Prolonging the time of progesterone supplementation to improve the pregnancy outcomes of single day 6 blastocyst transfer in frozen-thawed cycles: study protocol for a randomized controlled trial.

BACKGROUND: Infertility is one of the most important and underappreciated reproductive health problems in developing countries. Currently, in vitro fertilization and embryo transfer is the most effective treatment strategy for infertility. In a frozen-thawed cycle, single-blastocyst transfer can not only ensure relatively higher pregnancy and live birth rates but also effectively reduce the risk of maternal and neonatal complications. In frozen-thawed cycles, progesterone is initiated to promote the final phase of endometrial preparation prior to embryo transfer. However, the optimal duration of exposure to progesterone has remained inconclusive. Therefore, we designed a randomized controlled trial (RCT) to compare the effects of different prolonged progesterone transformation times (P+6 and P+7) on the pregnancy outcomes of D6 single blastocyst transfer in a frozen-thawed cycle. METHODS: This is a single-center, prospective, randomized controlled clinical trial involving 900 patients with single blastocyst transfer in the frozen-thawed cycle, aged from 20 to 38 years, with less than three transfers, and with HRT-cycle single D6 blastocyst transfer in the current cycle. Participants will be randomly assigned (1:1) into two parallel groups: the transfer of day 6 blastocysts on the 7th day of progesterone supplementation and the transfer of day 6 blastocysts on the 6th day of progesterone supplementation. The primary outcome measure is the clinical pregnancy rate. Secondary outcome measures include the miscarriage rate and live birth rate. DISCUSSION: This is the first randomized controlled trial to compare the transfer of day 6 blastocysts on the 6th and 7th day of progesterone supplementation. The results of this study will provide evidence for whether to prolong the duration of exposure to progesterone prior to embryo transfer. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT04938011. Registered on 19 June 2021.

A high level of KLF12 causes folic acid-resistant neural tube defects by activating the Shh signaling pathway in mice†.

Although adequate periconceptional folic acid (FA) supplementation has reduced the occurrence of pregnancies affected by neural tube defects (NTDs), the mechanisms underlying FA-resistant NTDs are poorly understood, and thus NTDs still remain a global public health concern. A high level of Krüppel-like factor 12 (KLF12) exerts deleterious effects on heath in most cases, but evidence for its roles in development has not been published. We observed KLF12-overexpressing mice showed disturbed neural tube development. KLF12-overexpressing fetuses died in utero at approximately 10.5 days post-coitus, with 100% presenting cranial NTDs. Neither FA nor formate promoted normal neural tube closure in mutant fetuses. The RNA-seq results showed that a high level of KLF12 caused NTDs in mice via overactivating the sonic hedgehog (Shh) signaling pathway, leading to the upregulation of patched 1, GLI-Krüppel family member GLI1, hedgehog-interacting protein, etc., whereas FA metabolism-related enzymes did not express differently. PF-5274857, an antagonist of the Shh signaling pathway, significantly promoted dorsolateral hinge point formation and partially rescued the NTDs. The regulatory hierarchy between a high level of KLF12 and FA-resistant NTDs might provide new insights into the diagnosis and treatment of unexplained NTDs in the future.

Categorization of wheat genotypes for phosphorus efficiency.

Production of phosphorus efficient crop cultivars can increase food productivity and decrease environmental pollution. Categorization of existing germplasm is a prerequisite to develop P efficient crop cultivars. For first experiment, 30 wheat genotypes were grown in hydroponics with two P levels (i.e., deficit, 20 µm KH2PO4 and adequate, 200 µm KH2PO4). Genotypes differed significantly for various P efficiency parameters. Two genotypes (Dirk and Bhakkar-02) showed < 25% decrease in growth at P deficiency. Genotype Seher-06 proved to be inefficient. Twelve selected genotypes based on the first experiment were sown in soil with two P levels (0 and 30 mg P kg-1) till maturity. As expected, genotypes differed for grain yield at both P levels. The efficient cultivars selected on the basis of both absolute and relative dry matter production at both P levels such as Dirk. Genotypes were grouped into three, four and nine classes on the basis of various parameters for P efficiency as proposed by different researchers. Most genotypes behaved in a similar fashion by different categorization methods and also at different P supply. The method to categorize the genotypes into three classes and plotting them into 9 classes proposed by Gill and his coworkers, is the best to differentiate the minor differences in genotypes. At least three different parameters at both P regimes should be used. The parameters may vary as per objectives of the study and/or growth conditions.

Cotton S-adenosylmethionine decarboxylase-mediated spermine biosynthesis is required for salicylic acid- and leucine-correlated signaling in the defense response to Verticillium dahliae.

MAIN CONCLUSION: Cotton S-adenosylmethionine decarboxylase-, rather than spermine synthase-, mediated spermine biosynthesis is required for salicylic acid- and leucine-correlated signaling in the defense response to Verticillium dahliae. Spermine (Spm) signaling is correlated with plant resistance to the fungal pathogen Verticillium dahliae. We identified genes for key rate-limiting enzymes in the biosynthesis of Spm, namely S-adenosylmethionine decarboxylase (GhSAMDC) and Spm synthase (GhSPMS). These were found by screening suppression subtractive hybridization and cDNA libraries of cotton (Gossypium) species tolerant to Verticillium wilt. Both were induced early and strongly by inoculation with V. dahliae and application of plant hormones. Silencing of GhSPMS or GhSAMDC in cotton leaves led to a significant accumulation of upstream substrates and, ultimately, enhanced plant susceptibility to Verticillium infection. Exogenous supplementation of Spm to the silenced cotton plants improved resistance. When compared with the wild type (WT), constitutive expression of GhSAMDC in Arabidopsis thaliana was associated with greater Verticillium wilt resistance and higher accumulations of Spm, salicylic acid, and leucine during the infection period. By contrast, transgenic Arabidopsis plants that over-expressed GhSPMS were unexpectedly more susceptible than the WT to V. dahliae and they also had impaired levels of putrescine (Put) and salicylic acid (SA). The susceptibility exhibited in GhSPMS-overexpressing Arabidopsis plants was partially reversed by the exogenous supply of Put or SA. In addition, the responsiveness of those two transgenic Arabidopsis lines to V. dahliae was associated with an alteration in transcripts of genes involved in plant resistance to epidermal penetrations and amino acid signaling. Together, these results suggest that GhSAMDC-, rather than GhSPMS-, mediated spermine biosynthesis contributes to plant resistance against V. dahliae through SA- and leucine-correlated signaling.

The complex jujube genome provides insights into fruit tree biology.

The jujube (Ziziphus jujuba Mill.), a member of family Rhamnaceae, is a major dry fruit and a traditional herbal medicine for more than one billion people. Here we present a high-quality sequence for the complex jujube genome, the first genome sequence of Rhamnaceae, using an integrated strategy. The final assembly spans 437.65 Mb (98.6% of the estimated) with 321.45 Mb anchored to the 12 pseudo-chromosomes and contains 32,808 genes. The jujube genome has undergone frequent inter-chromosome fusions and segmental duplications, but no recent whole-genome duplication. Further analyses of the jujube-specific genes and transcriptome data from 15 tissues reveal the molecular mechanisms underlying some specific properties of the jujube. Its high vitamin C content can be attributed to a unique high level expression of genes involved in both biosynthesis and regeneration. Our study provides insights into jujube-specific biology and valuable genomic resources for the improvement of Rhamnaceae plants and other fruit trees.

GmPAP4, a novel purple acid phosphatase gene isolated from soybean (Glycine max), enhanced extracellular phytate utilization in Arabidopsis thaliana.

KEY MESSAGE: GmPAP4 , a novel plant PAP gene in soybean, has phytase activity. Over-expressing GmPAP4 can enhance Arabidopsis growth when phytate is the sole P source in culture. Phosphorus (P) is an important macronutrient for plant growth and development. However, most of the total P in soils is fixed into organic phosphate (Po). Purple acid phosphatase (PAP) can hydrolyze Po in the soil to liberate inorganic phosphate and enhance plant P utilization. We isolated a novel PAP gene, GmPAP4, from soybean (Glycine max). It had an open reading frame of 1,329 bp, encoding 442 amino acid residues. Sequence alignment and phylogenetics analysis indicated that GmPAP4 was similar to other plant PAPs with large molecular masses. Quantitative real-time PCR analysis showed that the induced expression of GmPAP4 was greater in P-efficient genotype Zhonghuang15 (ZH15) than in P-inefficient genotype Niumaohuang (NMH) during the periods of flowering (28-35 days post phytate stress; DPP) and pod formation (49-63 DPP). Moreover, peak expression, at 63 DPP, was about 3-fold higher in 'ZH15' than in 'NMH'. Sub-cellular localization showed that GmPAP4 might be on plasma membrane or in cytoplasm. Over-expressing GmPAP4 in Arabidopsis resulted in significant rises in P acquisition and utilization compared with the wild-type (WT). Under phytate condition, transgenic Arabidopsis plants showed increases of approximately 132.7 % in dry weight and 162.6 % in shoot P content compared with the WT. Furthermore, when phytate was added as the sole P source in cultures, the activity of acid phosphatase was significantly higher in transgenic plants. Therefore, GmPAP4 is a novel PAP gene that functions in plant's utilization of organic phosphate especially under phytate condition.

Production of viable male unreduced gametes in Brassica interspecific hybrids is genotype specific and stimulated by cold temperatures.

BACKGROUND: Unreduced gametes (gametes with the somatic chromosome number) may provide a pathway for evolutionary speciation via allopolyploid formation. We evaluated the effect of genotype and temperature on male unreduced gamete formation in Brassica allotetraploids and their interspecific hybrids. The frequency of unreduced gametes post-meiosis was estimated in sporads from the frequency of dyads or giant tetrads, and in pollen from the frequency of viable giant pollen compared with viable normal pollen. Giant tetrads were twice the volume of normal tetrads, and presumably resulted from pre-meiotic doubling of chromosome number. Giant pollen was defined as pollen with more than 1.5 × normal diameter, under the assumption that the doubling of DNA content in unreduced gametes would approximately double the pollen cell volume. The effect of genotype was assessed in five B. napus, two B. carinata and one B. juncea parents and in 13 interspecific hybrid combinations. The effect of temperature was assessed in a subset of genotypes in hot (day/night 30°C/20°C), warm (25°C/15°C), cool (18°C/13°C) and cold (10°C/5°C) treatments. RESULTS: Based on estimates at the sporad stage, some interspecific hybrid genotypes produced unreduced gametes (range 0.06 to 3.29%) at more than an order of magnitude higher frequency than in the parents (range 0.00% to 0.11%). In nine hybrids that produced viable mature pollen, the frequency of viable giant pollen (range 0.2% to 33.5%) was much greater than in the parents (range 0.0% to 0.4%). Giant pollen, most likely formed from unreduced gametes, was more viable than normal pollen in hybrids. Two B. napus × B. carinata hybrids produced 9% and 23% unreduced gametes based on post-meiotic sporad observations in the cold temperature treatment, which was more than two orders of magnitude higher than in the parents. CONCLUSIONS: These results demonstrate that sources of unreduced gametes, required for the triploid bridge hypothesis of allopolyploid evolution, are readily available in some Brassica interspecific hybrid genotypes, especially at cold temperatures.

Genotypic effects on the frequency of homoeologous and homologous recombination in Brassica napus × B. carinata hybrids.

We investigated the influence of genotype on homoeologous and homologous recombination frequency in eight different Brassica napus (AAC(n)C(n)) × B. carinata (BBC(c)C(c)) interspecific hybrids (genome composition C(n)C(c)AB). Meiotic recombination events were assessed through microsatellite marker analysis of 67 unreduced microspore-derived progeny. Thirty-four microsatellite markers amplified 83 A-, B-, C(n)- and C(c)-genome alleles at 64 loci, of which a subset of seven markers amplifying 26 alleles could be used to determine allele copy number. Hybrid genotypes varied significantly in loss of A- and B-genome alleles (P < 0.0001), which ranged from 6 to 22% between hybrid progeny sets. Allele copy number analysis revealed 19 A-C, 3 A-B and 10 B-C duplication/deletion events attributed to homoeologous recombination. Additionally, 55 deletions and 19 duplications without an accompanying dosage change in homoeologous alleles were detected. Hybrid progeny sets varied in observed frequencies of loss, gain and exchange of alleles across the A and B genomes as well as in the diploid C genome. Self-fertility in hybrid progeny decreased as the loss of B-genome loci (but not A-genome loci) increased. Hybrid genotypes with high levels of homologous and homoeologous exchange may be exploited for genetic introgressions between B. carinata and B. napus (canola), and those with low levels may be used to develop stable synthetic Brassica allopolyploids.

Flower numbers, pod production, pollen viability, and pistil function are reduced and flower and pod abortion increased in chickpea (Cicer arietinum L.) under terminal drought.

Terminal drought during the reproductive stage is a major constraint to yield of chickpea in many regions of the world. Termination of watering (WS) during podding in a small-seeded desi chickpea (Cicer arietinum L.) cultivar, Rupali, and a large-seeded kabuli chickpea cultivar, Almaz, induced a decrease in predawn leaf water potential (LWP), in the rate of photosynthesis, and in stomatal conductance. Compared to well-watered (WW) controls, the WS treatment reduced flower production by about two-thirds. In the WW treatment, about 15% of the flowers aborted and 42% (Rupali) and 67% (Almaz) of the pods aborted, whereas in the WS treatment 37% and 56% of the flowers aborted and 54% and 73% of the pods aborted, resulting in seed yields of 33% and 15% of the yields in WW plants in Rupali and Almaz, respectively. In vitro pollen viability and germination in Rupali decreased by 50% and 89% in the WS treatment, and pollen germination decreased by 80% in vivo when pollen from a WS plant was placed on a stigma of a WW plant. While about 37% of the germinated pollen tubes from WW plants and 22% from the WS plants reached the ovary in the WW plants, less than 3% of pollen grains reached the ovary when pollen from either WS or WW plants was placed on a stigma of a WS plant. It is concluded that, in addition to pod abortion, flower abortion is an important factor limiting yield in chickpea exposed to terminal drought and that water deficit impaired the function of the pistil/style more than the pollen.

Microspore culture preferentially selects unreduced (2n) gametes from an interspecific hybrid of Brassica napus L. x Brassica carinata Braun.

We analysed the products of male meiosis in microspore-derived progeny from a Brassica napus (AAC(n)C(n)) x Brassica carinata (BBC(c)C(c)) interspecific hybrid (ABC(n)C(c)). Genotyping at 102 microsatellite marker loci and nuclear DNA contents provided strong evidence that 26 of the 28 progeny (93%) were derived from unreduced (2n) gametes. The high level of C(n)C(c) marker heterozygosity, and parallel spindles at Anaphase II in the ABC(n)C(c) hybrid, indicated that unreduced gametes were formed by first division restitution. The frequency of dyads at the tetrad stage of pollen development (2.6%) suggested that unreduced gametes were preferentially selected in microspore culture. Segregation of marker alleles in the microspore-derived progeny was consistent with homologous recombination between C(n) and C(c) chromosomes and homoeologous recombination involving A-, B- and C-genome chromosomes during meiosis in the ABC(n)C(c) hybrid. We discuss the potential for using microspore culture of unreduced gametes in interspecific hybrids to map Brassica centromeres through half-tetrad analysis.