A deletion in the PHYD gene of the Arabidopsis Wassilewskija ecotype defines a role for phytochrome D in red/far-red light sensing.

The PHYD gene of the Wassilewskija (Ws) ecotype of Arabidopsis contains a 14-bp deletion (the phyD-1 mutation) beginning at amino acid 29 of the reading frame, resulting in translation termination at a nonsense codon 138 nucleotides downstream of the deletion end point. Immunoblot analyses showed that Ws lacks phyD but contains normal levels of phyA, phyB, and phyC. By backcrossing into the Ws and Landsberg erecta genetic backgrounds, we constructed sibling pairs of PHYD+ and phyD-1 lines and of phyB- PHYD+ and phyB- phyD- lines. Hypocotyl lengths after growth under white or red light increased sequentially in strains that were B+D+, B+D-, B-D+, and B-D-. In the Ws genetic background, an increase in petiole length, a reduction in cotyledon area and in anthocyanin accumulation in seedling stems, a diminished effect of an end-of-day pulse of far-red light on hypocotyl elongation, and a decrease in the number of rosette leaves at the onset of flowering were also seen sequentially in these lines. Thus, phyD, which is approximately 80% identical in amino acid sequence to phyB, acts in conjunction with phyB in regulating many shade avoidance responses. The existence of the apparently naturally occurring phyD-1 mutation indicates that phyD is not essential in some natural environments.     

Different Mechanisms of Four Aluminum (Al)-Resistant Transgenes for Al Toxicity in Arabidopsis

We have characterized the mechanism of action of four transgenes (AtBCB [Arabidopsis blue copper-binding protein], parB [tobacco (Nicotiana tabacum) glutathione S-transferase], NtPox [tobacco peroxidase], and NtGDI1 [tobacco GDP dissociation inhibitor]) that independently Al resistance on transgenic Arabidopsis. All four transgenic lines showed lower deposition of callose after Al treatment than the Landsberg erecta ecotype of Arabidopsis, confirming that the four genes function to ameliorate Al toxicity. Influx and efflux experiments of Al ions suggested that the AtBCB gene may suppress Al absorption, whereas expression of the NtGDI1 gene promotes a release of Al in the root tip region of Arabidopsis. The total enzyme activities of glutathione S-transferases or peroxidases in transgenic lines carrying either the parB or NtPox genes were significantly higher than in the Landsberg erecta ecotype of Arabidopsis, and these enzyme activities were maintained at higher levels during Al stress. Furthermore, lipid peroxidation caused by Al stress was repressed in these two transgenic lines, suggesting that overexpression of these two genes diminishes oxidative damage caused by Al stress. Al-treated roots of transgenic plants were also stained by 4',6-diamino-2-phenylindole to monitor cell death caused by Al toxicity. The result suggested that cell death is repressed in the NtPox line. Analysis of F(1) hybrids between the four transgenic lines suggests that more resistant transgenic plants can be constructed by combinations of these four genes.     

Combined genetic and modeling approaches reveal that epidermal cell area and number in leaves are controlled by leaf and plant developmental processes in Arabidopsis

Both leaf production and leaf expansion are tightly linked to cell expansion and cell division, but the functional relationships between all these variables are not clearly established. To get insight into these relationships, a quantitative genetic analysis was performed in 118 recombinant inbred lines derived from a cross between the Landsberg erecta and Antwerp accessions and was combined with a structural equation modeling approach. Main effects and epistatic interactions at the quantitative trait locus (QTL) level were detected for rosette area, rosette leaf number, leaf 6 area, epidermal cell area and number. A QTL at ERECTA marker (ER) controlled cell expansion and cell division, in interaction with two other QTLs at SNP295 and SNP21 markers. Moreover, both the screening for marker association involved in the variation of the relationships between leaf growth variables and the test of alternative functional models by structural equation modeling revealed that the allelic value at ER controlled epidermal cell area and epidermal cell number in a leaf. These effects are driven both by a whole plant mechanism associated with leaf production and by a single leaf mechanism associated with leaf expansion. The complex effects of the QTL at ER were validated in selected heterogeneous inbred families. The ERECTA gene, which is mutated in the Landsberg erecta parental line, was found to be a putative candidate responsible for these mapped effects by phenotyping mutants of this gene at the cellular level. Together, these results give insight into the complex determination of leaf epidermal cell number and area.     

Mapping quantitative trait loci associated with selenate tolerance in Arabidopsis thaliana

Selenium is essential for many organisms, but is toxic at higher levels. To investigate the genetic basis of selenate tolerance in Arabidopsis thaliana, quantitative trait loci (QTL) associated with selenate tolerance in accessions Landsberg erecta and Columbia were mapped using recombinant inbred lines (RILs). The selenate tolerance index (TI(D10) = root growth + 30 microm selenate/root growth control x 100%) was fourfold higher for parental line Col-4 (59%) than for parent Ler-0 (15%). Among the 96 F8 RILs, TI(D10) ranged from 11 to 75% (mean 37%). Using composite interval mapping, three QTL were found on chromosomes 1, 3 and 5, which together explained 24% of variation in TI(D10) and 32% of the phenotypic variation for the difference in root length +/- Se (RL(D10)). Highly significant epistatic interactions between the QTL and markers on chromosome 2 explained additional variation for both traits. Potential candidate genes for Se tolerance in each of the QTL regions are discussed. These results offer insight into the genetic basis of selenate tolerance, and may be useful for identification of selenate-tolerance genes.     

Variations in xanthophyll composition in etiolated seedlings of Arabidopsis thaliana correlate with protochlorophyllide accumulation

Protochlorophyllide (Pchlide) accumulation and xantophyll composition were studied in 5-day old etiolated seedlings of three ecotypes of Arabidopsis thaliana: Columbia (Col-0), Landsberg erecta (Ler) and Wassiliewska (Ws). The total Pchlide level as measured by fluorescence spectroscopy varied significantly between ecotypes. A rapid HPLC method revealed quantitative differences in carotenoid composition. It was found that in the Ler ecotype any enhanced accumulation of Pchlide correlates with an increased level of lutein, suggesting the role of enzymes involved in lutein synthesis in cross-regulation between chlorophyll and carotenoid biosynthetic pathways. The function of the dark-accumulated carotenoid pool in seedling de-etiolation is discussed.     

Growth and development in Arabidopsis thaliana through an entire life cycle under simulated microgravity conditions on a clinostat.

The effects of simulated microgravity conditions produced by a horizontal clinostat on the entire life cycle of Arabidopsis thaliana ecotype Columbia and Landsberg erecta were studied. Horizontal clinorotation affected little germination of seeds, growth and development of rosette leaves and roots during early vegetative growth stage, and the onset of the bolting of inflorescence axis and flower formation in reproductive growth stage, although it suppressed elongation of inflorescence axes. The clinorotation substantially reduced the numbers of siliques and seeds in Landsberg erecta, and completely inhibited seed production in Columbia. Seeds produced in Landsberg erecta on the clinostat were capable of germinating and developing rosette leaves normally on the ground. On the other hand, growth of pin formed mutant (pin/pin) of Arabidopsis ecotype Enkheim, which has a unique structure of inflorescence axis with no flower and extremely low levels of auxin polar transport activity, was inhibited and the seedlings frequently died during vegetative stage on the clinostat. Seed formation and inflorescence growth of the seedlings with normal shape (pin/+ or +/+) were also suppressed on the clinostat. These results suggest that the growth and development of Arabidopsis, especially in reproductive growth stage, is suppressed under simulated microgravity conditions on a clinostat. To complete the life cycle probably seems to be quite difficult, although it is possible in some ecotypes.     

Involvement of ethylene and gibberellin signalings in chromosaponin I-induced cell division and cell elongation in the roots of Arabidopsis seedlings

Chromosaponin I (CSI), a triterpenoid saponin isolated from pea, stimulates the growth of roots in Arabidopsis thaliana seedlings on wetted filter paper in the light for 14 d. The growth rates of roots in Columbia (Col) and Landsberg erecta (Ler) wild-types were 0.92 and 0.26 mm d(-1), respectively, and they were accelerated to 3.46 (Col) and 2.20 (Ler) mm d(-1) by treating with 300 microM CSI. The length of mature epidermal cells was increased by 1.8-fold (Col) and 2.81-fold (Ler) compared with control and the number of epidermal cells was increased by a factor of 1.65 (Col) and 2.12 (Ler). Treatment with 2-aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, also increased cell length but not cell number. The effects of CSI on root growth were not detected in the ethylene-insensitive mutant ein2-1. CSI did not inhibit ethylene production but stimulated the growth of roots in ctr1-1, the constitutive triple response mutant for ethylene, indicating that CSI inhibits ethylene signaling, especially downstream of CTR1. In the GA-insensitive mutant gai and the mutant spy-3, in which the basal level of GA signaling is activated, CSI did not increase cell number, although both CSI and AVG stimulated cell elongation in these mutants. These results suggest that the inhibition of ethylene signaling is the cause of CSI-induced cell elongation. A possible involvement of both GA and ethylene signalings is discussed for the CSI-induced cell division.     

The TASTY locus on chromosome 1 of Arabidopsis affects feeding of the insect herbivore Trichoplusia ni

The generalist insect herbivore Trichoplusia ni (cabbage looper) readily consumes Arabidopsis and can complete its entire life cycle on this plant. Natural isolates (ecotypes) of Arabidopsis are not equally susceptible to T. ni feeding. While some are hardly touched by T. ni, others are eaten completely to the ground. Comparison of two commonly studied Arabidopsis ecotypes in choice experiments showed that Columbia is considerably more resistant than Landsberg erecta. In no-choice experiments, where larvae were confined on one or the other ecotype, weight gain was more rapid on Landsberg erecta than on Columbia. Genetic mapping of this difference in insect susceptibility using recombinant inbred lines resulted in the discovery of the TASTY locus near 85 cM on chromosome 1 of Arabidopsis. The resistant allele of this locus is in the Columbia ecotype, and an F(1) hybrid has a sensitive phenotype that is similar to that of Landsberg erecta. The TASTY locus is distinct from known genetic differences between Columbia and Landsberg erecta that affect glucosinolate content, trichome density, disease resistance, and flowering time.     

The VLF loci, polymorphic between ecotypes Landsberg erecta and Columbia, dissect two branches of phytochrome A signal transduction that correspond to very-low-fluence and high-irradiance responses

Phytochromes play a key role in the perception of light signals by plants. In this study, the three classical phytochrome action modes, i.e. very-low-fluence responses (VLFR), low-fluence responses (LFR) and high-irradiance responses (HIR), were genetically dissected using phyA and phyB mutants of Arabidopsis thaliana (respectively lacking phytochrome A or phytochrome B) and a polymorphism between ecotypes Landsberg erecta and Columbia. Seed germination and potentiation of greening, hypocotyl growth inhibition and cotyledon unfolding in etiolated seedlings of the ecotype Landsberg erecta showed biphasic responses to the calculated proportion of active phytochrome established by one light pulse or repeated light pulses. The first phase, i.e. the VLFR, was absent in the phyA mutant, normal in the phyB mutant (both in the Landsberg erecta background) and severely deficient in Columbia. The second phase, i.e. the LFR, was present in the phyA mutant, deficient in the phyB mutant and normal in Columbia. Under continuous far-red light, HIR of etiolated seedlings were absent in phyA and normal in phyB and Columbia. The segregation of VLFR in recombinant inbred lines derived from a cross between Landsberg erecta and Columbia was analysed by MAPMAKER/QTL. Two quantitative trait loci, one on chromosome 2 ( VLF1 ) and another on chromosome 5 ( VLF2 ), were identified as responsible for the polymorphism. Phytochrome A is proposed to initiate two transduction pathways, VLFR and HIR, involving different cells and/or different molecular steps. This is the first application of the analysis of quantitative trait loci polymorphic between ecotypes to dissect transduction chains of environmental signals.     

Analysis of leaf proteins in late flowering mutants of Arabidopsis thaliana

Late flowering monogenic mutants of Arabidopsis thaliana (L.) Heynh. at the loci co, gi, fca, fve, fwa, fha, fpa, fy and their corresponding wild type, Landsberg erecta , were analysed by two-dimensional gel electrophoresis. All plants were grown under continuous light and proteins were extracted from leaves of the same age (20-day-old). The polypeptide patterns of the mutants at the loci co, gi, fca, fve, fwa, fha, fpa , and Landsberg erecta were identical. The mutant at the fy locus showed a qualitative difference with Landsberg erecta . Crosses were made between this line and the wild type Landsberg erecta . F2 plants, resulting from autopollination of the hybrid, were analysed and showed no cosegregation between the observed protein and the flowering phenotype, indicating that these two lines differ by more than a single mutation.     

Peroxisomal ascorbate peroxidase resides within a subdomain of rough endoplasmic reticulum in wild-type Arabidopsis cells

Previously we reported (R.T. Mullen, C.S. Lisenbee, J.A. Miernyk, R.N. Trelease [1999] Plant Cell 11: 2167-2185) that overexpressed ascorbate peroxidase (APX), a peroxisomal membrane protein, sorted indirectly to Bright Yellow-2 cell peroxisomes via a subdomain of the endoplasmic reticulum (ER; peroxisomal endoplasmic reticulum [pER]). More recently, a pER-like compartment also was identified in pumpkin (Cucurbita pepo) and transformed Arabidopsis cells (K. Nito, K. Yamaguchi, M. Kondo, M. Hayashi, M. Nishimura [2001] Plant Cell Physiol 42: 20-27). Here, we characterize more extensively the localization of endogenous Arabidopsis peroxisomal APX (AtAPX) in cultured wild-type Arabidopsis cells (Arabidopsis var. Landsberg erecta). AtAPX was detected in peroxisomes, but not in an ER subcompartment, using immunofluorescence microscopy. However, AtAPX was detected readily with immunoblots in both peroxisomal and ER fractions recovered from sucrose (Suc) density gradients. Most AtAPX in microsomes (200,000g, 1 h pellet) applied to gradients exhibited a Mg2+-induced shift from a distribution throughout gradients (approximately 18%-40% [w/w] Suc) to > or =42% (w/w) Suc regions of gradients, including pellets, indicative of localization in rough ER vesicles. Immunogold electron microscopy of the latter fractions verified these findings. Further analyses of peroxisomal and rough ER vesicle fractions revealed that AtAPX in both fractions was similarly associated with and located mostly on the cytosolic face of the membranes. Thus, at the steady state, endogenous peroxisomal AtAPX resides at different levels in rough ER and peroxisomes. Collectively, these findings show that rather than being a transiently induced sorting compartment formed in response to overexpressed peroxisomal APX, portions of rough ER (pER) in wild-type cells serve as a constitutive sorting compartment likely involved in posttranslational routing of constitutively synthesized peroxisomal APX.     

Development and growth of several strains of Arabidopsis seedlings in microgravity

Growth and development of dark-grown Arabidopsis thaliana seedlings were studied in microgravity during space shuttle mission STS-84. The major purpose of this project was to determine if there were developmental differences among the four ecotypes studied--Wassilewskija (Ws), Columbia (Col), Landsberg erecta (Ler), and C24--and to evaluate whether particular ecotypes are better suited for spaceflight experimentation compared with others. A secondary goal was to study the growth of three starch-deficient strains of Arabidopsis by extending the observations made in a previously published report. For all strains, seed germination was not affected by microgravity, but seedlings were smaller in the spaceflight samples compared with the ground controls. The starch-deficient strains continued to exhibit vigorous growth until the termination of the experiment at 121 h after imbibition of seeds. However, ethylene effects, i.e., reduced growth and exaggerated hypocotyl hooks, were observed in all strains studied. Nevertheless, the Ler and C24 ecotypes seem to be more suitable for spaceflight research, compared with the other two ecotypes, based on measurements of their relative and absolute growth. This type of information should aid in the design of plant experiments for the International Space Station.     

The survey on cellular and tissue-engineered therapies in Europe and neighboring Eurasian countries in 2014 and 2015

Background aims

With the support of five established scientific organizations, this report, the seventh of its kind, describes activity in Europe for the years 2014 and 2015 in the area of cellular and tissue-engineered therapies, excluding hematopoietic stem cell (HSC) treatments for the reconstitution of hematopoiesis.

Ethylene-induced differential growth of petioles in Arabidopsis. Analyzing natural variation, response kinetics, and regulation

Plants can reorient their organs in response to changes in environmental conditions. In some species, ethylene can induce resource-directed growth by stimulating a more vertical orientation of the petioles (hyponasty) and enhanced elongation. In this study on Arabidopsis (Arabidopsis thaliana), we show significant natural variation in ethylene-induced petiole elongation and hyponastic growth. This hyponastic growth was rapidly induced and also reversible because the petioles returned to normal after ethylene withdrawal. To unravel the mechanisms behind the natural variation, two contrasting accessions in ethylene-induced hyponasty were studied in detail. Columbia-0 showed a strong hyponastic response to ethylene, whereas this response was almost absent in Landsberg erecta (Ler). To test whether Ler is capable of showing hyponastic growth at all, several signals were applied. From all the signals applied, only spectrally neutral shade (20 micromol m(-2) s(-1)) could induce a strong hyponastic response in Ler. Therefore, Ler has the capacity for hyponastic growth. Furthermore, the lack of ethylene-induced hyponastic growth in Ler is not the result of already-saturating ethylene production rates or insensitivity to ethylene, as an ethylene-responsive gene was up-regulated upon ethylene treatment in the petioles. Therefore, we conclude that Ler is missing an essential component between the primary ethylene signal transduction chain and a downstream part of the hyponastic growth signal transduction pathway.     

Identification of a locus in arabidopsis controlling both the expression of rhizobacteria-mediated induced systemic resistance (ISR) and basal resistance against Pseudomonas syringae pv. tomato.

Selected nonpathogenic rhizobacteria with biological disease control activity are able to elicit an induced systemic resistance (ISR) response that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR). Ten ecotypes of Arabidopsis thaliana were screened for their potential to express rhizobacteria-mediated ISR and pathogen-induced SAR against the leaf pathogen Pseudomonas syringae pv. tomato DC3000 (Pst). All ecotypes expressed SAR. However, of the 10 ecotypes tested, ecotypes RLD and Wassilewskija (Ws) did not develop ISR after treatment of the roots with nonpathogenic Pseudomonas fluorescens WCS417r bacteria. This nonresponsive phenotype was associated with relatively high susceptibility to Pst infection. The F1 progeny of crosses between the non-responsive ecotypes RLD and Ws on the one hand, and the responsive ecotypes Columbia (Col) and Landsberg erecta (Ler) on the other hand, were fully capable of expressing ISR and exhibited a relatively high level of basal resistance, similar to that of their WCS417r-responsive parent. This indicates that the potential to express ISR and the relatively high level of basal resistance against Pst are both inherited as dominant traits. Analysis of the F2 and F3 progeny of a Col x RLD cross revealed that inducibility of ISR and relatively high basal resistance against Pst cosegregate in a 3:1 fashion, suggesting that both resistance mechanisms are monogenically determined and genetically linked. Neither the responsiveness to WCS417r nor the relatively high level of basal resistance against Pst were complemented in the F1 progeny of crosses between RLD and Ws, indicating that RLD and Ws are both affected in the same locus, necessary for the expression of ISR and basal resistance against Pst. The corresponding locus, designated ISR1, was mapped between markers B4 and GL1 on chromosome 3. The observed association between ISR and basal resistance against Pst suggests that rhizobacteria-mediated ISR against Pst in Arabidopsis requires the presence of a single dominant gene that functions in the basal resistance response against Pst infection.     

Genetic control of storage oil synthesis in seeds of Arabidopsis

Quantitative trait loci (QTL) that control seed oil content and fatty acid composition were studied using a recombinant inbred population derived from a cross between the Arabidopsis ecotypes Landsberg erecta and Cape Verdi Islands. Multiple QTL model mapping identified two major and two minor QTL that account for 43% of the variation in oil content in the population. The most significant QTL is at the bottom of chromosome 2 and accounts for 17% of the genetic variation. Two other significant QTL, located on the upper and lower arms of chromosome 1, account for a further 19% of the genetic variation. A QTL near to the top of chomosome 3 is epistatic to that on the upper arm of chromosome 1. There are strong QTL for linoleic (18:2) and linolenic (18:3) acids contents that colocate with the FAD3 locus, another for oleic acid (18:1) that colocates with FAD2 and other less significant QTL for palmitic (16:0), stearic (18:0), and eicosaenoic (20:1) acids. The presence of the QTL for seed oil content on chromosome 2 was confirmed by the generation of lines that contain a 22-cM region of Landsberg erecta DNA at the bottom of chromosome 2 in a background containing Cape Verdi Islands in other regions of the genome that had been shown to influence oil content in the QTL analysis.     

Hydrolysis of isoflavone glucosides in soymilk fermented with single or mixed cultures of Lactobacillus paraplantarum KM, Weissella sp. 33, and Enterococcus faecium 35 isolated from humans

Lactobacillus paraplantarum KM (Lp), Weissella sp. 33 (Ws), and Enterococcus faecium 35 (Ef) were used in single (Lp, Ws, Ef) or mixed cultures (Lp+Ws, Lp+Ef, Ws+Ef) for soymilk fermentation (37 degrees C, 12 h). After 12 h, the cell numbers, pH, and TA of soymilk were 7.4x108 -6.0x109 CFU/ ml, 3.8-4.5, and 0.59-0.70%, respectively. Changes in the contents of glycitin and genistin in soymilk fermented with Ef were not significant (p<0.05). The contents of isoflavone glucosides in soymilk fermented with the other cultures decreased significantly with an increase of aglycone contents (p<0.05). It corresponded well with a sharp increase in beta- glucosidase activity during fermentation. About 92-100% of the daidzin and 98-100% of the genistin in soymilk were converted to corresponding aglycones by Lp, Ws, or Lp+Ef within 12 h.     

Temporal and Spatial Development of the Cells of the Expanding First Leaf of Arabidopsis thaliana (L.) Heynh

The three-dimensional quantitative leaf anatomy in developingyoung (9–22 d) first leaves of wild type Arabidopsis thalianacv. Landsberg erecta from mitosis through cell and leaf expansionto the cessation of lamina growth has been studied. The domainsof cell division, the relative proportion of the cell typespresent during development and the production of intercellularspace in the developing leaf have been determined by image analysisof entire leaves sectioned in three planes. Mitotic activityoccurs throughout the youngest leaves prior to unfolding andcell expansion is initiated firstly at the leaf tip with a persistentzone of mitotic cells at the leaf base resulting in a gradientof development along the leaf axis, which persists in the olderleaves. Major anatomical changes which occur during the developmentare, a rapid increase in mesophyll volume, an increase in thevein network, and expansion of the intercellular spaces. Thepattern of cell expansion results in a 10-fold variation inmesophyll cell size in mature leaves. In the youngest leavesthe plan area of mesophyll cells varies between 100 µm²and 400 µm² whereas in mature leaves mesophyll cells rangein plan area from 800 µm² to 9500 µm². The volumesof mesophyll tissue and airspace under unit leaf area increase3-fold and 35-fold, respectively, during leaf expansion. Thevolume proportions of tissue types mesophyll:airspace:epiderrnal:vascularin the mature leaf are 61:26:12:1, respectively. This studyprovides comparative information for future identification andanalysis of leaf development mutants of Arabidopsis thaliana. Key words: Arabidopsis, quantitative leaf anatomy, leaf expansion, image analysis     

Effects of brief postponement of the preovulatory LH surge on ovulation rates and embryo formation in eCG/prostaglandin-treated heifers

The aim of this study was to investigate whether prolongation of the period of preovulatory follicular development after superovulation reduces heterogeneity of oocytes of stimulated follicles with respect to the potential to mature, to ovulate, to be fertilized and to develop into embryos. Heifers were treated with eCG on Day 10 and prostaglandin (PG) 48 h later. At the time of eCG administration some of the heifers received a norgestomet implant (N) to suppress the LH surge. After 96 to 104 h, N was removed and an LH surge was induced with GnRH (G) (N/G); the other animals served as controls. Matured oocytes (Experiment A: n=9, 139 [N/G] and 11, 125 [Control] heifers, oocytes), zygotes and oviducts (Experiment B: n=8, 44 [N/G] and 9, 72 [Control] heifers, zygotes) and embryos (Experiment C: n=11, 205 [N/G] and 11, 165 [Control] heifers, embryos) were collected at 22 to 26 h, 38 to 52 h and 7 days after the LH surge, respectively. Hatched blastocyst formation of matured oocytes (Experiment A) was analyzed after 11 days of IVC after IVF. In vivo fertilization rate of zygotes, the presence of periodic acid-Schiff (PAS) positive granules in the oviduct (Experiment B) and stage of development of embryos (Experiment C) were analyzed stereomicroscopically. The mean interval between PG and the LH surge was 53.8+/-3 (SD) (N/G) vs. 42.4+/-4 h (Control). The maximum peripheral estradiol-17beta concentration (529+/-36 [SEM] [N/G] vs. 403+/-17 pmol/L [Control]) and the response to superovulation (25.4+/-2 [N/G] vs. 18.7+/-2 [Control]) were higher in N/G than in Control heifers. Hatched blastocyst formation rate (37.4 [N/G] vs. 33.6% [Control]), in vivo fertilization rate (69.0+/-14 [N/G] vs. 73.0+/-10% [Control]) and the yield of total embryos (3.8+/-1 [N/G] vs. 5.6+/-2 [Control]) did not differ between groups. The percentage of heifers with abundant PAS-positive granules in the distal ampulla (0 [N/G] vs. 31% [Control]) was reduced after N/G treatment. Prolongation of the period of preovulatory follicular development increased the number of mature follicles and ovulations but did not result in higher embryo yield, possibly because of an impaired oviductal environment.