Effects of protein source and co-culture on bovine embryo development in synthetic oviductal fluid medium

Experiment 1 compared the development of 2- to 4-cell bovine embryos cultured in synthetic oviductal fluid with 20% fetal calf serum or 3.2% BSA and in the presence of oviductal cells, cumulus cells, or medium alone. More embryos developed in medium with serum, regardless of culture method (P = 0.063). Oviductal cell co-culture resulted in more embryos developing to at least the morula stage (P /= 0.400). Addition of serum to oviductal cell co-culture medium increased the number of excellent or good quality embryos (P = 0.019). Experiment 2 further compared the development of 2-cell or 3- to 4-cell embryos co-cultured with oviductal cell suspensions in serum-supplemented synthetic oviductal fluid or M-199 medium. More 3- to 4-cell than 2-cell embryos developed to at least the morula stage (P < 0.001). More embryos developed to at least the morula stage in synthetic oviductal fluid (P = 0.083). Neither initial embryo cell stage nor medium type influenced the percentage of developing embryos that achieved the blastocyst stage or final morphological quality of embryos (P >/= 0.535).     

Prostaglandin E2 secretion by oviductal transport-stage equine embryos.

This study was conducted to identify embryonic products whose secretion was temporally associated with the oviductal transport period of the mare. Chemicals secreted by oviductal-transport-stage equine embryos were identified by incubating Day 6 or Day 7 early uterine embryos with 35S-methionine/cysteine, 3H-progesterone, or 3H-arachidonic acid for 24 h, and subsequently identifying radioactively labeled proteins (SDS-PAGE; n = 3 embryos), steroids (HPLC; n = 3 embryos), or prostaglandins (HPLC; n = 3 embryos) in the culture medium. Early uterine embryos secreted 116.1 +/- 45.5 pg of prostaglandin (PG) E2/embryo, 1.0 +/- 0.2 pg of 17 alpha-hydroxy progesterone/embryo, 4.8 +/- 0.6 pg of androstenedione/embryo, and 11.5 +/- 4.5 pg of PGF2 alpha/embryo. They did not secrete detectable quantities of protein, testosterone, or estradiol-17 beta. A second experiment was conducted to measure temporal changes in embryonic PGE2 secretion during the oviductal and early uterine period. Day 3, Day 4, Day 5, and Day 6 embryos (n = 8 embryos/day) were incubated with 3H-arachidonic acid for 24 h, and the concentration of 3H-PGE2 in the culture medium was subsequently measured by HPLC. Embryos did not secrete detectable amounts of PGE2 prior to the expected time of oviductal transport (Day 3 and Day 4). They secreted 5.7 +/- 1.0 pg of PGE2/embryo immediately before and during the expected time of oviductal transport (Day 5), and they secreted significantly of PGE2/embryo immediately before and during the expected time of oviductal transport (Day 5), and they secreted significantly (p less than 0.01) higher amounts (42.0 +/- 11.5 pg) of PGE2/embryo immediately after uterine entry (Day 6).(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro development of day-2 equine embryos co-cultured with oviductal explants or trophoblastic vesicles

This study compared the in vitro development of Day-2 equine embryos co-cultured with either trophoblastic vesicles or oviductal explants. Embryos were collected surgically from the oviducts of pony mares 2 d after ovulation and assessed for stage of development. Culture medium was Ham's F12 and Dulbecco's Modified Eagle's Medium (50:50 v/v) in a humidified atmosphere of 5% CO(2) in air at 38.5 degrees C with either trophoblastic vesicles or oviductal explants. The quality score of embryos was assessed daily. After 4 d in culture, embryos were stained (Hoechst 33342) and evaluated with epifluorescence to determine the number of nuclei present. Six of seven embryos co-cultured with oviductal exmplants developed to the morula/blastocyst stage, while four of seven embryos co-cultured with trophoblastic vesicles developed to the morula stage. More (P = 0.1) embryos co-cultured with oviductal explants reached the blastocyst stage than embryos co-cultured with trophoblastic vesicles (3 7 vs 0 7 , respectively). The number of cells was higher (P = 0.1) for embryos co-cultured with oviductal explants than for embryos co-cultured with trophoblastic vesicles (162.6 +/- 32 vs 87.3 +/- 28, respectively). The number of cells for embryos co-cultured with either oviductal explants or trophoblastic vesicles appeared to be lower than for embryos matured in vivo that were recovered from the uterus at Day 6 (378, 399, >1000). The co-culture of early equine embryos in a completely defined medium with either trophoblastic vesicles or oviductal explants can support development to at least the morula stage. The co-culture of embryos with oviductal explants resulted in superior development of four-to eight-cell embryos, as indicated by the proportion that reached the blastocyst stage and by the number of cells.     

In vitro development of bovine one-cell embryos: Influence of glucose, lactate, pyruvate, amino acids and vitamins

To elucidate the effect of nutrient substrates on embryo development, in vitro fertilized bovine one-cell embryos were cultured in a medium similar to synthetic oviduct fluid (SOF) but without glucose and containing 3.3 mM lactate, 0.3 mM pyruvate and 3 mg/ml bovine serum albumin (BSA) at 39 degrees C in 5% CO(2) in air. Results indicated that addition of glucose was not only unnecessary, but it also had a deleterious effect on embryo development to the morula stage. Lactate supported embryo development up to the morula stage as well as pyruvate. Supplementation with 20 amino acids contained in basal medium Eagle's (BME) and minimum essential medium (MEM) improved development to the morula stage dramatically and increased the cell number compared with that of the controls. Addition of the vitamins from MEM to SOF had no beneficial effect. The SOF with amino acids did not increase the frequency of blastocysts 7 days after in-vitro fertilization but did increase the total number of cells compared with that of the controls. Frequency of blastocysts at Day 7 in SOF with amino acids was equivalent to that of co-culture although the total cell number was lower. These results demonstrate that a semi-chemically defined medium can successfully support the development of bovine embryos to the morula stage to a limited extent, but the medium lacks some nutrients or growth factors to fully support development through the blastocyst stage.     

Culture of one-cell bovine embryos in explanted mouse oviduct and bovine oviductal epithelial cells

One-cell bovine embryos fertilized in vivo were cultured in TCM-199 and bovine oviductal epithelial cells, in TCM-199, or in explanted immature mouse oviducts supported by TCM-199 to compare development to the blastocyst stage. The morphological stage of development and cell number were determined following 144 hours of culture. Of the embryos that cleaved at least once, 52.6, 30.4 and 0.0% developed to the morula/blastocyst stage after culture in oviductal epithelial cells, in TCM-199 alone, or in explanted mouse oviducts, respectively. The mean total cell number for embryos cultured in oviductal epithelial cells (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<0.05). The mean cell number of embryos cultured in TCM-199 or in explanted mouse oviducts did not differ. The explanted immature mouse oviduct supported by TCM-199 did not provide an environment adequate for development of one-cell bovine embryos to the blastocyst stage. Development of one-cell bovine embryos was best supported by co-culture with oviductal epithelial cells in TCM-199 medium.     

Differential growth, cell proliferation, and apoptosis of mouse embryo in various culture media and in coculture

Sequential culture and coculture are two methods of improving the development of preimplantation embryos in vitro. Direct comparison of the efficiency of these methods is limited. Proliferation and apoptosis determine the total number of blastomere in preimplantation embryo, which is a sensitive parameter for evaluation of the development of embryo in vitro. In this study, we compared the proliferation and apoptosis of mouse embryo in different culture media, including CZB, KSOM, MTF, G1.2/G2.2 sequential culture media, and in human oviductal cell coculture. Sequential culture using G1.2/G2.2 was superior to KSOM, MTF, and CZB/CZB + G with respect to the formation of 3-4 cell embryos, morula, and blastocyst. G1.2/G2.2 cultured blastocyst had significantly more proliferating blastomeres and higher total cell number per blastocyst than those cultured in KSOM or CZB/CZB + G. Compared to embryos cultured in G1.2/G2.2, embryos cocultured in G1.2/G2.2 hatched more frequently. Cocultured blastocysts also had significantly higher percentage of proliferating cell and lower percentage of apoptotic cell per blastocyst than those cultured in G1.2/G2.2. It was concluded that G1.2/G2.2 facilitated the proliferation of blastomere whilst human oviductal cell coculture suppressed apoptosis in addition to stimulating proliferation of blastomere.     

Transfer and localisation of maternal serum antigens by mouse preimplantation embryos and oviductal epithelium.

Two hours after systemic injection of bovine plasma albumin (BPA) into pregnant mice, albumin-like antigen was detected by indirect immunohistological methods within the cytoplasm of oviductal and preimplantation uterine embryos whether ovulation was spontaneous or induced by hormone injection. Although fluorescence, localising antigen similar to or identical with the systemically injected foreign protein, was present in embryos in all oviductal regions and at all cleavage stages, the intraembryonic location of the transferred serum molecules differed from embryo stage. Most ootids and two-celled blastomeres contained large intracytoplasmic areas of intense fluorescence randomly associated with non-fluorescent or dimly fluorescent areas in the same cell. By four- and eight-celled stages, albumin-like antigen was localised at the periphery of blastomeres; less was found deep within embryos. By morula and blastocyst stages, blastomeres differed from each other in fluorescence intensity although intracellular fluorescence was homogeneous. Transferred BPA antigen, present in both pronuclei, probably was absent from blastomere nuclei. Ootid zonae pellucidae contained BPA antigen but none was detected in zonae surrounding cleaving embryos. Little foreign albumin was detected in oviductal epithelium. It is concluded that morphological, as well as biochemical, differentiation occurs during mammalian cleavage and it is suggested that maternal macromolecular contributions to mammalian preimplantation embryos may be necessary for normal development in vivo.     

Oviductal fluid and growth factors failed to enhance development of porcine embryos

Porcine embryos (1-, 2- and 4-cell) were cultured in a basal medium consisting of Krebs-Ringer bicarbonate buffer supplemented with oviductal fluid and several growth factors and observed for further development. Oviducts were flushed at either 48 h (Experiment 1) or 96 h (Experiment 2) after the onset of estrus. Observations were made every 48 h (Experiment 1) or 12 h (Experiment 2) until failure of the embryos to develop for 2 consecutive observations. Embryos were scored 0 = no development, 1 = cleavage, 2 = morula, 3 = blastocyst, or 4 = hatched blastocyst. In the first experiment, development of 1-, 2- and 4-cell embryos (n=282) in the basal medium supplemented with oviductal fluid (4:1) or 3 sets of growth factors, was less or equal to one cleavage stage. Those embryos cultured in the basal medium supplemented with bovine serum albumin (fatty acid free) (BSA) advanced to the blastocyst stage. In the second experiment, 96 h aged embryos (n=142) were cultured in the basal medium supplemented with IGF-1 and - 2 and EGF, or with BSA alone or with BSA and the three growth factors. In the treatments without BSA, the embryonic development was less than one cleavage, whereas in those treatments with BSA, embryos advanced beyond hatching and began to expand. We conclude that for culture of porcine embryos, supplementation with several growth factors or with oviductal fluid, in the concentration used in this study, was of little benefit at this stage of development. However, the type of BSA significantly affected development. More than 90% of the embryos reached the morula and blastocyst stages in medium than included BSA (fatty acid free).     

Concentration and composition of free amino acids and osmolalities of porcine oviductal and uterine fluid and their effects on development of porcine IVF embryos

The concentration of free amino acids and the osmolalities in porcine oviductal (OF) and uterine fluids (UFs) on day 3 (D3) and day 5 (D5) were measured by HPLC and Vapor Pressure Osmometer, respectively. Based on these measurements we designed new media based on PZM3 by modifying the amino acid composition and osmolality. The effectiveness of the modified PZM3 on the development of porcine IVF embryos was then investigated. A total of 24 free amino acids were measured, including 20 protein and 4 nonprotein amino acids (beta-alanine, taurine, ornithine, and citrulline). There was no significant difference in the total concentration of amino acids among D3OF (13.06 +/- 3.63 mmol/L), D3UF (10.54 +/- 5.16 mmol/L), or D5UF (10.23 +/- 6.69 mmol/L). But the total concentration of amino acids in D5OF (5.89 +/- 1.47 mmol/L) was significantly lower than the three fluids above. Some individual amino acids varied significantly depending on where they were collected and from which day. The blastocyst rates of porcine IVF embryos were not improved when embryos were cultured in PZM3 with amino acids at D3OF (PZM3-D3OF, 20.3 +/- 7.9%) or D5UF (PZM3-D5UF, 14.3 +/- 10.7%) concentrations or in PZM3-D3OF for the first 48 (20.5 +/- 15.1), 72 (25.6 +/- 10.4), and 96 (18.7 +/- 10.0) hr and then transferred into PZM3-D5UF compared with PZM3 with Sigma amino acid solution (PZM3-SAA) (30.8 +/- 9.1%). However, when IVF embryos were cultured in PZM3-D5UF, the average nuclear number per blastocyst (57.6 +/- 8.3) was increased compared to PZM3-SAA (40.5 +/- 3.5). The osmolalities in D3OF, D3UF, D5OF, and D5UF were 318 +/- 8, 320 +/- 32, 321, and 293 +/- 8 mOsM, respectively. When the IVF embryos were cultured in PZM3-SAA and PZM3-D3OF at a variety of osmolalities (150-360 mOsM), higher blastocyst rates were obtained at 270-300 mOsM in the PZM3-SAA group (24.6-33.9%) and 270-290 mOsM in PZM3-D3OF group (22.4-24.2%). The blastocyst rate gradually decreased when the osmolality was increased or decreased in both groups. When the embryos were cultured in PZM3-SAA at 330 mOsM for the first 72 hr and then transferred to 250 mOsM (33.3 +/- 3.4%), the blastocyst rate was higher than original PZM3 (21.2 +/- 2.2%) (288 mOsM).     

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)     

Birth of lambs after in vitro maturation, fertilization, and coculture with oviductal cells.

Control ovine oocytes matured and fertilized in vitro were transferred to intermediate recipient ewes. After 5 days, 59% of eggs were recovered. Thirty-one (38%) reached morula/blastocyst stage. Twenty-one embryos at the morula or blastocyst stage were transferred to six recipient ewes, resulting in five pregnancies, of which four were maintained. Nine lambs were born (43%). In the experiment, 72 ooctyes matured and fertilized in vitro were cocultured for 5 days with sheep oviductal epithelial cells. Thirty-one eggs (43%) developed to the noncompacted morula stage. Transfer of 26 embryos to 11 recipient ewes resulted in two pregnancies (18%). Two male lambs were born. The result indicates that the coculture of in vitro matured and fertilized ovine eggs with sheep oviductal epithelial cells throughout the preimplantation period is compatible with further development to term.     

Early embryonic development in Thai swamp buffalo (Bubalus bubalis )

A total of 33 nonsurgical embryo collections was carried out to investigate early embryo development in Thai swamp buffalo. Collections were performed on Days 5.5, 6.0, 6.5, 7.0 and 7.5. The different stages of embryo development on these days were the 16-cell stage, compact morula, blastocyst, hatched blastocyst and hatched expanding blastocyst, respectively. In addition, some degenerating embryos and unfertilized ova were also recovered. A higher recovery rate was obtained with single embryo collection after natural estrus than after induced estrus or superovulation, 78% (7 9 ) vs 46% (6 13 ) vs 54.5% (6 11 ), respectively. A higher percentage of normal embryos was also obtained with single embryo collection after either natural or induced estrus than after superovulation, 71% (5 7 ), 83% (5 6 ) and 38% (6 16 ), respectively.     

Effects of low molecular weight oviductal factors on the development of mouse one-cell embryos in vitro.

The relationship between the oviduct and embryo development in the mouse was investigated and the period at which the influence of oviduct can be concerned in the development of mouse embryos in vitro was identified. In addition, the relative molecular weight of oviductal factors that promote embryo development was demonstrated. Mouse zygotes developed to the blastocyst stage when co-cultured with ampulla. The period of embryo co-culture significantly affected the further development of the embryos. Fewer one-cell embryos co-cultured with dissected ampullae for less than 24 h developed to blastocysts than those co-cultured for more than 28 h (P < 0.001). A high percentage of embryos co-cultured with ampullae after 24 h of culture in vitro developed to the blastocyst stage, which suggests that the influences of ampulla on the development of mouse embryos are restricted to a specific period at the two-cell stage (about 55-56 h after hCG injection) in vitro. Mouse ova that were cultured in media conditioned by ampullae could also develop to the blastocyst stage. The fractionated medium that contained low molecular weight fractions was more effective (P < 0.001) on the development of embryos to the blastocyst stage than that containing high molecular weight fractions. These results suggest that the low molecular weight oviductal factors play an important role in the development of mouse embryos at a certain critical age in vitro.     

Uptake and metabolism of pyruvate and glucose by individual sheep preattachment embryos developed in vivo

The uptake of pyruvate and glucose by individual sheep oocytes and preattachment sheep embryos at each state of development up to the hatching blastocyst was determined using a microfluorescence technique. After an initial increase at fertilization, pyruvate uptake was relatively constant (?15 pmol/embryo/h) from the zygote through to the morula. Upon blastocyst formation and hatching, there were significant increases in uptake (39 pmol/embryo/h, P < 0.001; and 53 pmol/embryo/h, P < 0.001, respectively). In contrast to that of pyruvate, glucose uptake was very low (?1 pmol/embryo/h) up to the time of genome activation (eight- to 16 cell stage), after which there were significant increases in uptake at each successive stage of development. By the hatching blastocyst stage, glucose uptake had reached 54 pmol/embryo/h. The ability of day-7 hatching blastocysts to oxidize pyruvate and glucose was determined indirectly by measuring the production of lactate when either substrate was present as the sole energy source. Unlike the mouse blastocyst, which has a considerable oxidative capacity for both pyruvate and glucose, the day-7 sheep blastocyst showed limited ability to oxidise either substrate. Rather, in the sheep blastocyst, 65% of pyruvate and 98% of glucose taken up could be accounted for as lactate. Such low levels of substrate oxidation appear to be inconsistent with the energy requirements of the proliferating preattachment ruminant blastocyst. The utilization of alternative substrates at the blastocyst, such as amino acids, is proposed. © 1993 Wiley-Liss, Inc.     

Amino acid concentrations in fluids from the bovine oviduct and uterus and in KSOM-based culture media.

Amino acids in bovine oviductal and uterine fluids were measured and compared with those in modified simplex optimized medium (KSOM) supplemented with either fetal calf serum or Minimum Essential Medium amino acids in addition to bovine serum albumin, fetal calf serum or polyvinyl alcohol. Concentrations of cysteine, threonine, tryptophan, alanine, aspartate, glycine, glutamate, proline, beta-alanine, and citrulline were higher in oviductal fluids than in KSOM-based culture media. Nonessential and essential amino acids were present in ratios of 5:1 and 2:1 in oviductal and uterine fluids, respectively. Concentrations of alanine (3.7 mM), glycine (14.1 mM) and glutamate (5.5 mM) were high in oviductal fluids, comprising 73% of the free amino acid pool. Of the amino acids measured in uterine fluids, alanine (3.1 mM), glycine (12.0 mM), glutamate (4.2 mM), and serine (2.7 mM) were highest in concentration, and the first three comprised 43% of the free amino acid pool. In conclusion, amino acid concentrations in the bovine reproductive tract were substantially higher than those in embryo culture media. Certain amino acids, particularly alanine, glutamate, glycine and taurine, are present in strikingly high concentrations in both oviductal and uterine fluids, suggesting that they might play important roles in early embryo development. The particular pattern of amino acid concentrations may be an important factor to be considered for the improvement of embryo culture media.     

Development of in vitro matured and fertilized bovine embryos cocultured with bovine oviductal epithelial cells obtained from oviducts ipsilateral to cystic follicles.

The present experiment was conducted to clarify the effect of bovine oviductal epithelial cells (BOEC) collected from oviducts ipsilateral to cystic follicles (CFs) using an in vitro coculture system on the development of in vitro matured/fertilized (IVM/IVF) bovine embryos. In the first comparison, the effect of the presence of CF on the development of the embryos cocultured with BOEC derived from the cows with CF (n = 18) and corpus hemorrhagicum (CH, n = 10) was examined. In the second comparison, the effect of the type of cyst [progesterone (P4)-dominant; n = 9, estradiol-17beta (E2)-dominant; n = 5] on the development of the embryos cocultured with BOEC derived from the cystic cows was examined. No difference was observed between CF and CH (control) groups in the mean developmental rates of embryos developed to > or =2-cell (86.3% vs. 86.4%), 8-16 cells (53.0% vs. 56.2%), blastocyst (24.2% vs. 24.8%) and hatched blastocyst (12.0% vs. 14.6%). However, the blastocyst production rate was significantly different (P<0.05) between the P4-dominant (19.8%) and E2-dominant (32.6%) groups. The rate of development from cleavage stage embryo to blastocyst was significantly different between P4-dominant (22.9%) and E2-dominant (37.9%) groups. Moreover, the blastocyst rate from 8-16 cells of E2-dominant group (61.6%) was significantly higher than that of P4-dominant one (39.5%). These results indicate that the effects of BOEC collected from oviduct ipsilateral to CFs on embryo development are variable, and the variability is closely associated with the steroid hormone profiles of the follicular fluid.     

Porcine oviductal cells support in vitro bovine embryo development

This study was designed to investigate the developmental competency of in vitro-matured and in vitro-fertilized bovine embryos co-cultured with a) medium alone, b) bovine oviductal cells (BOC), c) bovine conditioned medium (BCM), d) porcine oviductal cells (POC), and porcine conditioned medium (PCM). Follicular oocytes collected from cattle at local slaughterhouses were matured and fertilized in vitro. Epithelial cells were scraped from the luminal surface tissue of either bovine or porcine oviducts collected after ovulation, cultured in TALP + 10% heat-treated fetal calf serum, and the conditioned media were collected following a 3- to 5-d incubation period. After 18 to 22 h of sperm-ova co-incubation, the fertilized and/or cleaved ova were randomly assigned to 1 of 5 co-culture groups. The results revealed that the efficiency of medium alone in supporting embryo development from the 16- to 32-cell stage up to the blastocyst stage was significantly (P<0.01) lower than of embryos co-cultured with either bovine or porcine epithelial cells, or with conditioned media from such cells. Epithelial cell co-culture, regardless of cell source, was more effective (P<0.01) than culture with conditioned medium. Co-culture in medium containing or conditioned by porcine cells was more effective in supporting bovine embryo development than co-culture with bovine-derived cells or conditioned medium. These data support the concept that oviductal cells produce a soluble component which enhances embryo development to the blastocyst stage in vitro and that the effect is not species-specific.     

Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova.

Almost 30 years after the first successful in vitro fertilization (IVF) in golden hamsters (Mesocricetus auratus), we report that IVF hamster embryos can develop in a chemically defined, protein-free culture medium into morulae and blastocysts, and produce normal offspring after transfer to recipients. When examined 96 h post-insemination, 82% (160/200) of IVF ova had cleaved to at least 2 cells, 55% (97/200) had developed beyond the 4-cell stage, and 22% (38/200) had developed into morulae/blastocysts. In vitro development of IVF embryos to greater than or equal to 8 cells was absolutely dependent on hypotaurine. Twenty living offspring were produced from transfer of IVF embryos to recipients, with an overall success rate of 5% and 17% for oviductal (2-cell) and uterine (8-cell/morulae) transfers, respectively. In vivo-fertilized pronucleate embryos collected 3 h after egg activation were less able to develop in vitro than embryos collected only 6 h later, revealing a critical influence of the oviduct within the first hours of embryo development. Hypotaurine partly compensated for the decreased oviductal exposure of early 1-cell embryos. Establishment of a key role for hypotaurine in hamster embryo development, support of IVF embryos to morula/blastocyst stages in vitro, and production of living offspring after IVF embryo transfer are significant steps towards the goal of obtaining comparative data on preimplantation embryogenesis.     

Development of in vitro fertilized oocytes from pregnant and nonpregnant cows in oviductal epithelial and cumulus cell co-culture systems

The objectives of this study were 1) to measure cleavage, blastocyst formation, and blastocyst hatching after in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes aspirated from pregnant versus nonpregnant cows, and 2) to compare embryo development in co-culture with bovine oviductal epithelial cells versus cumulus cells. No differences in cleavage (38 versus 40%), blastocyst formation (13 versus 13%), or blastocyst hatching (53 versus 51%) were observed for in vitro-matured, fertilized, and cultured oocytes from pregnant versus nonpregnant cows, respectively (P > 0.05), indicating that nonpregnant and early-pregnant cows are equally acceptable donors of oocytes for IVM/IVF/IVC procedures. Cleavage (36 versus 40%), blastocyst formation (11 versus 12%), and blastocyst hatching (50 versus 55%) were not different for embryos co-cultured with oviductal epithelial cells versus cumulus cells (P > 0.05). Thus, equivalent embryo development can be obtained with co-culture systems commonly used for in vitro-derived bovine embryos. These results help to define variables that affect comparison of results across laboratories and that are relevant to the practical application of IVM/IVF/IVC procedures to cattle.     

Temporal effect of human oviductal cell and its derived embryotrophic factors on mouse embryo development.

Mouse embryos at different stages of development were cocultured with human oviduct cells or cultured in the presence of oviduct-derived embryotrophic factor-1, -2, and -3 (ETF-1, -2, and -3) for various amounts of time within the preimplantation period. Cocultures that included the period from 48 to 72 h post-hCG stimulated cell division and increased the cell numbers in the inner cell mass (ICM) of the exposed blastocyst. Exposure of embryos to oviductal cells from 96 to 120 h post-hCG increased the cell number in the trophectoderm (TE), blastocyst size, hatching rate, attachment, and in vitro spreading of the blastocyst. ETF-1 and ETF-2 affected embryos between 48 and 72 h post-hCG by increasing the number of cells in the ICM. In contrast, ETF-3 had a more profound effect on embryos that were exposed from 96 to 120 h post-hCG, where it mostly affected the development of TE cells, leading to higher hatching rate. Human oviductal cells improved mouse embryo development partly by the production of high molecular weight embryotrophic factors. These factors had differential effects on mouse embryo development.