asynchronous conditions. Blastocyst development was influenced by the progestational stage of the recipient. At 1 day after transfer into asynchronous older uteri, blastocyst diameters were larger and cell proliferation was increased compared with all other groups, suggesting an attempt of the blastocyst to adjust to the more advanced maternal milieu. Development in asynchronous younger uteri was delayed. No comparable differences in development were found in cultured embryos for which the media had been supplemented with flushings from the same progestational uterine stages as used for transfer. Thymidine incorporation in cultured embryos did not differ between the various supplements (P greater than 0.05) and was generally lower than in chronologically aged asynchronously transferred embryos (P less than 0.05 for Day-3 and P less than 0.001 or P greater than 0.05 for Day-4 embryos).     

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.     

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.     

Culturing in vitro produced blastocysts in sequential media promotes ES cell derivation

Embryonic stem (ES) cell lines are routinely derived from in vivo produced blastocysts. We investigated the efficiency of ES cells derivation from in vitro produced blastocysts either in monoculture or sequential culture. Zygotes from hybrid F1 B6D2 mice were cultured in vitro to the blastocyst stage in Potassium (K(+)) simplex optimised medium (KSOM) throughout or in KSOM and switched to COOK blastocyst medium on day 3 (KSOM-CBM). Blastocysts were explanted on a feeder layer of mitomycin C-inactivated murine embryonic fibroblasts (MEF) in TX-WES medium for ES cell derivation. Sequential KSOM-CBM resulted in improved blastocyst formation compared to KSOM monoculture. ES cells were obtained from 32.1% of explanted blastocsyts cultured in KSOM-CBM versus 18.4% in KSOM alone. ES cell lines were characterized by morphology, expression of SSEA-1, Oct-4 and alkaline phosphatase activity, and normal karyotype. These results indicate that in vitro culture systems to produce blastocysts can influence the efficiency of ES cell line derivation.     

Prediction of porcine blastocyst formation using morphological, kinetic, and amino acid depletion and appearance criteria determined during the early cleavage of in vitro-produced embryos

The determination for early cleavage-stage embryos of noninvasive morphologic and metabolic criteria that are predictive of blastocyst development and/or full-term viability remains an important research target. We describe the derivation of a logistic regression model that predicts the probability of porcine blastocyst formation in vitro. Pig zygotes, derived by in vitro maturation and fertilization of slaughterhouse oocytes, were cultured in NCSU-23 medium that was supplemented with a mixture of 20 amino acids (NCSU-23(aa)). On Day 1, at 21, 23, 25, 27, 29 and 31 h postinsemination, cleaving embryos were evaluated morphologically in terms of the: i) number of blastomeres, ii) evenness of division, and iii) degree of fragmentation. These embryos were then placed in 1.5-microl drops of NCSU-23(aa) for 24 h, after which time the three morphologic criteria were re-evaluated and 1.2 microl of spent medium were removed for analysis by HPLC, in order to determine the net rates of amino acid depletion and appearance. Embryos were then cultured singly in NCSU-23(aa) by placing them between the filaments of a woven polyester mesh until Day 6, in order to permit the identification of individual embryos. Of 256 cleaved embryos, 28.7 +/- 6.2% (n = 5 replicates) developed into blastocysts. Discriminant analysis was used to select a subset of amino acids (threonine, valine, lysine, and phenylalanine) that discriminated optimally between embryos that became blastocysts or degenerated. These discriminant scores were entered into the logistic regression. Significant univariate relationships were established between the probability of blastocyst development and amino acid score (odds ratio [OR] 0.53, 95% confidence interval [CI] 0.40-0.69, P < 0.001), cleavage time (OR 0.79, 95% CI 0.71-0.87, P < 0.001), degree of fragmentation on Day 1 (OR 0.55, 95% CI 0.35-0.84, P = 0.009) and Day 2 (OR 0.53, 95% CI 0.35-0.78, P = 0.002), evenness of division on Day 2 (OR 0.66, 95% CI 0.46-0.96, P = 0.028), and categorical values of blastomere number on Day 2 (all P < 0.02), although no single variate could accurately predict blastocyst formation. However, multivariate analysis of the cell numbers on Day 1 and Day 2 correctly classified 51.9% of the predicted blastocysts. The inclusion of cleavage time in the regression analysis raised this rate to 63.5%, which was increased to 66.2% by the addition of evenness of division and degree of fragmentation. Finally, the full logistic regression model, which incorporated amino acid score together with all the other morphologic and kinetic variables, correctly classified 80.8% of the predicted blastocysts. This represented 51.2% of the observed blastocysts. Our data are novel in that they not only define in a quantitative manner the influence of previously undescribed predictors of porcine blastocyst formation, but they also provide a simple model of preimplantation development with reasonable predictive accuracy. The present study also provides a basic model for the examination and incorporation of additional early morphologic and metabolic correlates of developmental competence and could potentially be applied to the selection of human embryos for transfer in clinical IVF.     

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)     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.     

Mouse blastocyst surface expression of galactose-containing epitopes coinciding with trophoblast differentiation

A galactose-containing cell surface epitope of mouse blastocysts was identified and partially characterized by means of immuno- and lectincytochemistry, using a mouse IgM anti-blastocyst monoclonal antibody (mAb N63) and four different galactose-binding lectins (BSL-1, DBA, PNA and SBA) as molecular probes. The mAb was produced by syngeneic intrasplenic immunization with adhesive mouse blastocysts, obtained 18 h after estrogen reactivation from facultative delay of implantation. Labelling of different mouse embryonic stages collected by uterine flushings revealed that the labelling of the epitope by monoclonal antibodies was restricted to the blastocyst stage. A peak labelling intensity was observed on late blastocysts. When examining blastocyst outgrowths, both trophoblast and embryoblast were weakly stained by mAb N63. Direct antigen characterization performed on blastocysts indicated that the mAb N63 recognized a galactose-containing glycolipid antigen. Immunochemistry of cryosectioned, unfixed mouse tissues including ovary, testis, uterus in delay and at implantation, Day 12 and term placenta, liver, kidney, brain, intestine, heart, striated muscle, and skin was negative. In addition, labelling of rat and hamster blastocysts was negative. In vitro experiments demonstrated that the galactose-containing blastocyst surface epitope was not involved in blastocyst attachment to plastic culture dishes. The appearance of the epitope at the embryonic surface in vivo coincides with the time of trophoblast differentiation and implantation in the mouse.