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.     

Quantitation of deoxycorticosterone and its relationship to progesterone in the prepartum bovine.

A method and its validation is described for the radioimmunological measurement of deoxycorticosterone (DOC) in bovine serum. Levels of DOC and progesterone were determined in six pregnant heifers from one to three weeks before and during parturition. Levels of these steroids fluctuated widely from day to day and tended to be inversely related (r = -0.24). High levels of DOC in conjunction with low levels of progesterone at or near parturition are suggestive that DOC is involved in the parturition process.     

The non-genomic rapid acidification in peripheral T cells by progesterone depends on intracellular calcium increase and not on Na+/H+-exchange inhibition

Progesterone is an endogenous immunomodulator that is able to suppress T cell activation during pregnancy. An increased intracellular free calcium concentration ([Ca(2+)](i)), acidification, and an inhibition of Na(+)/H(+)-exchange 1 (NHE1) are associated with this progesterone rapid non-genomic response that involves plasma membrane sites. Such acidification, when induced by phytohemagglutinin, is calcium dependent in PKC down-regulated T cells. We investigated the relationship between this rapid response involving the [Ca(2+)](i) increase and various membrane progesterone receptors (mPRs). In addition, we explored whether the induction of acidification in T cells by progesterone is a direct result of the [Ca(2+)](i) increase. The results show that the intracellular calcium elevation caused by progesterone is inhibited by SKF96365, U73122, and 2-APB, but not by pertussis toxin or U73343. The elevation is enhanced by the protein tyrosine kinase inhibitor staurosporine and the protein kinase C inhibitors Ro318220 and Go6983. These findings suggest that progesterone does not stimulate the [Ca(2+)](i) increase via the Gi coupled mPR(α). Furthermore, progesterone-induced acidification was found to be dependent on Ca(2+) entry and blocked by the inorganic channel blocker, Ni(2+). However, BAPTA, an intracellular calcium chelator, was found to prevent progesterone-induced acidification but not the inhibition of NHE1. This implies that acidification by progesterone is a direct result of the [Ca(2+)](i) increase and does not directly involve NHE1. Taken together, further investigations are needed to explore whether one or more mPRs or PGRMC1 are involved in bringing about the T cell rapid response that results in the [Ca(2+)](i) increase and inhibition of NHE1.     

A chemical quenching apparatus for studying rapid reactions.

A chemical quench-flow apparatus is described for studying enzymatic reactions with half-lives of 0.005 sec or longer. The syringe pistons are driven by a stepping motor which provides precise control over the volume and rate of flow of reactants. The drive mechanism also ensures a rapid approach to a steady flow velocity and thus minimizes the amount of material used per stroke. Improved mixing efficiency is accomplished by means of ball mixers which utilize the zone of turbulence in the wake of a sphere as the mixing mechanism. The instrument was used to follow the presteady state time course of phosphorylation and dephosphorylation of a microsomal preparation of (Na⁺ + K⁺)-stimulated ATPase.     

Caffeine-stimulated ATP-reactivated motility in a detergent-treated bovine sperm model.

Mammalian sperm cells contain most of the components of a cyclic AMP-mediation system. To determine if the cyclic AMP-dependent protein kinase has a role in the control of bovine sperm motility, a sperm model was developed that was permeable to exogenously added ATP. Treatment of bovine epididymal spermatozoa with dithiothreitol and Brij-35 (polyoxyethylene alcohol), a nonionic surfactant, resulted in a sperm model with caffeine-stimulated, ATP-reactivatable motility. The results of the data obtained using this sperm model can be summarized as follows. (1) Brij partially solubilized the cyclic AMP-dependent protein kinase activity and released nearly half of the total acid-extractable nucleotides of the cells. (2) Brij treatment severely damaged the sperm mitochondria as judged by their lack of respiration. (3) Brij-treated spermatozoa lose their motility but were reactivated with ATP; the reactivated motility was stimulated by caffeine. (4) Despite the caffeine stimulation of motility in Brij-treated spermatozoa, increased protein phosphorylation did not accompany reactivation of motility, nor could a cyclic AMP effect be demonstrated on reactivated motility or on kinase activity in the sperm model.     

Two binding proteins for progesterone in the bovine corpus luteum

The cytosolic supernatant of bovine corpus luteum contains two proteins which bind progesterone specifically. Bovine luteal cytosol was fractionated on hydroxylapatite and the peaks of protein obtained subjected to equilibrium dialysis against progesterone. Progesterone-binding activities (Ka approx. 10(6) 1/mol) was eluted at 40 mM (Binding Protein 1) and 100 mM phosphate (Binding Protein 2). They sedimented differently (3.95 and 4.65, respectively) on sucrose gradients. In contrast to Binding Protein 1, Binding Protein 2 bound R5020 better than progesterone on sucrose gradients. Purification of the binding activity eluted by 40 mM phosphate from the hydroxylapatite column showed that it resided in a single protein (molecular weight 65,000 daltons). The function of these proteins is presently unknown, but they may participate in the biosynthesis and/or secretion of progesterone from bovine luteal cells.     

Quorum sensing is not required for twitching motility in Pseudomonas aeruginosa

It has been reported that mutations in the quorum-sensing genes lasI and rhlI in Pseudomonas aeruginosa result in, among many other things, loss of twitching motility (A. Glessner, R. S. Smith, B. H. Iglewski, and J. B. Robinson, J. Bacteriol. 181:1623-1629, 1999). We constructed knockouts of lasI and rhlI and the corresponding regulatory genes lasR and rhlR and found no effect on twitching motility. However, twitching-defective variants accumulated during culturing of lasI and rhlI mutants. Further analysis showed that the stable twitching-defective variants of lasI and rhlI mutants had arisen as a consequence of secondary mutations in vfr and algR, respectively, both of which encode key regulators affecting a variety of phenotypes, including twitching motility. In addition, when grown in shaking broth culture, lasI and rhlI mutants, but not the wild-type parent, also accumulated unstable variants that lacked both twitching motility and swimming motility and appeared to be identical in phenotype to the S1 and S2 variants that were recently reported to occur at high frequencies in P. aeruginosa strains grown as a biofilm or in static broth culture (E. Deziel, Y. Comeau, and R. Villemur, J. Bacteriol. 183:1195-1204, 2001). These results indicate that mutations in one regulatory system may create distortions that select during subsequent culturing for compensatory mutations in other regulatory genes within the cellular network. This problem may have compromised some past studies of regulatory hierarchies controlled by quorum sensing and of bacterial regulatory systems in general.     

Level and distribution of progesterone in bovine milk in relation to storage in the mammary gland.

The study investigated the effect of the place of storage of milk in the mammary gland on progesterone concentrations in whole milk, skim milk and milk fat. Skim milk, milk fat and whole milk progesterone concentrations were lower (P < 0.05) in milk fractions obtained from the cisternal part of the mammary gland compared to those in the milk fractions from the alveoli. Mean milk fat concentrations did not mirror the changes in the mean skim milk, milk fat and whole milk progesterone concentrations. After administration of oxytocin, milk fat concentrations rose significantly (P < 0.01). At the same time, skim milk and milk fat progesterone concentrations remained unchanged (P > 0.05), compared to those in the milk fractions of alveolar origin, obtained before oxytocin administration. Skim milk and whole milk progesterone concentrations were higher (P < 0.01) in composite milk and in milk samples collected 1 h after milking, compared to concentrations in the milk samples collected before morning milking and at 3, 5, 7 and 9 h after milking. The results suggest that defatted milk, milk fat and whole milk progesterone concentrations were affected by the place of storage of the milk in the mammary gland, and that this effect is independent of milk fat content. Time of milk sampling, not the milk fat concentration, in relation to time of milking, was a critical factor in determining skim milk, milk fat and whole milk progesterone. The study also revealed that the concentrations of the other milk components, somatic cell count, lactose and protein were affected by the place of storage of milk in the mammary gland.     

Proteomic changes in Debaryomyces hansenii upon exposure to NaCl stress

The proteome of the highly NaCl-tolerant yeast Debaryomyces hansenii was investigated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), and 47 protein spots were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) followed by mass spectrometry (MS). The influence of NaCl on the D. hansenii proteome was investigated during the first 3 h of NaCl exposure. The rate of protein synthesis was strongly decreased by exposure to 8% and 12% (w/v) NaCl, as the average incorporation rates of l-[(35)S]methionine within the first 30 min after addition of NaCl were only 7% and 4% of the rate in medium without NaCl. In addition, the number of protein spots detected on 2D gels prepared from cells exposed to 8% and 12% (w/v) NaCl exceeded less than 28% of the number of protein spots detected on 2D gels prepared from cells without added NaCl. Several proteins were identified as being either induced or repressed upon NaCl exposure. The induced proteins were enzymes involved in glycerol synthesis/dissimilation and the upper part of glycolysis, whereas the repressed proteins were enzymes involved in the lower part of glycolysis, the route to the Krebs cycle, and the synthesis of amino acids. Furthermore, one heat shock protein (Ssa1p) was induced, whereas others (Ssb2p and Hsp60p) were repressed.     

Alterations in primate sperm motility with maturation and during exposure to theophylline

Micropuncture was used to collect pure suspensions of sperm from the caput and cauda regions of chimpanzee epididymides, which were analyzed with a Motion Analysis VP-110. Sperm recovered from the caput region showed no forward motility. Incubation of these sperm with cauda epididymal fluid affected motility in 62%–90% of the sperm. Dilution of cauda sperm into buffer containing >50 mM theophylline resulted in immediate initiation of progressive forward motility. Although this motility was maintained by at least 50% of the sperm for over 5 hr, these “activated” caput sperm did not penetrate zona-free hamster ova. These data show that sperm from the caput epididymis of the chimpanzee have the capacity for normal motility but do not have the capacity to bind to and penetrate an ovum. Cauda epididymal chimpanzee sperm were motile at the time of recovery and this motility was maintained for over 5 hr. These sperm penetrated both hamster zona-free ova and intact chimpanzee ova. These data show that sperm from the cauda epididymis of the chimpanzee have the capacity for normal motility and also have the capacity to bind to and penetrate an ovum. This is the first use of computer assisted analysis to quantify motility in maturing nonhuman primate sperm.     

Hormonal regulation of oestrogen and progesterone receptors in cultured bovine endometrial cells.

Changes in the number of progesterone and oestradiol receptors in the endometrium are thought to play a role in the induction of luteolysis. The effect of oestradiol and progesterone on the regulation of their receptors in cultured bovine uterine epithelial and stromal cells was examined to determine the mechanisms involved in this process. Cells were obtained from cows at days 1-3 of the oestrous cycle and were cultured for 4 or 8 days in medium alone (RPMI medium + 5% (v/v) charcoal-dextran stripped newborn calf serum) or with oestradiol, progesterone or oestradiol and progesterone. At the end of culture, receptor binding was measured by saturation analysis. Specific binding of both [3H]ORG 2058 (16 alpha-ethyl-21-hydroxy-19-nor (6,7-3H) pregn-4-ene-3,20-dione) and [3H]oestradiol to epithelial and stromal cells showed high affinities (Kd = 1.1 x 10(-9) and 6 x 10(-10) mol l-1, respectively, for progesterone receptors; Kd = 5.5 x 10(-9) and 7 x 10(-10) mol l-1, respectively, for oestradiol receptors). In the stromal cells, oestradiol (0.1-10 nmol l-1) increased the number of oestradiol receptors from 0.21 +/- 0.06 to 0.70 +/- 0.058 fmol microgram-1 DNA and the number of progesterone receptors from 1.4 +/- 0.83 to 6.6 +/- 0.70 fmol microgram-1 DNA in a dose-dependent manner after 4 days of culture (P < 0.01). After culture for 8 days, the stimulatory effect of oestradiol increased. Progesterone (50 nmol l-1) had no effect on the number of oestradiol or progesterone receptors (P > 0.05). However, progesterone inhibited the stimulatory effect of oestradiol. In epithelial cells, the lower concentrations of oestradiol (0.1 and 1 nmol l-1) stimulated the number of progesterone receptors (P = 0.05) after 4 days culture, whereas the highest concentration of oestradiol (10 nmol l-1), progesterone (50 nmol l-1) and progesterone (50 nmol l-1) plus oestradiol (1 nmol l-1) had no effect. After culture for 8 days, the stimulatory effect of oestradiol decreased. In contrast to progesterone receptors, the number of oestradiol receptors increased with oestradiol concentration (P < 0.01). These data show that the number of progesterone receptors was higher in the stromal cells than in epithelial cells, whereas the number of oestradiol receptors was higher in the epithelial cells than in stromal cells. Oestradiol upregulates its own receptor and increases the number of progesterone receptors in both cell types in vitro, whereas progesterone has little effect, but inhibits the effects of oestradiol on progesterone receptors.     

Trypsin treatment of bovine embryos after in vitro exposure to infectious bovine rhinotracheitis virus or bovine herpesvirus-4

The objectives of this study were to evaluate the efficacy of trypsin treatment for the removal/inactivation of infectious bovine rhinotracheitis virus (IBRV) adhering to zona pellucida-intact (ZP-I) bovine embryos and to determine if bovine herpesvirus-4 (BHV-4) adheres to ZP-I bovine embryos. When adherence of BHV-4 was demonstrated, an additional objective was to determine whether trypsin treatment removes or inactivates this virus. A total of 139 ZP-I embryos was collected from superovulated donor cows at 7 d after estrus. Embryos were exposed to 10(6) to 10(7) plaque-forming units (pfu) of either IBRV or BHV-4 for 1 to 2 h. Subsequently, approximately equal numbers of embryos exposed to each virus were either washed 12 times and the washes and embryos examined for the presence of infectious virus, or they were treated with trypsin and the embryos examined for the presence of infectious virus. Although the fourth wash was the last positive wash, an average of 18 pfu of virus was detected from each of six groups (a total of 24 embryos) after exposure to IBRV and washing. Infectious bovine rhinotracheitis virus was not isolated from any of nine trypsin-treated groups (a total of 43 embryos). The seventh wash was the last positive wash for any group after exposure to BHV-4, yet an average of 2 pfu of virus was detected from each of six groups (a total of 29 embryos) after washing. No BHV-4 was isolated from any of eight trypsin-treated groups (a total of 43 embryos). The study confirmed previous reports that IBRV adheres to the bovine ZP after in vitro exposure and that trypsin treatment is effective in keeping ZP-I embryos free of this virus. Adherence of BHV-4 to ZP-I bovine embryos was demonstrated for the first time. Trypsin treatment was also effective in removing this herpesvirus.     

The lipoxygenase pathways are involved in LH-stimulated progesterone production in bovine corpus luteum.

We have examined the effects of endogenous lipoxygenase products on basal progesterone (P4) production by cultured bovine mid-luteal cells. The involvement of lipoxygenase products in the stimulatory effect of LH on luteal cAMP accumulation and P4 production was also examined. Bovine luteal cells from mid-cycle corpora lutea (CL) were exposed for 16 h to a lipoxygenase inhibitor (nordihydroguaiaretic acid: NDGA; 0.33-33 microM). For the last 4 h of incubation, the cells were exposed to LH and/or three different lipoxygenase products, 5-, 12- and 15-hydroxyeicosatetraenoic acid (HETE). NDGA inhibited P4 production by the cells in a dose-dependent manner (P < 0.05). NDGA-reduced P4 production was reversed by the addition of 12-HETE, but not 5- or 15-HETE, whereas 5-, 12- and 15-HETE alone showed no significant effect on P4 production in the intact cells. Furthermore, NDGA (33 microM) blocked the stimulatory action of LH on P4 production (P < 0.05), without changing cAMP accumulation (P > 0.1). When the cells were exposed to 5-, 12- or 15-HETE with LH and NDGA, only 15-HETE maintained the stimulatory effect of LH on P4 production in the cells (P < 0.05). These results suggest that endogenous lipoxygenase products play important roles in P4 production by bovine CL, i.e. basal P4 production is supported by 12-HETE, and LH-stimulated P4 production is partially mediated via the activation of lipoxygenase and subsequent 15-HETE formation downstream of the LH-activated cAMP-PKA-phosphorylation pathway.     

Bone marrow hemopoiesis at emotional stress reactions of various intensity upon a background of exposure to low dose ionizing radiation

Rats preliminary exposed to 0.9 Gy of ionizing radiation showed disturbances in the development of adaptive reactions of blood system to emotional stress, compensatory capacity of the blood system being decreased. A degree of the disturbances directly depended on the duration and intensity of the emotional stress: at the prolonged intensive emotional stress a sharp decrease in the adaptive and compensatory capacity of blood system was found; at short intensive or moderate stress the changes in the adaptive and compensatory capacity of hemopoiesis were less pronounced. A combined action of low-dose ionizing radiation and short weak emotional stress did non affect the adaptive and compensatory capacity of hemopoietic system.     

Evidence for thermal reactions following exposure of Didinium to intermittent ultraviolet radiations

1. The nature of ultraviolet injury and its variation with the same dose given at different intensities and wave lengths have been investigated in the protozoan Didinium nasutum, using time to the fourth division as a measure of injury. 2. The injury has been found to consist of a "slowdown" of division rate, which always occurs, and a "stasis," usually at the second division after irradiation, which appears in varying degrees among more severely injured samples. 3. Injury was found to be almost independent of intensity at three wave lengths out of four studied over a wide range of intermediate and high intensities, but was found to rise sharply with lower intensity at all except the longest wave length. 4. Flashed UV of high intensity is much more effective than the same dose of continuous radiation at high intensity and shorter total time of treatment. It is also more effective than the same dose at low intensity and equal time of treatment, though only slightly so. 5. An increase of injury with rise of temperature and with increase of dark period clearly indicates that injury depends on thermochemical reactions following the absorption of UV in Didinium. 6. The most reasonable assumption is that a similar conclusion applies to other organisms as well, and that its general application may be useful in the investigation of UV effects on protoplasm.     

Stability of bacterial populations in tropical soil upon exposure to Lindane

The effect of the pesticide Lindane on microbial populations was analyzed in soil with a history of contamination with various chemicals, including this pesticide. Soil microcosms were amended with 100 mg Lindane/kg soil and microbial populations were monitored for 70 days. Bacterial cell concentrations, metabolic versatility (whole community Biolog), and genetic diversity (16S rDNA/denaturing gradient gel electrophoresis) were used to monitor microbial communities. Results show the persistence of Lindane in the soil environment; at the end of the experiment, 70% of the added Lindane remained undegraded. A reduction of 50% in bacterial cell concentration was observed in Lindane-amended microcosms during the 2nd week of the experiment. This reduction was correlated with a reduction in the rate of substrate utilization as observed by Biolog. Overall, no effect of Lindane was observed on the metabolic versatility and genetic diversity in these soils, demonstrating the ability of these bacterial populations to tolerate the pressure caused by the addition of pesticides.