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.     

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.     

Prostaglandin release by zygotes and endometria of pregnant rabbits

When 4-day rabbit zygotes were incubated for 1 h at 37 degrees C in vitro, very little prostaglandin (PG) was released into the medium, and the concentration of PGs in the zygotes after incubation was also low. The release of prostaglandin E (PGE) and prostaglandin F (PGF) into the medium, and their concentration in the zygotes after incubation, increased sharply on Days 6 and 7 of pregnancy, reaching, by Day 7, values close to 200 ng of each PG released in 1 h per mg of protein. By contrast, endometrial samples on Days 4 and 5 of pregnancy released more PGF and less PGE than the zygotes of the same ages on a per mg of protein basis, and on Days 6 and 7, less of both PGs. Furthermore, endometrial concentrations of PGs after incubation, except for PGF on Day 4, were always lower than values for zygotes. Endometrial concentrations of PGs on Day 6 were lower before than after incubation. Although there was a slight upward trend in PG release by endometrial samples with increasing length of pregnancy, the changes were minimal and, in the case of PGE, none of the mean values exceeded 1 ng per mg of protein. In 7-day blastocysts, high levels of both PGF and PGE were found in the blastocoelic fluid, and these did not change during the 1-h incubation. The release of PGF and PGE during in vitro incubation of ruptured and washed Day 6 blastocysts was stimulated by arachidonic acid, and that of PGF, but not PGE, inhibited by indomethacin. The release of PGE, but not of PGF, from Day 6 blastocysts was inhibited by low temperature, and the same conditions inhibited release of both PGF and PGE from endometrial cell suspensions. It seems that both blastocysts and endometria have capability to synthesize PGs, the blastocysts being particularly active in this regard on Days 6 and 7 of pregnancy. It is hypothesized that, in vivo, Day 6 and 7 blastocysts release large quantities of PGs which trigger some of the local endometrial changes associated with pregnancy.     

An abnormal pattern of embryonic development during early pregnancy in aging rats

There is recent evidence that a decline in fertility and litter size precedes the cessation of regular estrous cyclicity in middle-aged female rats. This decline in litter size is related to a decrease in the number of normal blastocysts that are present on Day 5 of gestation, immediately prior to implantation. Thus, the pattern of embryonic development during the first 5 days of pregnancy may be altered in middle-aged rats, resulting in fewer implanting embryos and smaller litter sizes. The present study examined the ovulation rates, fertilization rates, and the patterns of embryonic development in regularly cyclic, young and middle-aged females during the first 5 days of pregnancy. Examination of the numbers of ovulated ova revealed that the ovulation rate was significantly reduced in 12- to 14-mo-old females (13 mo; 9.0 +/- 1.0/rat), but not in 9- to 11-mo-old females (10 mo; 12.2 +/- 0.8/rat), as compared to that in young animals (12.8 +/- 1.0/rat). However, there was no decrease in fertilization rate in either the 10-mo or 13-mo group. While the total numbers of embryos present on Days 2-5 were similar among all 3 groups, embryos from 10-mo females displayed a delayed pattern of development and an increased incidence of morphological abnormalities. These changes in embryo development were even more pronounced in the 13-mo group. By Day 5 of pregnancy there was a significant reduction in normal blastocysts in 10-mo (7.3 +/- 1.2/rat) and 13-mo (6.0 +/- 1.6/rat) rats, as compared to young females (10.6 +/- 0.9/rat).(ABSTRACT TRUNCATED AT 250 WORDS)     

Uterine protein synthesis during the early stages of pregnancy in the rat.

The synthesis of uterine-soluble proteins during early pregnancy in the rat has been examined by means of dual-isotope labelling techniques and subsequent electrophoretic analysis. A protein of similar electrophoretic mobility to the uterine oestrogen-induced protein was observed, and synthesis of this 'presumptive induced protein' was maximal on Day 4 and Day 6 of pregnancy but low on day 5. Pregnancy associated protein synthesis was observed in many regions on polyacrylamide gels, including the beta-lipoprotein, alpha2-macroglobulin, post-transferrin and albumin regions. Synthesis of the post-transferrin species rapidly increased from Day 4 to reach a maximum on Day 6 in the implantation tissue. The temporal pattern of synthesis of post-transferrin protein and and of 'presumptive induced region' suggests involvement in the processes of cell proliferation and decidualization.     

Effect of flushing of blastocysts on days 10-13 on the life-span of the corpora lutea in the pig

Blastocysts were flushed out of both uterine horns of gilts on Days 10, 11, 12 or 13. In mated non-pregnant gilts flushing had no effect on progesterone profile or cycle length (20.8 +/- 0.4 versus 20.6 +/- 0.6 days in the preflush cycle, N = 6, mean +/- s.e.m.). Flushing the blastocysts out of the uterine horns on Day 10 resulted in a cycle with a normal progesterone profile and a normal length (21.2 +/- 0.4 days, N = 5). Flushing on Days 11, 12 or 13 resulted in a normal cycle or in maintenance of the CL for 3-13 days as indicated by elevated progesterone concentrations and an increased interoestrous interval of, respectively, 22.0 +/- 1.2 versus 19.8 +/- 0.6 days (Day 11; N = 6), 24.8 +/- 1.4 versus 21.0 +/- 0.6 days (Day 12; N = 5; P less than 0.05) and 26.3 +/- 2.3 versus 20.5 +/- 0.4 days (Day 13; N = 6; P less than 0.05). There was a positive relationship between the change in interoestrous interval and the interval between the first observed standing oestrus and flushing of the blastocysts (rs = 0.350; n = 22; P less than 0.1). There was a large variation in the diameter of the blastocysts flushed on the same day. Only in those gilts in which the blastocysts were greater than or equal to 8 mm or filamentous were the CL maintained for 3 or more days. These results indicate that a first signal for maternal recognition of pregnancy is generated on Day 12 and that blastocysts greater than or equal to 8 mm are required for prolongation of CL function for 3 or more days. Since CL function is only extended for a maximum of 13 days (mean 7.4 +/- 1.0), a second signal seems necessary to maintain the CL for the whole period of pregnancy.     

Estradiol production by preimplantation blastocysts and increased serum progesterone following estradiol treatment in llamas

Estradiol is a potential candidate for the blastocyst signal responsible for maternal recognition of pregnancy in the llama (Lama glama). Two experiments were conducted to determine if the llama blastocyst produces estradiol during the presumed period of maternal recognition of pregnancy and if exogenous estradiol can extend the luteal phase. In Experiment 1, llamas were superovulated with eCG and mated 7 days later (Day 0=day of mating). Blastocysts were collected nonsurgically on Days 7, 9, or 11 or at necropsy on Days 13 and 15 post-mating and cultured for 48h. Conditioned medium was recovered, replaced with fresh medium at 24-h intervals, and assayed for estradiol-17beta. Estradiol production (pg/blastocyst) over the 48-h culture increased (P<0.05) by day of gestation where more estradiol (P<0.05) was produced by Day 11 compared to Day 7 blastocysts, Day 13 compared to Days 7-11 blastocysts, and Day 15 compared to Days 7-13 blastocysts. A dramatic increase was observed between Days 11 and 13 when estradiol production by Day 13 blastocysts increased (P<0.05) more than 50-fold. In Experiment 2, 30 females were induced to ovulate with hCG (Day 0=day of hCG injection). Starting on Day 7 and continuing through Day 15, animals received daily injections i.m. of 0 (n=11), 5 (n=7), or 10mg (n=12) estradiol benzoate (EB) dissolved in isopropylmyristate. Sera were collected immediately prior to each injection and on Days 16, 17, 18, 20, and 22 and analyzed for progesterone. Progesterone concentrations were greater (P<0.05) on Days 14, 15, 16, and 17 in llamas treated with 10mg EB compared to llamas treated with 0mg EB. These results demonstrate that llama blastocysts produce estradiol and exogenous estradiol can enhance and transiently extend luteal progesterone production. Estradiol produced by the preimplantation llama blastocyst may play a role in maternal recognition of pregnancy and early luteal support.     

Two-dimensional polyacrylamide gel electrophoresis of proteins synthesized and released by conceptuses and endometria from pony mares

Conceptuses were obtained from pony mares on each day of pregnancy between Days 12 and 28, and on Days 39, 45, 65 and 100. Endometrium was obtained from mares at Days 12, 14, 16, 18, 39, 45, 65 and 100 of pregnancy, and from non-pregnant mares during anoestrus, during transition into the breeding season, at oestrus, or during dioestrus. Tissues were incubated in vitro for 24 h with L-[3H]leucine. Proteins synthesized and released into the culture medium were analysed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and fluorography. Conceptuses obtained before Day 14 after ovulation released a characteristic pattern of labelled proteins. These included two groups of apparent isoelectric variants of relative molecular weights (Mr) 30,000-40,000 (pI values 4.5-5.5 and 6-7), one group of Mr approximately 22,000 (pI 6.5-7), and large protein(s) that did not enter the 10% polyacrylamide gel. After Day 14 the array of labelled proteins had changed and resembled that produced by isolated yolk sac at the later stages of pregnancy studied. Included amongst these were several acidic polypeptides with Mr 20,000 (pI 5-6). The endometrial samples released an array of non-dialysable polypeptides into the culture medium. Fluorograms could be assigned to one of three general groups, with endometrium from mares within each group producing similar patterns of labelled proteins. The first group consisted of anoestrous, transitional and ovariectomized mares, and mares at oestrus or Day 1 or Day 18 after ovulation. The second group was comprised of mares at Days 12-16 of dioestrus or Days 12-18 of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of rabbit endometrial plasma membranes from receptive and non-receptive uteri

We have developed a method for isolation of plasma membranes from rabbit endometrium, with high yield and purification. Endometrial homogenates are precipitated with calcium chloride and the resulting supernatant is fractionated by centrifugation in a self-forming gradient of 20% Percoll. Before fractionation, the intact luminal epithelial surface was labelled with 125I-labelled soyabean agglutinin. Between buoyant densities of 1.015 and 1.017 g/ml, a discrete peak of surface label was obtained, which coincided with activities for 5'-nucleotidase and alkaline phosphatase, enzyme markers for the plasma membrane. This peak was well separated from the majority of cellular protein, and from marker enzyme activities for mitochondria and microsomes (NADH cytochrome C reductase) and lysosomes (acid phosphatase). Electron microscopy of the purified membranes showed membrane sheets and vesicles free from other cellular organelles. Analysis of detergent-soluble membrane proteins, fractionated by concanavalin A-affinity chromatography, revealed differences in the protein pattern of membranes from uteri of rabbits receptive (Day 6 of pregnancy) and non-receptive (Day 3) for implantation. The method will be useful for generation of immunological and affinity probes for surface antigens involved in ovoimplantation.     

Pregnancy rates following timed embryo transfer with fresh or vitrified in vitro produced embryos in lactating dairy cows under heat stress conditions

Timed embryo transfer (TET) using in vitro produced (IVP) embryos without estrus detection can be used to reduce adverse effects of heat stress on fertility. One limitation is the poor survival of IVP embryos after cryopreservation. Objectives of this study were to confirm beneficial effects of TET on pregnancy rate during heat stress as compared to timed artificial insemination (TAI), and to determine if cryopreservation by vitrification could improve survival of IVP embryos transferred to dairy cattle under heat stress conditions. For vitrified embryos (TET-V), a three-step pre-equilibration procedure was used to vitrify excellent and good quality Day 7 IVP Holstein blastocysts. For fresh IVP embryos (TET-F), Holstein oocytes were matured and fertilized; resultant embryos were cultured in modified KSOM for 7 days using the same method as for production of vitrified embryos. Excellent and good quality blastocysts on Day 7 were transported to the cooperating dairy in a portable incubator. Nonpregnant, lactating Holsteins (n = 155) were treated with GnRH (100 microg, i.m., Day 0), followed 7 days later by prostaglandin F2alpha (PGF2alpha, 25 mg, i.m.) and GnRH (100 microg) on Day 9. Cows in the TAI treatment (n = 68) were inseminated the next day (Day 10) with semen from a single bull that also was used to produce embryos. Cows in the other treatments (n = 33 for TET-F; n = 54 for TET-V) received an embryo on Day 17 (i.e. Day 7 after anticipated ovulation and Day 8 after second GnRH treatment). The proportion of cows that responded to synchronization based on plasma progesterone concentrations on Day 10 and Day 17 was 67.7%. Pregnancy rate for all cows on Day 45 was higher (P < 0.05) in the TET-F treatment than for the TAI and TET-V treatments (19.0 +/- 5.0,6.2 +/- 3.6, and 6.5 +/- 4.1%). For cows responding to synchronization, pregnancy rate was also higher (P < 0.05) for TET-F than for other treatments (26.7 +/- 6.4, 5.0 +/- 4.3, and 7.4 +/- 4.7%). In the TET-F treatment group, cows producing more milk had lower (P < 0.05) pregnancy rates than cows producing less milk. In conclusion, ET of fresh IVP embryos can improve pregnancy rate under heat stress conditions, but pregnancy rate following transfer of vitrified embryos was no better than that following TAI.     

Differential expression of ezrin/radixin/moesin (ERM) and ERM-associated adhesion molecules in the blastocyst and uterus suggests their functions during implantation

Development of the blastocyst to implantation competency, differentiation of the uterus to the receptive state, and a cross talk between the implantation-competent blastocyst and the uterine luminal epithelium are all essential to the process of implantation. In the present investigation, we examined the possibility for a potential cross talk between the blastocyst and uterus involving the ezrin/radixin/moesin (ERM) proteins and ERM-associated cytoskeletal cross-linker proteins CD43, CD44, ICAM-1, and ICAM-2. In normal Day 4 blastocysts and after rendering dormant blastocysts to implantation-competent by estrogen in vivo (activated), the outer surface of mural trophectoderm cells showed much higher levels of radixin as compared to those in the polar trophectoderm cells, inner cell mass (ICM), and primitive endoderm. In contrast, ezrin was present on both the mural and the polar trophectoderm cell surfaces of normal Day 4 and activated blastocysts at higher intensity than dormant blastocysts. A distinct localization was noted in the primitive endoderm of dormant blastocysts that was not apparent in activated or normal Day 4 blastocysts. The expression of moesin was modestly higher at the mural trophectoderm of implantation-competent blastocysts, while the localization appeared to be present primarily on the polar trophectoderm cell surface of Day 4 blastocysts. The localization of ERM-associated adhesion molecules CD43, CD44, and ICAM-2 was more intense in the implantation-competent blastocysts compared with the dormant blastocysts. However, while CD44 was present both in the trophectoderm and in ICM, CD43 and ICAM-2 were localized primarily to the trophectoderm. The signal for ICAM-1 was very intense in the ICM but was modest in the trophectoderm. No significant changes in fluorescence intensity were noted between activated and dormant blastocysts. In the receptive uterus on Day 4 of pregnancy, ERM proteins were localized to the uterine epithelium, while on Day 5 the localization, especially of radixin and moesin, extended to the stroma surrounding the implantation chamber. With respect to ERM-associated adhesion molecules, while CD44 and ICAM-1 were exclusively localized in the stroma on Day 4, CD43 and ICAM-2 were localized to the epithelium. On Day 5, the localization of CD44 and ICAM-1 became highly concentrated in the antimesometrial stroma of the implantation chamber. The localization of CD43 and ICAM-2 remained mostly epithelial, although some stromal localization of CD43 was noted on Day 5. These results suggest that differential expression and distribution of ERM proteins and ERM-associated adhesion molecules are involved in the construction of the cellular architecture necessary for blastocyst activation and uterine receptivity leading to successful implantation.     

Plasma patterns of prolactin, progesterone, and estradiol during early pregnancy in aging rats: relation to embryonic development

Regularly cyclic, middle-aged female rats exhibit a decreased incidence of fertility, and those females that are fertile produce smaller litters. This decreased litter size is directly related to a reduced number of normal blastocysts available for implantation. Recent evidence indicates that embryonic abnormalities in middle-aged rats become apparent as early as Day 2 of pregnancy. Inasmuch as the semicircadian secretion of prolactin (PRL) is essential for the rescue of corpora lutea during early gestation and luteal production of progesterone (P) and estradiol (E2) in sufficient quantities is obligatory for embryonic development and implantation, the present study examined the profiles of plasma PRL, P, and E2 during the first 3 days of pregnancy in both young and middle-aged rats and assessed the embryonic development in these same animals. Regularly cyclic, middle-aged (9-11 mo) and young (4-5 mo) rats were cannulated via the right jugular vein on Diestrus Day 2 and mated with fertile males on proestrus. The next morning, sperm in the vaginal lavage confirmed mating, and that day was designated Day 1 of pregnancy. Beginning at 1400 h on Day 1 and continuing to 2400 h on Day 2, serial blood samples were taken at 2-h intervals for PRL assay. In the first experiment, samples were also collected at 8-h intervals during Days 1-3 for measurement of plasma P.(ABSTRACT TRUNCATED AT 250 WORDS)     

The culture of human cleavage stage embryos alone or in groups: effect upon blastocyst utilization rates and implantation

The effect of cleavage-stage group culture (CGC; embryos cultured in groups of three or more for the first 3 days and then individually to blastocyst) was compared to extended single embryo culture (ESC; embryos cultured individually to the blastocyst stage). While implantation and ongoing pregnancy rates were similar between groups, the blastocyst utilization rate (number of blastocysts suitable for freezing and thawing/total number of embryos cultured to Day 5 and 6) was significantly higher when embryos were cultured in CGC for women ≤35 yrs thereby increasing the number of embryos available for clinical use for the younger women. This strategy of group culture to Day 3 would seem an ideal protocol to capitalize on an overall embryo quality in two particular settings, namely programmes wishing to (i) undertake Day 3 transfers, and (ii) keep embryos separate from Day 3 to Day5/6 for the purposes of selection. The culture system can also be applied to the embryos of older women without adverse effect, enabling the same system to be used for all embryos.     

Transbilayer mapping of membrane proteins using membranes isolated on polylysine-coated polyacrylamide beads

Erythrocyte and HeLa cell plasma membranes were isolated on polylysinecoated polyacrylamide beads and the transbilayer disposition of their proteins was investigated.When membranes of intact erythrocytes were isolated on beads and then labelled by lactoperoxidase-catalysed iodination, their labelling pattern was similar to that of inside-out vesicles in solution.When the membranes of intact HeLa cells were isolated on beads and then labelled by galactose oxidase-[³H]borohydride treatment, no glycoprotein or glycolipid sugars were accessible. On the other hand, when the HeLa cell membranes were isolated on beads and then labelled by the lactoperoxidase-catalysed iodination, all of the major membrane proteins were iodinated. These experiments confirmed for HeLa cell membranes what had previously been shown for erythrocyte membranes: when the membranes of intact cells are isolated on beads, the accessibility of their surfaces to enzymatic probes is the same as would be expected of inside-out vesicles in suspension. Double-label experiments, in which the HeLa cell membranes were labelled first on the intact HeLa cells and again after isolation on beads, identified several     

Acrylamide Neuropathy: Changes in the Composition of Proteins of Fast Axonal Transport Resemble Those Observed in Regenerating Axons

Proteins conveyed by fast axonal transport along sensory and motor axons of rat sciatic nerve were labelled with L-[35S]methionine and characterized by one- and two-dimensional electrophoresis on polyacrylamide gels, followed by fluorography. Nerves from normal or bis-acrylamide-treated animals were compared with nerves from acrylamide-treated animals and nerves regenerating after a crush axotomy. In both sensory and motor axons significant changes in the pattern of labelled bands on one-dimensional gels occurred after 10 days of acrylamide treatment (50 mg/kg daily, i.p.). These changes resembled those seen in regenerating axons, but were less pronounced. No changes were detectable after shorter periods of treatment, even though the onset of the neuropathy, assessed by a behavioral test, occurred on days 4-6 of treatment. Two-dimensional separations of the labelled proteins revealed increased labelling of growth-associated protein 43 in acrylamide-treated animals, but again this was less pronounced than in regenerating nerves. Acrylamide treatment induces changes in composition of fast-transported protein that are qualitatively similar to those seen after axotomy. Since these changes are not detectable until the neuropathy is advanced, it is unlikely that they are causative factors. Instead, they are most likely a result of the cell body reaction previously observed in acrylamide intoxication, a reaction that resembles that produced by axotomy.     

Effect of resynchronization with GnRH on day 21 after artificial insemination on pregnancy rate and pregnancy loss in lactating dairy cows

The objectives of the present study were to determine the effects of resynchronization with GnRH on Day 21 after artificial insemination (AI) on pregnancy rate and losses of pregnancy in lactating dairy cows. Holstein cows (n=585) on two dairy farms were assigned to one of two treatments in a randomized complete block design. On Day 21 after a pre-enrollment AI, animals assigned to the resynchronization (RES) group received 100 microg of GnRH i.m., whereas animals in the control (CON) group received no treatment. All animals were examined ultrasonographically on Days 21 and 28 after AI, and blood samples were taken for progesterone measurement on Day 21. Pregnancy was diagnosed on Day 28 and reconfirmed 14 days later. Nonpregnant cows on Day 28 were inseminated using timed AI after the completion of the Ovsynch protocol 10 and 17 days after enrollment in the study for RES and CON groups, respectively. Progesterone concentration > or =2.35 ng/ml was used as an indicator of pregnancy on Day 21. For RES and CON cows, pregnancy rate at Days 21 (70.9% versus 73.0%, P<0.56), 28 (33.1% versus 33.6%; P<0.80) and 42 (27.0% versus 26.8%; P<0.98) after the pre-enrollment AI did not differ. Administration of GnRH on Day 21 after AI had no effect on pregnancy loss in RES and CON groups from days 21 to 28 (53.2% versus 53.5%; P<0.94) and days 28 to 42 (17.9%; P<0.74) after AI. Pregnancy rate after the resynchronization period was similar for both treatment groups. Resynchronization with GnRH given on Day 21 after AI for initiation of a timed AI protocol prior to pregnancy diagnosis does not affect pregnancy rate and pregnancy loss in lactating dairy cows.     

Equine cloning: in vitro and in vivo development of aggregated embryos

The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.     

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.     

Maternal recognition of pregnancy in the goat: effects of conceptus removal on interestrus intervals and characterization of conceptus protein production during early pregnancy

Goat conceptuses were surgically removed from the uterus at different days during early pregnancy and cultured for 24-30 h in the presence of L-[3H]leucine to determine the effects of embryo removal on the interestrus interval and to characterize in vitro synthesis and release of conceptus proteins. Normal cyclic and animals (controls) exhibited interestrus intervals of 20.44 +/- 0.89 days. Removal of conceptuses on Days 13 and 15 did not alter interestrus intervals compared to cyclic animals. Removal of conceptuses on Day 17 and times thereafter resulted in significant (p less than 0.05) prolongation of interestrus intervals. These results demonstrate that maternal recognition of pregnancy in the goat occurs between Days 15 and 17. Proteins synthesized and released into the medium by conceptuses were first detectable at Day 16 by the analytical method employed (two-dimensional polyacrylamide gel electrophoresis followed by fluorography). The major protein synthesized at this time was acidic (pI = 5.2-5.7) and consisted of two isotypes with molecular weights of about 17,000. Although patterns of protein production became more complex with conceptus development, this protein remained as a major product through Day 21 but not afterwards. This protein, as well as two other low molecular weight acidic proteins (Mr approximately equal to 21,000, 23,000; pI = 5.7-6.0) were shown by immunoprecipitation to react with anti-ovine trophoblast protein-1 (oTP-1) serum. Hence, these products may comprise a caprine trophoblast protein-1 (cTP-1) complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of heparin/heparan sulfate interacting protein/ribosomal protein l29 during the estrous cycle and early pregnancy in the mouse

Using a variety of approaches, we have examined the expression of the heparin/heparan sulfate (Hp/HS) interacting protein/ribosomal protein L29 (HIP/RPL29) in mouse uteri during the estrous cycle and early pregnancy. HIP/RPL29 selectively binds heparin and HS and may promote HS-dependent embryo adhesion. HIP/RPL29 was prominently expressed in both luminal and glandular epithelia under almost all conditions, including the phase of embryo attachment. In contrast, differences were noted in HIP/RPL29 expression in the stromal compartment both during the estrous cycle and during early pregnancy. Most notably, HIP/RPL29 accumulated in decidua, where it displayed a pattern complementary to that of pericellular deposition of the HS proteoglycan, perlecan. HIP/RPL29 protein was detected in implanted embryos at both initial and later stages of implantation; however, embryonic HIP/RPL29 mRNA accumulation was more pronounced at later stages (Day 7.5 postcoitum). In situ hybridization revealed similar spatial changes for HIP/RPL29 mRNA during these different physiological states. Whereas differences in the spatial pattern of HIP/RPL29 protein and mRNA expression were demonstrable, little change was detected in the level of HIP/RPL29 mRNA or protein in total endometrial extracts. Mouse blastocysts attached, but did not outgrow, on surfaces coated with recombinant murine HIP/RPL29. Surprisingly, soluble glycosaminoglycans including heparin, low molecular weight heparin, or chondroitin sulfate were not able to inhibit embryo attachment to HIP/RPL29-coated surfaces. These latter observations indicate that embryonic cell surface components other than HS proteoglycans can promote binding to HIP/RPL29 expressed by uterine cells.