Immunocytochemical localization of BP, a conceptus secretory protein, in trophectoderm during pig trophoblast elongation

Tissue sections of periimplantation pig conceptuses (days 9-15 of pregnancy) were incubated with antiserum to the basic protein (BP), a major secretory protein of filamentous pig blastocysts. Bound antibody was detected by the peroxidase-antiperoxidase method. BP was restricted to trophectoderm in conceptuses which had made the transition from a spherical to an ovoid shape having a diameter of greater than 5 mm (day 11). Tubular (day 11, 10-20 mm) and filamentous (day 11-15) conceptus trophectoderm contained BP. These results suggest that BP synthesis commences at the time of rapid trophoblast growth.     

Protein secretion by the mouse trophoblast during attachment and outgrowth in vitro

Protein secretion from mouse blastocysts undergoing attachment and trophoblast outgrowth in vitro was assessed. When Day 5 blastocysts were cultured in serum-containing medium, secretion of several 'attachment-associated' proteins (PAS) was initiated within 24 h, coincident with attachment and outgrowth. Those proteins characteristic of the pre-attachment blastocyst disappeared or made-up only a small portion of the secretions once attachment began. The major secreted protein from attached embryos, PA1, is a 35,000-45,000 Mr acidic glycoprotein with multiple isoelectric forms. PA2, a group of basic 40,000 Mr proteins and PA3 a group of 72,000 Mr proteins were also produced during outgrowth. PAS were secreted during outgrowth on fibronectin-coated plastic in serum-free medium, but not by blastocysts held in a non-attachment state during culture in serum-free medium on uncoated plastic. In pre-attachment blastocysts, secreted proteins were produced by trophoblast vesicles, but not by isolated inner cell masses. Both trophoblast vesicles growing out in vitro and surgically isolated trophoblast from spreading blastocysts had secreted protein patterns qualitatively similar to those of intact blastocyst outgrowths. The results indicate that development of trophoblast protein secretion continues through the period of outgrowth and giant cell transformation. These changes are apparently dependent on attachment of the blastocyst to a suitable substrate, but not dependent on any other serum influence.     

Characterization of feline conceptus proteins during pregnancy

This study characterized proteins secreted de novo by feline conceptuses collected on Days 10, 12, and 15 (n = 22, preimplantation blastocysts); Days 15, 16, 17, 19, 21, and 25 (n = 6, postimplantation zonary girdle [ZG] i.e. trophoblast and endometrium); and Days 30, 36, 39, and 50 (n = 5, postimplantation ZG and free chorioallantois [CA]) and cultured in Minimal Essential Medium. De novo secretion was shown by incorporation of 3H-leucine into proteins detected in culture media by 2D-PAGE and fluorography. Western blotting, and NH2-terminal amino acid microsequencing. Major radiolabeled proteins identified as they appeared temporally on fluorographs were as follows: feline conceptus protein 1 (fCP1), Mr = 20,000, pI 5.0-5.3; fCP2, Mr = 80,000, pI 6.5-7.2; fCP3a, Mr = 67,000, pI 6.3-6.5; fCP3b, Mr = 67,000, pI 5.9-6.3; fCP4, Mr = 56,000, pI 5.0-6.0; and fCP5, Mr = 29,000, pI 5.0-5.8. The fCP1 was produced by blastocysts on Days 10-15, ZG on Days 16-25, and CA on Day 30; on Days 39-50, CA synthesized 5 proteins, possibly fCP1 isomers. The fCP2, fCP3a and b, and fCP4 were produced by blastocysts on Day 15, ZG on Day 25, and CA on Days 30-50. The fCP5 was made by ZG on Days 16-36 and by CA on Days 30-39. Western blotting identified fCP1 as retinol-binding protein (RBP), fCP2 as alpha fetoprotein, fCP3a as albumin, and fCP3b as transferrin. Amino acid sequence homologies between fCP1 and rabbit and human plasma RBP and porcine conceptus RBP2 were 93, 96, and 100%, respectively, at the first 37 NH2-terminal amino acids. The identities of fCP4 and fCP5 have not been established. Antiviral activity detected in all media was less than 3 units/ml when tested with feline fibroblast cells infected with vesicular stomatitis virus.     

Rapid growth and elongation of bovine blastocysts in vitro in a three-dimensional gel system

The aim of this study was to establish an in vitro system that supports development and differentiation of bovine blastocysts. Agar gel tunnels were covered with modified synthetic oviduct fluid medium supplemented with 10% fetal calf serum and 3g/l D-glucose. Of the total 67 blastocysts loaded individually into the tunnels, 46 continued expansion to 1mm and reached the walls of the gel on Day 10. On Day 12, 35 blastocysts elongated to minimum 1.6 mm while filling completely the space between the walls of the tunnel, and 16 still continued growth and reached an average of 4.3 mm length on Day 14. The largest blastocyst on Day 16 was 12 mm long. On Day 12, in 31 of the 35 elongated blastocysts a second cell layer occurred beneath the trophoblast and formed a complete cover in surviving Day 14 embryos. In most proliferating embryos the inner cell mass was prominent, however, the detection of signs of embryonic disc formation will require further studies. The established system was suitable to induce in vitro elongation, rapid growth and further differentiation, and may have considerable theoretical and practical value for studies of development and differentiation of bovine embryos.     

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.     

Electrophoretic characterization of the plasminogen activator produced by bovine blastocysts.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and zymography were used to determine the tissue source and to characterize the types of plasminogen activator (PA) produced by bovine blastocysts. Day 12-14 blastocysts were collected at slaughter from oestrus-synchronized, superovulated and artificially inseminated Holstein cows. In Expt 1, blastocysts were cultured for 24 h in Ham's F-12 in a humidified atmosphere of 5% CO2 in air at 37 degrees C. After culture, blastocysts and medium were recovered and stored separately at -20 degrees C. In Expt 2, embryonic discs were separated from trophoblast by microdissection. Intact blastocysts, embryonic discs and trophoblast were then cultured for 24 h and recovered as in Expt 1. In both experiments, embryonic tissues and media were electrophoresed with PA and molecular mass standards. Polyacrylamide gels were laid onto casein-agar gel plates (zymograms) and incubated at room temperature for 24-48 h. Caseinolytic zones in zymograms containing plasminogen were evidence of PA. In Expt 1, bovine blastocysts contained and secreted light and heavy forms of PA (47.0 +/- 1.0 and 86.1 +/- 0.7 kDa, respectively). In Expt 2, intact blastocysts and trophoblast produced both forms of PA (41.5 +/- 1.5 and 92.2 +/- 2.7 kDa) but PAs were not detected in embryonic discs. The results suggest that Day 12-14 bovine blastocysts produce urokinase-type PA (41.5-47.0 kDa) and a form of high molecular mass (86.1-92.2 kDa) that is either a novel tissue-type PA or a PA inhibitor which complexes with the lighter form.     

Effect of presence of a dominant follicle on the superovulatory response in buffalo (Bubalus bubalis)

Ten buffalo were superovulated by administration of 8 doses of FSH in a descending schedule spread over 4 d (5.5/5.5, 4.5/4.5, 3.5/3.5 and 2.5/2.5 mL, i.m.; total dose of 64 AU in 32 mL) beginning on Day 10 of an unstimulated estrous cycle, and 30 and 20 mg Lutalyse was given alongwith the 5th and 6th injections of FSH, respectively, to induce luteolysis. The number of corpora lutea (CL) was determined on 6 d post estrus. The ovaries were examined daily by ultrasonography from Day -5 to Day 5 (Day 0 = day of start of superovulation). The animals were retrospectively classified into 2 groups depending upon the presence (n = 4) or absence of a dominant follicle (n = 6). The mean diameter of the largest follicle (F1) increased from 8.25 +/- 0.48 mm on Day -5 to 10.75 +/- 0.25 mm on Day 0 in the dominant group, whereas in the nondominant group the F1 follicle exhibited a progressive decrease from 9.00 +/- 0.45 mm to 7.00 +/- 0.65 mm during the same period, the difference in profiles between the 2 groups was significant (P = 0.042). The profile of the diameter of the second largest follicle (F2) and the difference in diameters between largest and second largest follicles (F1-F2) were not significantly different between the 2 groups. The profile of mean number of large (> or = 10 mm diameter), but not small (2 to 5 mm diameter) or medium (6 to 9 mm diameter) follicles differed significantly (P = 0.001) between the 2 groups from Day -5 to Day 5 (P = 0.030). The number of CL was not significantly different between nondominant (4.00 +/- 0.97) and dominant groups (3.25 +/- 1.31). The number of CL was positively correlated (P < 0.01) with the number of medium follicles and the total number of follicles on the day of initiation of superovulation, but not with follicles of any size category or total number of follicles on any previous day. The results of this study indicate that following the use of morphological criteria based on the size of the largest follicle alone, the superovulation response is not affected by the presence of a dominant follicle at the initiation of superovulation in buffalo.     

A serum-free, cell-free culture system for development of bovine one-cell embryos up to blastocyst stage with improved viability

With the aim of developing a serum-free, cell-free culture system for embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 with the following supplements: 1) BSA alone (10 mg/ml); 2) BSA with ITS (5 mug/ml insulin, 5 mug/ml transferrin and 5 ng/ml selenium; BSAITS medium); 3) estrous cow serum alone (ECS; 10%); or 4) ECS with BOEC (bovine oviduct epithelial cells) (Experiment 1). In Experiment 2, embryos were cultured in BSAITS medium with or without feeding with fresh medium on Day 4 (day of insemination = Day 0). Embryos were evaluated on Day 2 for first cleavage, on Day 7 for morulae and blastocysts, and on Day 8 for blastocysts. Blastocysts from Experiment 1 were frozen in 10% glycerol in PBS, thawed and further cultured in ECS medium with BOEC for 48 h, and evaluated for formation of a distinct blastocoel, or expansion and hatching of blastocysts. In vivo-developed, Grade-1 and Grade-2, 7-d-old embryos served as control for the freezing, thawing and subsequent culture procedures. The percentage of first cleavage did not differ between the treatments (74 to 79% in Experiment 1 and 80 to 83% in Experiment 2). The percentage of blastocysts developed in BSAITS medium did not differ from that in ECS medium whether BOEC were present or not. However, medium with BSA alone had fewer blastocysts than any other culture system (P<0.05). Feeding embryos with fresh BSAITS medium on Day 4 did not lead to any further increase in the proportion of blastocysts. The culture systems had a significant effect on the post-thaw viability of blastocysts developed in them (P<0.001). Blastocysts developed in BSAITS medium had better (P<0.05) viability (14/38) than those from medium with ECS alone (1/27) or with ECS and BOEC (3/37). The post-thaw survival of control embryos was 80% (n=30). One of the three transfers of BSAITS-treated, frozen-thawed blastocysts resulted in a pregnancy. The results indicate that a serum-free, cell-free culture system can support the development of IVM-IVF bovine oocytes up to the blastocyst stage with better viability than a complex co-culture system.     

Isolation, characterization and immunocytochemical localization of bovine trophoblast protein-1

Bovine trophoblast protein-1 (bTP-1) was isolated to 90% purity from culture medium of Day 18-20 conceptuses incubated in vitro (in the presence of L-[3H]leucine) by a combination of Sephacryl S-200 gel filtration chromatography and O-(diethylaminoethyl) (DEAE) anion-exchange high-performance liquid chromatography (DEAE-HPLC). The radiolabeled protein had an Mr of 21,200 +/- 800 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had three isoelectric variants (pI 5.7-6.5) by two-dimensional SDS-PAGE. DEAE-HPLC-enriched bTP-1 cross-reacted with anti-o TP-1 serum on Western blots. A monospecific antiserum against bTP-1 was produced by excising the bTP-1 polypeptide band from preparative SDS-PAGE gels. Antiserum reacted with a single polypeptide with an Mr of 21,200 as determined by Western blotting of SDS-PAGE-separated conceptus medium proteins and by immunoprecipitation from L-[35S]methionine-labeled medium proteins followed by SDS-PAGE and autoradiography. Bovine trophoblast protein-1 was localized by immunocytochemistry in the cytoplasm of both mono- and binuclear trophectoderm cells of Day 20 bovine conceptuses, indicating that it is a product of the trophoblast.     

Developmental competence of bovine oocytes: effects of follicle size and the phase of follicular wave on in vitro embryo production

Developmental competence of bovine oocytes collected from follicles of different size categories (in either the growth or the dominant phase of the first follicular wave) was studied, with the aim of improving in vitro embryo production. Estrus and ovulation of 39 cyclic Holstein dairy cows were synchronized by two prostaglandin F2alpha treatments at 11-day intervals and one hCG treatment on the day of onset of estrus (Day 0). Cows with follicles in either the growth (Day 3, n=25) or the dominant phase (Day 7, n=14) were slaughtered, and follicles >5 mm were counted. Three oocyte populations were recovered separately from large (11-15 mm), medium (6-10 mm) and small (2-5 mm) follicles in both follicular phases. All collected cumulus-oocyte complexes (COC), except for markedly atretic oocytes without cumulus cells, were used in experiments. Oocytes were matured, fertilized and cultured by standard methods. There were no significant differences between the growth and the dominant phases for mean numbers of large follicles, usable oocytes and embryos per donor. Generally, those numbers were low, but the development rates of oocytes into blastocysts were high, particularly in the growth phase (60.0%). Mean (+/- S.E.M.) numbers of medium follicles, oocytes and embryos per donor were higher in the growth as compared with the dominant phase; in the usable oocytes and embryos, this difference was significant (9.6 +/- 1.4 and 3.5 +/- 0.6 versus 3.9 +/- 0.6 and 1.1 +/- 0.3; P<0.01). The development rates of oocytes into blastocysts, however, did not differ significantly between the growth and the dominant phases (36.7% versus 27.8%). Mean numbers of usable oocytes and embryos per donor recovered from small follicles in both follicular wave phases were similar. The development rate of oocytes into blastocysts was generally low, but higher (P<0.01) in the growth than in the dominant phase (24.5% versus 11.7%). Comparison between the two phases showed that mean number of all counted follicles and all usable oocytes collected per donor were similar, but the mean number of embryos per donor and the development rate of oocytes into blastocysts were higher in the growth phase than in the dominant phase (8.0 +/- 1.2 versus 3.8 +/- 2.4; P=0.012 and 30.3% versus 14.9%; P<0.01). The interaction between follicle size and the phase of follicular wave affected the efficiency of embryo production. The yield of embryos was primarily influenced by the number of oocytes collected from medium follicles and the developmental competence of oocytes from small follicles. The growth phase was more effective for oocyte collection; the number of oocytes from medium follicles and the developmental competence of oocytes from small follicles decreased in the dominant phase.     

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.     

Retinol-binding protein: a major secretory product of the pig conceptus

Pig conceptuses and endometrial explants recovered from gilts between Days 10 and 15 of pregnancy were cultured in leucine-deficient or methionine-deficient medium supplemented with 3H-leucine or 35S-methionine, respectively, for 30 h. Conceptus and endometrial tissues from Day 15 of pregnancy were fixed in Bouin's fixative for immunocytochemistry and light microscopy. Conceptus culture medium from Day 15 of pregnancy was pooled, dialyzed, and fractionated by anion exchange and gel filtration chromatography. A family of 3-5 low molecular weight (Mr) acidic (Mr = 19,000-22,000; pI = 5.6-6.5) 3H-leu-labeled proteins were isolated and identified by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), electroblotting, and fluorography. The two major proteins (pCSP-1 and pCSP-2) were excised from a polyvinylidene difluoride transfer membrane, and NH2-terminal amino acids were sequenced. One peptide was sequenced through 33 amino acids and the second, which shared 100% homology, was sequenced through 22 amino acids. Analysis of the larger sequence indicated that it shared 93.9% and 90.9% homology with the first 33 amino acids of human and rabbit plasma retinol-binding protein (RBP), respectively. Analyses of culture medium from pig conceptus incubations by 2D-PAGE and immunoprecipitation with rabbit anti-human RBP serum indicated that immunoreactive RBP was produced between Days 10 and 15 of pregnancy and was present in Day 30 allantoic fluid. Western blotting of enriched fractions of Day 15 conceptus RBP followed by immunostaining indicated that five isoforms of radiolabeled RBP were present. Immunoreactive RBP was detected in trophectoderm and yolk sac of conceptuses and endometrial surface and glandular epithelium at Day 15 of pregnancy. Results from this study demonstrate that pig conceptuses secrete RBP prior to onset of conceptus elongation and throughout the peri-implantation period, which suggests that RBP and associated retinoids influence conceptus development.     

Fibroblast growth factor requirements for in vitro development of bovine embryos

The overall goal was to describe the importance of fibroblast growth factors (FGFs) during development of the bovine embryo. An inhibitor of FGF receptor kinase activity (SU5402) was used to examine whether FGF signaling is required for embryo development. Addition of 20 μM SU5402 on Day 0 (Day of IVF) reduced (P = 0.04) the percentage of oocytes becoming blastocysts on Day 7 compared to controls (5.9 ± 2.1 vs 16.9 ± 2.4; average ± SEM). Also, Day-8 blastocysts placed into individual culture drops of medium containing SU5402 tended to have decreased (P = 0.08) blastomere numbers at Day 11 (211.1 ± 27.5 vs 297.8 ± 25.0). A second series of studies determined if supplemental FGF2 enhances development in vitro. There was no effect of FGF2 on cleavage or blastocyst development rates when 5 or 100 ng/mL FGF2 was provided immediately after fertilization. Also, FGF2 supplementation beginning on Day 5 post-fertilization did not significantly affect blastocyst rates or the number of trophoblast and inner cell mass cells. However, addition of 500 ng/mL FGF2 at both Day 0 and Day 4 increased (P = 0.03) the percentage of oocytes that became blastocysts on Day 7 compared with controls (27.4 ± 1.3 vs 19.7 ± 1.3). In a final study, the thermal-protective ability of FGF2 was examined by adding FGF2 1 h before exposing Day 5 embryos to heat shock. Addition of FGF2 did not significantly influence embryo thermal-tolerance. In conclusion, FGF receptor activation was important for optimal blastocyst formation and FGF2 supplementation increased bovine blastocyst formation when provided at high concentrations.     

Porcine conceptuses secrete an interferon during the preattachment period of early pregnancy

Maternal recognition of pregnancy in sheep and cattle is thought to be initiated by the conceptus secretory proteins ovine trophoblast protein-1 (oTP-1) and bovine trophoblast protein-1 (bTP-1), respectively. Recently, these proteins have been shown to be members of the interferon-alpha (IFN-alpha) family. In this study, we have examined whether pig conceptuses also produce IFN during early pregnancy. conceptuses were collected at Days 8, 10, 11, 12, 13, 15, and 17 of pregnancy and cultured in serum-free medium for 24 h. Day 11 conceptuses secreted a dominant 22,000-24,000 Mr cluster of acidic proteins (pI 5.2-5.4), that appeared to cross-react on immunoblots with antiserum against human IFN-alpha but not against oTP-1. Antiviral activity characteristic of an IFN was present in conceptus culture medium and uterine flushings from Day 11 through Day 17 of pregnancy, but was absent in flushings prior to Day 11 of pregnancy and in flushings from Day 12 nonpregnant gilts. The antiviral activity coeluted with a 22,000-24,000 Mr protein during partial purification through a gel filtration column. The activity was extremely labile, but could be restored by sequential protein denaturation, reduction, and renaturation. We conclude that production of IFN by early conceptuses is not restricted to ruminant species, and may therefore represent a more general phenomenon.     

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.     

Metabolism of progesterone by preimplantation mouse blastocysts in culture

This study examined the question whether or not preimplantation mouse blastocysts can metabolize progesterone (P). When young (Day 4) and implanting (Day 5) blastocysts were cultured in supplemented Eagle's minimum essential medium containing 0.4 microM [3H]P, metabolism of P and formation of metabolites were noticed at 10 h of culture. The metabolites accumulated in medium as the culture continued to 118 h. Three of the four metabolite fractions were identified, by crystallization to constant sp. act., to be 5 alpha-pregnane-3,20-dione and 3 beta-hydroxy-5 alpha-pregnan-20-one (or allopregnanolone), accounting for 22 and 57% of radioactivity, respectively, and a small amount (1-10%) of 3 alpha-hydroxy-5 alpha-pregnan-20-one. This suggests that both delta 4-5 alpha-reductase and 3 alpha- and 3 beta-hydroxysteroid dehydrogenase are active. Day 5 blastocysts were much more active than Day 4 blastocysts in P metabolism. It is suggested that the ability of blastocysts to metabolize P could produce the following effects in the adjacent endometrium: a lessening of P effects; and consequently a change in P-estrogen interaction; and possible effects from the metabolites. These local effects of embryos on the endometrium may be important for embryonic development and implantation.     

Differential patterns of blastulation in bovine morulae cultured in synthetic oviduct fluid medium containing FCS or BSA

This study examined the effects of fetal calf serum (FCS) supplementation of culture medium on blastulation and hatching of bovine morulae cultured in vitro. The presumptive zygotes derived from in vitro maturation and fertilization (IVM/IVF) were cultured in the modified synthetic oviduct fluid medium containing 3 mg/ml BSA (mSOF-BSA). At 120 h post insemination, morulae were randomly assigned to culture with mSOF-BSA (control) or mSOF containing 5% FCS (mSOF-FCS) instead of BSA. The replacement of BSA with FCS in mSOF significantly increased the percentage of blastocyst formation from Day 6 to Day 10 (Day 0 = the day of in vitro insemination) and the hatching rate of embryos on Days 8 and 9. The total number of cells in morulae and blastocysts on Day 6, in blastocysts on Day 7, and in blastocysts and hatched blastocysts on Day 8 were similar among the treatments. However, the replacement of BSA with FCS in mSOF significantly increased the total number of cells in hatched blastocysts on Day 10. Although the time of blastulation of embryos was significantly accelerated by the replacement of BSA with FCS in mSOF, the total number of cells in embryos at blastulation was lowered. The total number of cells in embryos at blastulation showed a time-dependent decrease when the embryos were cultured in mSOF-BSA. In contrast, the total number of cells in embryos that were cultured in mSOF-FCS depended little on the time after in vitro insemination. The results indicate that FCS supplementation of culture medium increased the percentage of embryos developing to the blastocyst stage without an increase in the total number of cells. However, an acceleration in the hatching rate and an increase in the total number of cells in hatched blastocysts were observed, compared with that in BSA-supplemented medium. It is suggested that FCS in the culture medium initiates earlier blastulation with fewer total numbers of cells in the morulae than BSA during in vitro culture of bovine embryos.     

Improved in vitro development of porcine embryos with different energy substrates and serum

The effect of replacing 5.5 mM glucose in North Carolina State University (NCSU)-23 medium with 0.5 mM pyruvate/5.0 mM lactate on porcine IVF embryo development was investigated in Experiment 1. Culturing embryos with pyruvate/lactate for 7 days or with pyruvate/lactate from Days 0 to 2, and then glucose from Days 2 to 7 improved cleavage rates. In Experiment 2, embryos were cultured for 7 days in pyruvate/lactate containing NCSU-23 medium supplemented with 0.05% PVA, 0.4% BSA or 10% fetal bovine serum (FBS). The BSA supplement increased the rates of cleavage, blastocyst formation, and the number of total cells in blastocysts. In Experiment 3, embryos were cultured in pyruvate/lactate containing NCSU-23 medium supplemented with 0.4% BSA for 7 days (BSA-PL), 0.4% BSA from Days 0 to 4 and then 10% FBS from Days 4 to 7 (BSA-PL-->F ) or 0.4% BSA from Days 0 to 7 with addition of 10% FBS (BSA-PL + F ) at Day 4. More blastocysts in BSA-PL--> F and hatching or hatched blastocysts in BSA-PL-->F and BSA-PL+F were obtained. Total cell number in blastocysts derived from BSA-PL-->F and BSA-PL+F were increased. Our results demonstrated that supplementing pyruvate/lactate containing NCSU-23 medium with 0.4% BSA for 4 days and replacing it with 10% FBS for another 3 days improved porcine IVF embryo development.