The effects of water-soluble vitamins on the expansion of rabbit blastocysts in vitro

The vitamin requirements for culture of rabbit morulae to expanded blastocysts were examined. Early morulae were cultured for 5 days either in a control complete medium containing all the 11 water-soluble vitamins of F10 culture medium (biotin, pantothenate, choline, inositol, niacinamide, pyridoxine, riboflavin, thiamine, folic acid, B12, and lipoic acid) or in media with each vitamin omitted individually. Blastocyst diameters were measured at the end of culture. The omission of inositol, pyridoxine, riboflavin, and niacinamide resulted in large statistically significant decreases in blastocyst expansion. The omission of B12 resulted in a significant increase in blastocyst expansion indicating that the level present in F10 is toxic to rabbit blastocysts.     

Prostaglandin synthesis by day-6 rabbit blastocysts in vitro

Day-6 rabbit blastocysts were recovered from superovulated donor animals, washed in ice-cold Krebs-Ringer-bicarbonate (KRB) buffer, pooled and randomly allocated to polypropylene incubation tubes, usually 10 blastocysts in 1 ml KRB. The blastocysts were ruptured with a dissecting needle and incubated at 37 degrees C for periods of 1-3 h with 10 microCi [3H]arachidonic acid/tube. A control tube without blastocysts was run in each experiment. At the end of the incubation, the samples were acidified, extracted with ethyl acetate, dried down and resuspended in h.p.l.c., using a solvent system for prostaglandins (PGs), was subtracted from each experimental run in the same experiment. The remaining radioactivity constituted 0.14% of the original [3H]arachidonic acid added to each incubation tube. This was considered to have been the result of conversion of the radiolabelled arachidonic acid to prostanoids. In the absence of 10 mM-EDTA no conversion occurred, whereas in its presence peaks of radioactivity co-eluting with [3H]PGF-2 alpha and [3H]PGE-2 were seen. A third peak that eluted was either 15-keto metabolites of these PGs or PGD-2. These 3 peaks were always significantly above background, and usually did not differ from each other. No differences in amount of conversion could be related to incubation time. Addition of indomethacin (100 micrograms/ml) or radioinert arachidonic acid (10 micrograms/ml) inhibited production of [3H]PG, even in the presence of EDTA. Removal of calcium from the incubation medium was per se without effect. Addition of atropine (0.15 mM) or carbachol (0.15 mM) in the presence or absence of EDTA did not change the pattern of conversion of [3H]arachidonic acid to [3H]PGs. These experiments demonstrate that rabbit blastocysts have the capacity for de-novo synthesis of PGs from exogenous substrate, when utilization of endogenous substrate is inhibited. The extent of conversion observed may not be a true reflection of the capacity for conversion of endogenous substrate.     

Production of steroids and release of prostaglandins by spherical pig blastocysts in vitro

Levels of pregnenolone and progesterone in spherical pig blastocysts (near 4 and 15 microM respectively) exceeded respective levels in histotroph by about 400-fold. When blastocysts were cultured for 5 days in a synthetic medium containing pregnenolone sulfate (1 microM), daily rates of release of pregnenolone, progesterone, androstenedione, testosterone, oestrone and oestradiol were determined to be near 320, 45, 26, 27, 0.8 and 9.2 fmol per blastocyst respectively. Daily outputs of progesterone and testosterone (fmol per blastocyst) diminished (P less than 0.05) to 1.3 and undetectable levels (less than 2) respectively in the presence of Trilostane (94 microM). Increasing the content of pregnenolone sulfate in the culture medium (to 4.5 microM) resulted in higher daily rates of release of pregnenolone and progesterone (to near 1740 and 380 fmol per blastocyst respectively), verifying activity of 3 beta-hydroxy-delta 5-steroid dehydrogenase, and of arylsulfatase, in tissues of intact spherical pig blastocysts. Prostaglandin E2 was the predominant prostaglandin (PG) released by cultured blastocysts (about 1 fmol per blastocyst per hour), hourly rates of release of PGH2 (derived) and PGF2 alpha being near 0.1 and less than 0.06 fmol per blastocyst respectively. The data establish a capacity for spherical pig blastocysts to release a range of steroids and PGs of possible significance to embryonic growth and development in vivo.     

Role of prostaglandins in development of porcine blastocysts

Rapid elongation of porcine blastocysts between Days 11 to 12 of pregnancy coincides with an increase in uterine luminal content of prostaglandins. The present study evaluated the effect of two prostaglandin synthesis inhibitors (indomethacin and flunixin meglumine) on elongation of porcine blastocysts from spherical to filamentous forms between Day 11 to 12 of pregnancy. Gilts were hemi-hysterectomized on Day 11 of prenancy. The excised uterine horn was flushed with 0.9% saline and diameter of blastocysts recovered were measured. Immediately following surgery, pregnant gilts were assigned to receive either: 1) vehicle every 4 h, 2) flunixin meglumine (banamine) every 4 h, or 3) indomethacin every 12 h. The remaining uterine horn was removed and flushed after the time of blastocyst elongation estimated for each gilt on basis of blastocyst development in the first horn. Uterine flushings were analyzed for total calcium, protein, acid phosphatase activity, estrone, estradiol-17β and prostaglandin F. Pretreatment blastocyst diameter was similar for all groups and ranged from 1 mm to 20 mm. Treatment of gilts with either banamine or indomethacin effectively inhibited (P<0.001) the increase in uterine luminal content of PGF. Total calcium, estrone and estradiol-17β were not influenced by treatment. Total uterine luminal protein and acid phosphatase activity were reduced (P<0.05) in banamine treated gilts compared to those receiving vehicle or indomethacin treatments. Although total PGF recovered in uterine flushings was reduced during the period of blastocyst elongation, treatment with PGF synthetase inhibitors failed to block rapid elongation of blastocysts from the spherical to filamentous forms.     

Role of prostaglandins in development of porcine blastocysts

Rapid elongation of porcine blastocysts between Days 11 to 12 of pregnancy coincides with an increase in uterine luminal content of prostaglandins. The present study evaluated the effect of two prostaglandin synthesis inhibitors (indomethacin and flunixin meglumine) on elongation of porcine blastocysts from spherical to filamentous forms between Day 11 to 12 of pregnancy. Gilts were hemi-hysterectomized on Day 11 of pregnancy. The excised uterine horn was flushed with 0.9% saline and diameter of blastocysts recovered were measured. Immediately following surgery, pregnant gilts were assigned to receive either: 1) vehicle every 4 h, 2) flunixin meglumine (banamine) every 4 h, or 3) indomethacin every 12 h. The remaining uterine horn was removed and flushed after the time of blastocyst elongation estimated for each gilt on basis of blastocyst development in the first horn. Uterine flushings were analyzed for total calcium, protein, acid phosphatase activity, estrone, estradiol-17 beta and prostaglandin F. Pretreatment blastocyst diameter was similar for all groups and ranged from 1 mm to 20 mm. Treatment of gilts with either banamine or indomethacin effectively inhibited (P less than 0.001) the increase in uterine luminal content of PGF. Total calcium, estrone and estradiol-17 beta were not influenced by treatment. Total uterine luminal protein and acid phosphatase activity were reduced (P less than 0.05) in banamine treated gilts compared to those receiving vehicle or indomethacin treatments. Although total PGF recovered in uterine flushings was reduced during the period of blastocyst elongation, treatment with PGF synthetase inhibitors failed to block rapid elongation of blastocysts from the spherical to filamentous forms.     

Analysis of the direct effects of prostaglandins on human sperm function

Time-exposure photomicrography and interspecies in-vitro fertilization procedures have been used to examine the influence of prostaglandins on human sperm function. An analysis of variance indicated that the presence of PGs in the incubation media was associated with a significant increase in sperm velocity and the frequency of sperm head rotation, although there were no differences between individual PGs in the degree of stimulation observed. Changes in the penetrating ability of human spermatozoa were detected after exposure to PGs, particularly PGE-1 and PGE-2. PGE-2 induced a sustained increase in penetration rates at all doses of greater than 8.4 micrograms/ml, while exposure to PGE-1 gave a bell-shaped dose-response curve which exhibited a peak between 8.4 and 33.3 micrograms/ml and progressively fell to reach control levels at the maximum concentration tested of 270 micrograms/ml. A combination of PGE-2 and PGE-1 produced a dose-response curve similar to that for PGE-1 alone, while exposure to PGF-2 alpha was without effect. Seminal extracts containing predominantly 19-hydroxy PGE-1, or equal amounts of 19-hydroxy PGE-1 + 2 induced a slight, but significant, rise in penetration rates while a combination of PGs representing the major components of human seminal plasma was without significant effect. We conclude that certain prostaglandins may have a direct action on the functional competence of human spermatozoa.     

Successful pregnancy after transfer of rabbit blastocysts grown in vitro from single-cell zygotes

To establish successful pregnancy in rabbits after the transfer of blastocysts cultured in vitro for 72 h, pregnancy rates were compared according to synchronization methods of recipient and embryo transfer sites. Also, the effect of RDH (1:1:1 mixture of RPMI, DMEM and Ham's F10) medium with additives such as BSA and taurine was evaluated for developmental capacity and cell number. Developmental capacity and cell number were considered important for implantation. When we evaluated the relative survival of rabbit one-cell embryos after culture in Ham's F10, in RD or in RDH for 72 h, embryos cultured in RDH and RD developed much better than in Ham's F10. When the effects of BSA and taurine in RDH medium were tested for rabbit embryo development, BSA or taurine promoted transition to the blastocyst stage and increased cell numbers of cultured embryos in RDH medium. The BSA and taurine together in RDH medium had a synergistic effect on embryo development. By transferring cultured blastocysts to the oviduct of the recipient doe synchronized one day behind the donor, live-born pups were obtained successfully. These results demonstrated that rabbit blastocysts can develop to normal pups after in vitro culture and embryo transfer.     

Effect of copper on isolated rabbit blastocysts

The effect of copper on the electrical membrane properties of the isolated-perfused 6-day rabbit blastocyst was studied to understand changes in the intrauterine environment caused by the copper IUD. Blastocysts were perfused in an environmental chamber containing Krebs-Ringer bicarbonate with 1 mg bovine serum albumin/ml. Electrical measurements made included short-circuit current (SCC) (the net result of currents produced by all net active ionic transport processes when there is no electrochemical gradient), transmural potential difference (p.d.), and conductance (computed from the ratio of open circuit p.d. to SCC). Control values were obtained and 9 experiments were performed in which 10 mcl aliquots of ?cuCl2 was added to the bathing solution. Electrical parameters of solutions containing 10-5M concentration CuCl2 remained essentially unchanged. 2.5 x 10-5 M reduced average p.d. 25% and average SCC 12%, WHILE 5 X 10-4C-5 M further reduced p.d. 48% and SCC 38% after 30 minutes. At 7.5 x 10-5 M p.d. was depressed 89% after 10 minutes with 1/3 of the values being positive, and SCC values decreased to 71% at 10 minutes and then increased to 77% of control values at 30 minutes. The subsequent changes in p.d. and SCC caused a 6-fold increase in membrane conductance. 9 experiments were performed on a 2nd group of blastocysts in which the effects of a single addition of CuCl2 at 10-4 M were studied. Average p.d. decreased reversing to positive values at 30 minutes. There was a biphasic response to SCC decreasing to 46% after 20 minutes then increasing to 1.7 times control values. Single additions of copper ions collapsed all blastocysts after a return to copper-free solutions. Serial additions showed only 3 out of 9 collapsing under similar conditions. Further experiments involving simultaneous SCC-isotope flux are necessary to determine which specific actively transported ions are affected by copper and to determine the effect on conductance. It is suggested that the action of copper in these experiments might have some bearing on the effectiveness of the copper IUD.     

Hexose transport in preimplantation rabbit blastocysts

Transtrophectodermal 3-0-methyl glucose (3-0MG) transport in the rabbit blastocyst at Days 6 and 7 post coitum was investigated to understand better how the trophectoderm can regulate inner cell mass growth by controlling substrate availability. 3-0MG rapidly traversed the trophectoderm and displayed saturation kinetics (Km = 4.3 +/- 0.5 mM, Vmax = 79 +/- 3.8 nmol.cm-2). The flux of 3-0MG was inhibited nearly 95% by 10(-4) M-phloretin, and only 15% by 10(-4) M-phlorizin. Furthermore, 3-0MG influx was inhibited by cytochalasin B (5 microM) and was unaffected by removal of sodium. The transport system had a high specificity for 2-deoxy-D-glucose and glucose, and a very low specificity for fructose and 4-alpha-methyl glucoside. Western blots probed with a polyclonal antibody to the human erythrocyte glucose transport protein and also with a polyclonal antibody to the C-terminus of the glucose transport protein of the rat brain revealed a broad band with a molecular weight of 55,000. Using immuno-gold labelling techniques, Na(+)-independent glucose transporters were localized to both the apical and basolateral borders of the trophectodermal cell. These results suggest that the mechanism in the trophectoderm responsible for transport of glucose is similar to other sodium-independent glucose transport systems. In addition, 3-0MG influx was unaffected by short-term incubation with progesterone, the progesterone antagonist mifepristone (RU-486), PGF-2 alpha, PGE-2, insulin, or cAMP. Day-7 p.c. embryos also transported hexoses by a similar system because the influx rate and the phlorizin/phloretin sensitivity were the same as in the Day-6 p.c. embryo.     

Absence of direct effects of GnRH on testicular steroid secretion in the ram

Effects of GnRH, administered via the testicular artery, on testicular steroidogenesis were studied in rams during the non-breeding season. Concentrations of testosterone and 17-hydroxyprogesterone in testicular venous blood showed similar profiles which were identical for GnRH-treated (0.5 ng infused over 60 min or 25 ng injected) and control testes. Increases of testicular venous concentration of both hormones were only marginally reflected in peripheral venous concentrations. Peripheral administration of hCG (200 i.u., i.v.) stimulated testosterone secretion to a larger extent than 17-hydroxyprogesterone secretion in 10/11 rams, GnRH-treated and control testes showing identical responses. High testicular venous concentrations of both hormones after administration of GnRH were paralleled by increased concentrations of endogenous LH. These LH peaks were evoked by 25 ng GnRH in 7/8 rams. The observed effects of GnRH treatment on testicular steroid secretion thus cannot be considered to be the result of direct stimulation of steroidogenesis by GnRH.     

Distribution of prostaglandins in rabbit kidney

Three prostaglandins (PGE(2), PGF(2alpha) and PGA(2)) are present in rabbit kidney medulla. An acidic lipid extract (0.165g) obtained from 2kg of frozen rabbit kidney cortex was separated by silicic acid chromatography to yield eluates containing fatty acids, possible non-polar prostaglandin metabolites, PGA, PGE and PGF compounds. Ultraviolet spectra of the eluates before and after treatment with sodium hydroxide did not yield chromophores typical of any known prostaglandins or related metabolites. By using more sensitive bioassay procedures (contraction of rabbit duodenum) weak activity equivalent to 60mug of PGE(2) and 10mug of PGF(2alpha) was detected in the PGE and PGF eluates respectively. Extraction and bioassay of fresh kidney cortex revealed no prostaglandin-like activity. Attempts to biosynthesize prostaglandins in fresh homogenates of rabbit kidney cortex from endogenous precursors and from added arachidonic acid were unsuccessful. When freshly prepared homogenates of rabbit kidney cortex were incubated with added PGE(1) no evidence of enzymic breakdown was obtained. It is concluded that rabbit kidney prostaglandins are present predominantly in the medulla and there are no cortical mechanisms for their biosynthesis or inactivation under normal conditions.