Effects of feeding OmniGen-AF® on superovulatory response in donor beef cows: I. Serum progesterone and cortisol, embryo recovery and quality.

Optimal results in cattle embryo transfer are limited by the variation in ova recovery, fertilization rate and embryo quality experienced with superovulation. Inflammation and immune dysregulation may be contributing factors. This study, evaluated feeding OmniGen-AF® (OG), a nutritional supplement that reduces inflammation and supports immune health, on superovulatory response and serum progesterone and cortisol concentrations in embryo donors treated with two different doses of Folltropin®-V (FSH). Angus cross-bred beef cows (n = 24) were assigned to four groups, fed OG at 0 or 56 g/animal/day for 49 days and were treated with 200 or 400 mg FSH to induce superovulation. Treatments for superovulation started after feeding OG for 28 days and ova were non-surgically recovered 7 days after estrus and graded for quality. More transferrable embryos (P < 0.05) were recovered from cows fed 56 g OG and treated with 400 compared with 200 mg FSH. Percent degenerate embryos recovered from cows treated with the 400 mg FSH dose was threefold greater (P < 0.05) when fed no OG compared with 56 g OG. Serum progesterone on day of embryo collection was greater (P < 0.05) in OG-supplemented cows and cows treated with 200 mg FSH. Serum cortisol was not affected (P > 0.10) by FSH dose or OG-feeding, but was greatest (P <  0.05) on Days 0 and 42 of the feeding period. In summary, the improvement in embryo quality with OG-feeding may relate to a greater serum progesterone concentration.

Factors affecting cellular outgrowth from porcine inner cell masses in vitro.

During early embryonic development, endodermal cells leave the inner cell mass (ICM) and migrate over an extracellular matrix located on the blastocoelic side of the trophectoderm to form extraembryonic endoderm. Two experiments were conducted to evaluate factors supporting porcine endodermal cell migration in vitro. In Exp. 1, porcine ICM were cultured on matrices of collagen IV, fibronectin, or laminin. Percentages of ICM generating cellular outgrowth on fibronectin (5/11; 45%) and laminin (4/10; 40%) were similar (P > 0.10); however, collagen IV (0/10; 0%) failed (P < 0.05) to support cellular outgrowth. Inner cell mass and outgrowth areas and numbers of cells in outgrowths were similar (P > 0.10) for fibronectin and laminin, and increased (P < 0.05) with time in culture. In Exp. 2, ICM were cultured on fibronectin or laminin in medium containing 0 or 500 microg/mL of the inhibitory tripeptide, arg-gly-asp (RGD), or on laminin in medium containing 0 or 10 microg/mL recombinant human tissue inhibitor of matrix metalloproteinases-2 (rhTIMP-2). Inner cell mass and outgrowth areas and numbers of cells in the outgrowths for ICM cultured on fibronectin did not differ (P > 0.10) due to the presence of RGD. Inner cell masses cultured on laminin in medium containing 500 microg/mL RGD had fewer cells in the outgrowths and slower rates of cell migration compared with 0 microg/mL (P < 0.05). No differences (P > 0.10) in ICM and outgrowth areas and numbers of cells in the outgrowths were observed for ICM cultured on laminin in medium containing 0 or 10 microg/mL rhTIMP-2. Both fibronectin and laminin supported porcine ICM outgrowth in vitro; however, because outgrowth on fibronectin was not inhibited by RGD, endodermal cells must express an integrin that recognizes an alternative sequence in fibronectin. Cell migration on laminin was inhibited by RGD, suggesting either RGD competes with laminin for binding sites on endodermal cells or binding RGD alters endodermal cell migration on laminin. Because rhTIMP-2 had no effect on cell outgrowth, porcine ICM do not appear to be responsive to the proliferative effects of rhTIMP-2.

Survival of oocytes recovered from vitrified sheep ovarian tissues.

The objective of this work was to develop an effective vitrification technique for cryopreserving oocytes in sheep ovarian tissues. Ovaries were surgically recovered from 15 pubertal ewes and the ovarian cortex was cut into sections. Ovarian tissues were placed in equilibration medium consisting of 4% (v/v) ethylene glycol (EG) and 20% (v/v) FBS in TCM-199 on ice for 30 min and transferred to vitrification solution (35% EG, 5% polyvinylpyrrolidone, 0.4M trehalose and 20% FBS in TCM-199) for 5 min. Ovarian tissues were vitrified by dropping the tissue on the surface of a steel cube cooled by liquid nitrogen. Cumulus-enclosed oocyte complexes (COC) were also collected and vitrified following the procedure used for ovarian tissues. After 2-3 weeks of storage in liquid nitrogen, ovarian tissues and COC were thawed at 37 degrees C in 0.3M trehalose and COC in ovarian tissues were mechanically and enzymatically isolated. Vitrified COC and freshly collected COC were washed twice in maturation medium (TCM-199 supplemented with 0.255 mM pyruvate and 10% heat-treated estrus cow serum) and cultured in 50 microl drops of maturation medium under paraffin oil for 23-25h at 39 degrees C in a humidified atmosphere of 5% CO(2) in air. After culture, cumulus cells were removed by hyaluronidase treatment and vortexing and oocytes were fixed and stained. No significant differences were observed between vitrified oocytes, oocytes recovered from vitrified ovarian tissues and non-vitrified control oocytes in the percentage of oocytes with acceptable staining per total number of oocytes fixed or with visible chromatin per total number of oocytes with acceptable staining. However, fewer (P<0.05) oocytes obtained from vitrified ovarian tissues (70%) reached metaphase II compared to vitrified oocytes (88%) and non-vitrified control oocytes (90%). In contrast, when oocytes with at least 3-5 layers of cumulus cells were considered from each of the three groups, no differences (P>0.05) were observed due to treatment in the percentages of oocytes developing to metaphase II. These results demonstrate that sheep oocytes can be successfully cryopreserved by vitrification of ovarian tissues and exhibit in vitro maturation rates similar to that of vitrified and non-vitrified oocytes.

Cathepsin L: a predominant heat-activated proteinase in arrowtooth flounder muscle.

Characterization of the autolytic profile of arrowtooth flounder (ATF) muscle indicated the involvement of heat-activated proteinases active at both acidic and alkaline pH values. Further assay of fish extract exhibited the maximum activity at 60 degrees C against casein used as a substrate at both pH 5.5 and 8.0. The maximum activity shifted to lower temperatures by the addition of urea with two distinctive patterns: activity reduction at pH 5.5 and activity enhancement at pH 8.0. The highest inhibition by E-64 indicated the proteinase belongs to the cysteine proteinase class. At pH 5.5, the proteinase hydrolyzed Z-Phe-Arg-NMec and all types of protein substrates tested at higher rate than that at pH 8.0. Activity bands, observed on the activity-stained substrate gels, indicated similar proteinases are responsible for the proteolytic activity observed at both pH values. When proteins of fish extract were separated by HPLC-SEC, only one proteolytic peak was observed at the retention time of 26 min with an estimated molecular weight of 39800 Da. The results implied cathepsin L is a predominant proteinase responsible for autolysis of ATF muscle at elevated temperatures.

Changes in the bovine zona pellucida induced by plasmin or embryonic plasminogen activator.

Effects of bovine plasmin and plasminogen activator recovered from bovine embryo-conditioned medium (bePA) on the polypeptide profile and solubility of bovine zonae pellucidae (ZP) were evaluated. ZP were isolated from bovine ovarian oocytes and incubated at 39 degrees C with 0, 100, or 200 microg/ml plasmin for 0, 24, or 48 hr or bePA with 0 or 100 microg/ml human plasminogen for 0 or 48 hr. ZP were evaluated either by SDS-PAGE or for changes in solubility using a zona pellucida dissolution time (ZPDT) assay. Two prominent polypeptides, molecular weight (MW) 76,000 and 65,000, and two minor polypeptides, MW 23,000 and 22,000, were resolved by SDS-PAGE. No changes occurred in the polypeptide profile for ZP incubated with 0 microg/ml plasmin for 0, 24, or 48 hr, and ZPDT did not differ (P > 0.10). Treatment with 100 or 200 microg/ml plasmin induced reductions in the MW 76,000, 23,000, and 22,000 polypeptides and the appearance of MW 45,000 and <10,000 polypeptides. ZPDT were less (P < 0.05) in 100 and 200 microg/ml compared with 0 microg/ml plasmin. Polypeptide profiles and ZPDT for ZP incubated with bePA were similar (P > 0.10) to ZP incubated with unconditioned medium. Addition of human plasminogen to ZP incubated with bePA reduced the MW 76,000, 23,000, and 22,000 polypeptides, caused the appearance of MW 45,000 and 20,000 polypeptides, and decreased ZPDT (P < 0.05). These results demonstrate that bovine plasmin is capable of proteolytically degrading the bovine ZP and that bePA can indirectly affect the ZP by converting plasminogen to plasmin.

Expression of proteinases and proteinase inhibitors during embryo-uterine contact in the pig.

Implantation in pigs is noninvasive and characterized by interdigitation of embryonic and endometrial epithelial cell processes. However, when pig embryos are transferred to ectopic sites, trophoblast becomes invasive. The objective of this study was to evaluate expression of proteinases and proteinase inhibitors in pig embryos and uteri at the time of endometrial attachment. RNA was extracted from Day 15.75 pig embryos and uteri and reverse transcribed, and cDNA was amplified by polymerase chain reactions using primers specific for urokinase-type plasminogen activator (uPA), matrix metalloproteinases-2 and -9 (MMP-2 and -9), and tissue inhibitors of MMP-1, -2, and -3 (TIMP-1, -2, and -3). Localization of transcripts for the genes of interest in embryos and uteri was performed using in situ hybridization with antisense riboprobes. Day 15.75 pig embryos and uteri expressed transcripts for uPA, MMP-2 and -9, and TIMP-1, -2, and -3. In situ hybridization revealed weak expression of uPA in the trophectoderm and moderate expression in the adjacent extraembryonic endoderm. TIMP-1 transcripts were abundant in extraembryonic endoderm and scattered throughout the trophectoderm. TIMP-2 appeared to be expressed in all cells of the embryo. TIMP-3 expression was observed in the trophectoderm and, to a lesser extent, in the extraembryonic endoderm. Specific localization of MMP-2 and -9 transcripts above background was not observed by in situ hybridization in either embryos or uterus. Uterine expression of uPA and TIMP-1, -2 and -3 was localized to the endometrial stroma. Transcripts of these genes were not observed in either the luminal or glandular endometrial epithelium. These results suggest that pig embryos and uteri express a wide array of proteinases and proteinase inhibitors during the period of uterine association. The abundant expression of proteinases and proteinase inhibitors during the period of uterine association. The abundant expression of TIMP in pig embryos may partially explain the absence of invasive implantation in this species in contrast to implantation typified by rodents and primates.

Effect of different concentrations of potassium on pig embryos developing in vitro.

The effects of varying the potassium concentration in Whitten's medium on the development of pig embryos in vitro was investigated. Osmolality was maintained by adjusting the NaCl concentration. In Expt I, 209 one-cell to early blastocyst stage embryos were individually cultured in 50 microliters microdrops of glucose-free Whitten's medium containing 0.4% (w/v) BSA and 1.5, 3.0, 6.0 or 12.0 mmol potassium l-1. The percentages of embryos undergoing blastocoel formation and expansion for each cell stage evaluated did not differ (P > 0.10) among the various concentrations of potassium. However, more four-cell embryos commenced hatching in 3.0 mmol potassium l-1 (53%) than in 1.5 mmol potassium l-1 (16%) (P < 0.05) and 12.0 mmol potassium l-1 (24%) (P < 0.10). The time required for embryos to develop blastocoels, expand and initiate hatching was not affected (P > 0.10) by the concentration of potassium in the medium. However, blastocoel formation by one- to two-cell embryos was delayed (P < 0.05) in medium with 1.5 mmol potassium l-1. In Expt 2, 89 four-cell embryos were cultured in medium containing 6.0 or 24.0 mmol potassium l-1. No differences (P > 0.10) were observed in the percentages of embryos undergoing blastocoel formation, expansion and hatching or in the time required to develop to these stages. The number of cells in embryos recovered at 96 h was greater (P < 0.05) in medium containing 6.0 compared with 24.0 mmol potassium l-1 (32.9 +/- 1.7 versus 26.9 +/- 2.1, respectively). These results demonstrate that pig embryos are tolerant to a wide range of potassium concentrations when the osmolality of the medium is maintained by adjusting the NaCl concentration. These results also suggest that limitations in development due to culture conditions are not due to inappropriate concentrations of this ion.

Evaluation of extracellular matrices and the plasminogen activator system in sheep inner cell mass and trophectodermal outgrowth in vitro.

Effects of extracellular matrices (ECM) and the plasminogen activator (PA) system on outgrowth of sheep inner cell masses (ICM) and trophectoderm in vitro were investigated. Experiment 1 evaluated the effects of plasminogen and ECM type on ICM and trophectodermal outgrowth, on glass Lab-Tek chamber slides coated with collagen IV, fibronectin, or laminin. ICM outgrowth areas were reduced (p < 0.05) by plasminogen and were greatest (p < 0.05) on fibronectin. Trophectodermal outgrowth was not supported in this system. Experiment 2 evaluated the effects of PA inhibitor-2 (PAI-2) or antiserum to urokinase-type PA (anti-uPA) on ICM outgrowth on fibronectin. Numbers of cells in the outgrowths were increased (p < 0.05) with PAI-2, and anti-uPA had no effect (p > 0.10). Experiment 3 evaluated the relationship between PA production and ECM type on ICM and trophectodermal outgrowth in microdrop cultures. PA production by ICM was greatest (p < 0.05) on fibronectin, but no differences (p > 0.10) were observed for trophectoderm. PA production was not correlated with ICM outgrowth areas (r = -0.12; p = 0.72) or numbers of cells in the ICM outgrowths (r = 0.09; p = 0.74) but was correlated with ICM areas (r = 0.75; p < 0.01) and numbers of cells in trophectodermal outgrowths (r = 0.57; p = 0.01). These results suggest that type of ECM, culture system, and alterations in the PA system influence cellular outgrowths by ICM and trophectoderm.

Effects of stimulators of protein kinases A and C and modulators of phosphorylation on plasminogen activator activity in porcine oocyte-cumulus cell complexes during in vitro maturation.

Effects of phorbol myristate acetate (PMA), dibutyryl cyclic AMP (dbcAMP), 6-dimethylaminopurine (6-DMAP), and okadaic acid (OA) on plasminogen activator (PA) activity in porcine oocyte-cumulus cell complexes (POCC) in vitro were determined. Cumulus cell-enclosed oocytes were collected from 1-4 mm antral follicles and cultured in TCM-199 with 0.3% polyvinylpyrrolidone for 48 hr. PA activities in POCC were quantified using SDS-PAGE, casein-agar zymography, and densitometry. Two plasminogen-dependent lytic zones (93-96 kD and 71-79 kD) were observed in POCC. Addition of amiloride to the zymography, a competitive inhibitor of urokinase-type PA, failed to reduce activities in either zone, suggesting that the 71-79 kD band is a tissue-type PA (tPA) and the 93-96 kD band is possibly a tPA-inhibitor complex. Changes in PA activity due to the various treatments were expressed relative to the PA activity in 40 POCC. Increasing dbcAMP increased PA (P < 0.05) activity in dose-dependent fashion, whereas 6-DMAP and 10 and 100 ng/ml PMA inhibited (P < 0.05) PA activity. PA activity increased (P < 0.05) in POCC treated with up to 25 nM OA; however, activity decreased (P < 0.05) at concentrations > 75 nM. Treatment with 25 nM OA also induced the expression of an amiloride-sensitive PA (49-52 kD). Germinal vesicle breakdown and progression to metaphase II were inhibited (P < 0.05) by 2.5 mM dbcAMP and 2 mM 6-DMAP, whereas 100 ng/ml PMA and 25 nM OA inhibited (P < 0.05) only progression to metaphase II.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of blastocoelic expansion and plasminogen activator activity on hatching and zona pellucida solubility in bovine embryos in vitro.

Two experiments were conducted to evaluate factors affecting zona pellucida (ZP) solubility in bovine embryos during hatching in vitro. In Exp. 1, the relationship between blastocoelic expansion and ZP solubility was determined. Day-6 embryos (n = 42) with good or excellent quality grades were cultured (Cultured embryos), whereas embryos of fair quality (Not-cultured embryos) were immediately placed in .2% SDS, and the time required for complete dissolution of the ZP (ZPDT) was determined. For Cultured embryos, ZPDT was determined after 192 h of in vitro development. Zona pellucida dissolution time was greater (P < .05) in Not-cultured embryos than in Cultured embryos and negatively correlated (P < .01) with changes in embryonic surface area. In Exp. 2, the effects of suppressing plasminogen activator (PA) activity and blastocoelic expansion on hatching and ZP solubility were examined. Day-6 embryos (n = 99) were cultured in medium containing 0, 10, or 100 IU/mL of human PA inhibitor-2 (PAI-2) and incubated for 24 to 44 h in medium containing 0, .1, or .5 nM ouabain. Percentages of embryos hatching were not different (P > .05) among 0, 10, and 100 IU/mL of PAI-2; however, more (P < .05) embryos hatched after exposure to 0 and .1 nM ouabain than to .5 nM ouabain. Embryonic PA activity was suppressed (P < .05) by PAI-2, whereas exposure to ouabain did not affect (P > .05) PA activity. Mean ZPDT did not differ (P > .05) following culture in medium containing PAI-2 or exposure to ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of plasminogen activator production in sheep embryos in vitro after treatment with cycloheximide or ouabain.

Effects of the metabolic inhibitors cycloheximide and ouabain on development in vitro and plasminogen activator production by sheep embryos were investigated. Embryos (n = 152) from the eight-cell to the morula stage were surgically collected from naturally mated, oestrus-synchronized and superovulated Polypay ewes. In Expt 1, embryos (n = 104) were grouped by cell stage, cultured in Whitten's medium with 1.5% BSA containing 0, 0.1 or 1.0 microgram cycloheximide ml-1 for 24 h, washed and cultured in this medium for 168 h. In Expt 2, morulae (n = 48) were cultured for 48 h in Whitten's medium with 1.5% BSA transferred to the same medium containing 0 or 1.0 mmol ouabain l-1 and cultured for 24 h, and then washed and cultured in this medium for 120 h. At 24 h intervals in both experiments, the medium was recovered and analysed for plasminogen activator. In Expt 1, eight-cell embryos underwent limited development; little difference in the production of plasminogen activator due to cycloheximide treatment was therefore observed. Compared with medium without cycloheximide, treatment with 1.0 microgram cycloheximide ml-1 reduced the number of 16-cell embryos (P < 0.05) and morulae (P < 0.05) (60% versus 10% and 77% versus 8%, respectively) that began to hatch. The mean production of plasminogen activator was greatest in embryos cultured initially as morulae compared with that of 16-cell and eight-cell embryos (P < 0.05). Cycloheximide treatment suppressed the mean production of plasminogen activator in a dose-dependent manner (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Plasminogen activator production and enhanced development in medium containing plasminogen or plasmin by rabbit embryos in vitro.

A series of experiments was conducted to evaluate plasminogen activator production and effects of supplementing culture medium with plasminogen or plasmin on development of rabbit embryos in vitro. In Expt 1, 495 one- to two-cell embryos were cultured in Ham's F-12 with 15 mg BSA ml-1 containing 0, 30, 60 or 120 micrograms porcine plasminogen ml-1 or 0, 75, 150 or 300 micrograms rabbit plasminogen ml-1. Percentages of embryos developing to the expanded blastocyst, hatching blastocyst and hatched blastocyst stages were greater (P < 0.05) in medium with 120 micrograms porcine plasminogen ml-1 than in the absence of plasminogen. More (P < 0.05) embryos developed to the blastocyst, expanded blastocyst and hatching blastocyst stages in medium with 300 micrograms rabbit plasminogen ml-1 than in the absence of plasminogen. In Expt 2, 216 one- to two-cell embryos were cultured in medium with 0, 30, 60 or 120 micrograms porcine plasminogen ml-1 for 96 h, fixed and strained with haematoxylin and eosin, and the number of cells determined. No differences (P > 0.05) were observed in number of cells of morulae but blastocysts developing in medium with 120 micrograms porcine plasminogen ml-1 had more (P < 0.05) cells (109.9 +/- 10.4) than did blastocysts in medium with either 0 (69.4 +/- 14.6) or 30 micrograms porcine plasminogen ml-1 (73.3 +/- 12.2). In Expt 3, 144 one- to two-cell embryos were cultured in medium with 0, 13 or 45 micrograms porcine plasmin ml-1 or 120 micrograms porcine plasminogen ml-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial characterization of the plasminogen activator produced by ovine embryos in vitro.

The type of plasminogen activator (PA) produced by Day 7, 9, and 11 ovine embryos in vitro was partially characterized. PA activity in the conditioned medium did not change (p > 0.05) up to equivalent gestational Day (EGD) 14 but increased to peak levels on EGD 15 (p < 0.05). Zymographic analysis of conditioned medium and embryos revealed one plasminogen-dependent lytic zone (48-51 kDa) within EGD 8-15 and a second plasminogen-dependent lytic zone that appeared after EGD 13 (79-83 kDa). Addition of the urokinase competitive inhibitor, amiloride, to the zymograph inhibited plasminogen-dependent lytic activity; and forms of both high and low molecular mass were immunoprecipitated with antiserum to bovine urokinase-type PA. PA activity in conditioned medium was completely suppressed by amiloride treatment (p < 0.05) but was increased (p < 0.05) after incubation with antibodies to human PA inhibitor (PAI)-2. Treatment with antibodies to human PAI-1 did not increase (p > 0.05) PA activity in conditioned medium. These results suggest that Day 7, 9, and 11 ovine embryos produce a urokinase-type PA (49.9 +/- 0.5 kDa) and possibly a PAI-2-like protein that associates with the PA and forms a PA-PAI complex of high molecular mass (81.4 +/- 1.2 kDa).

Steroids and plasminogen activator concentrations in follicular fluid of gilts at first and third estrus.

An experiment was conducted to determine whether morphological and functional characteristics of follicles differed at a similar stage of pubertal (first) and third estrus in the same gilts. Nine prepubertal gilts were checked three times daily for estrus and laparotomized 6 h after detected first and third estrus. Samples of vena cava and ovarian venous blood were collected, follicle numbers and diameters were recorded, and follicular fluid (FF) was aspirated from all follicles 8 to 12 mm in diameter. Sera and(or) FF were analyzed for progesterone (P4), estradiol-17 beta (E2), testosterone (T), androstenedione (A4), 5 alpha-dihydrotestosterone (DHT), plasminogen activator (PA), and plasmin (PLM). Overall mean number of follicles > or = 8 mm in diameter did not differ between gilts at first and third estrus (P > .05) but gilts at first estrus had more follicles 4 to 8 (P < .05) and 8.1 to 10 mm in diameter (P < .01) and fewer 10.1 to 12 mm in diameter (P < .07) than at third estrus. Mean FF concentrations of E2, T, and A4 at third estrus were significantly greater than at first estrus, whereas FF concentrations of P4, DHT, PA, and PLM were similar at first and third estrus (P > .05). Mean concentrations of E2 in systemic and ovarian venous sera were also greater in gilts at third than at first estrus (both P < .05). Systemic concentrations of P4 in gilts at first and third estrus did not differ (P > .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of effect of hormones and inducers of intracellular messengers on plasminogen activator production by bovine embryos in vitro.

Several hormones and inducers of intracellular messengers, known to affect plasminogen-activator (PA) production in other systems, were investigated for putative effects on bovine embryos. Day 8 embryos were cultured for 5 days in a humidified atmosphere of 5% CO2 in air at 37 degrees C in media containing different concentrations of progesterone, oestradiol, dexamethasone, retinoic acid, dibutyryl cyclic AMP (dbcAMP) and phorbol myristate acetate (PMA). At intervals of 24 h, the medium was recovered for PA analysis and overall embryonic diameter was measured. While none of the hormones and agents tested affected PA production (P > 0.05), dimethyl sulfoxide, which was used to dissolve PMA, inhibited PA production during the first 72 h of culture (P < 0.05). PA production was affected by duration of culture (P < 0.05). Concentrations of plasminogen activator in the media were low during the first 48 h, had increased after 72 and 96 h in culture, and either remained high or decreased slightly toward the end of the culture period. With the exceptions of dbcAMP and PMA, the hormones tested in this study did not affect embryonic size. Dibutyryl cAMP caused a progressive decrease in embryonic diameter. PMA resulted in embryo death at high concentrations but at lower concentrations it enhanced overall embryonic diameter throughout the time of culture (P < 0.05). These results suggest that cultured bovine embryos produce PA in a fixed, time-dependent manner, independent of exogenous hormonal regulation.

Superovulation of cattle with equine pituitary extract and porcine ESH.

Superovulation has been practiced in cattle for more than 50 years but the results have been highly variable. Scientists at six locations compared a horse pituitary extract (HAP) with a single batch of porcine FSH (pFSH) to determine the efficacy of these hormones to induce superovulation and to test for variability in the superovulatory response. Acetone-dried equine pituitaries were suspended in 40% ethanol containing 6% ammonium acetate, and the supernatant was mixed with 2.5 volumes of cold ethanol. The resulting precipitate was washed with cold ether and dried. Total doses of 18 mg of HAP and 36 mg of pFSH were injected intramuscularly (i.m.) over 4 days, two injections per day, and prostaglandin (PGF(2)alpha; 25 mg, i.m.) was administered on Day 3. Injections were begun on Days 6 to 13 of the estrous cycle. The overall ovulation rates (mean +/- SEM) for HAP and FSH were 8.8 +/- 0.7 and 15.1 +/- 1.0, respectively (n=231; P<0.01). Location interacted (P<0.01) with the type of gonadotropin for the ovulation rate. When expressed as a proportion of the number of corpora lutea, the total number of embryos recovered was greater (P=0.03) for pFSH than for HAP, but there was no difference in the number of Quality 1 and 2 embryos. The results show that HAP can induce a satisfactory superovulatory response, but there was no evidence of reduced variability of response to HAP compared with pFSH.

Bovine embryos produce a urokinase-type plasminogen activator.

The type of plasminogen activator (PA) secreted by bovine embryos was identified. Day 12-14 embryos were collected from estrus-synchronized, superovulated, and naturally mated crossbred beef cows. Embryos were left intact (E) or microdissected into component embryonic discs (ED) and trophoblastic vesicles (TV). Intact embryos, ED, and TV were pre-cultured for 2 days in Minimum Essential Medium Alpha (MEM alpha) with 10% heat-inactivated fetal calf serum, washed in serum-free MEM alpha, and cultured individually for 5 days in 50 microliters microdrops of MEM alpha with 15 mg/ml bovine serum albumin. At 24 hr intervals, E, ED, and TV were observed for tissue morphology and transferred to fresh microdrops, and medium was recovered and frozen at -20 degrees C. At the end of culture, blastocoelic fluid (BF) and embryonic tissues were recovered and frozen at -20 degrees C. Plasminogen activator concentrations in medium, tissues, and BF were determined by using a caseinolytic assay. Antibodies to urokinase-type PA (anti-uPA) and tissue-type PA (anti-tPA), and the urokinase inhibitor, amiloride (AMR), were used to identify the type of PA produced by bovine embryonic tissues. Intact embryos and TV released more PA (P less than 0.05) than ED, and tissues exhibiting expanded blastocoels released less PA (P less than 0.05) than tissues with collapsed blastocoels. Blastocoelic fluid from TV exhibited more PA (P less than 0.05) activity than from ED. Treatment with anti-uPA decreased PA activity (P less than 0.05) in pooled medium and tissues from E compared to treatment with nonspecific immunoglobulins and anti-tPA.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Sodium/potassium adenosine triphosphatase alpha- and beta-subunit and alpha-subunit mRNA levels during mouse embryo development in vitro.

Changes in sodium/potassium adenosine triphosphatase (Na+/K+ ATPase) and Na+/K+ ATPase mRNA content during preimplantation mouse embryo development were determined. Western blotting, using polyclonal antiserum against guinea pig Na+/K+ ATPase, was used to detect changes in Na+/K+ ATPase alpha- and beta-subunit content during mouse embryo development. Total RNA from mouse embryos was analyzed using Northern and slot blots hybridized with random-primer-labeled cDNA for Na+/K+ ATPase alpha-subunit from sheep kidney. Northern blots exhibited a single mRNA band (3.65 kb) in sheep and mouse kidneys and mouse embryos. Although Na+/K+ ATPase alpha-subunit mRNA content of mouse embryos increased 45-fold between Day 1 and Day 4 of development, Na+/K+ ATPase alpha-subunit content remained constant, and beta-subunit content increased 9-fold. The Na+/K+ ATPase alpha-subunit and alpha-subunit mRNA content did not increase in a similar manner. The results suggest that, in mouse embryos, blastocoel formation is not triggered by an increase in Na+/K+ ATPase alpha-subunit content. Changes in beta-subunit content may be important in regulating Na+/K+ ATPase activity and blastocoel formation.