Endocrinology: Nuclear maturity and oocyte morphology after stimulation with highly purified follicle stimulating hormone compared to human menopausal gonadotrophin

Several studies have shown that high concentrations of luteinizing hormone (LH) in the follicular phase of stimulation can have a negative effect on oocyte quality, pregnancy rate and incidence of miscarriage. The aim of the present study was to examine the effects of highly purified follicle stimulating hormone (FSH HP) on ovarian stimulation and particularly on nuclear maturity and morphological appearance of the oocyte in intracytoplasmic sperm injection (ICSI) therapy and to compare the results with human menopausal gonadotrophin (HMG) stimulation. For this purpose, 50 patients for ICSI (HMG: 30; FSH HP: 20) and 26 patients for in-vitro fertilization (TVF; HMG: 14, FSH HP: 12) were stimulated with either HMG of FSH HP using a short-term protocol. Patients were divided into the two groups according to the first letter of their family name. No differences were observed among the groups in relation to patient age, duration of stimulation, number of aspirated oocytes or maturity of the oocyte-cumulus complex. After removal of the cumulus-corona cells in the ICSI oocytes, a significantly higher proportion of oocytes in the FSH HP group were nuclear mature (metaphase II) than in the HMG group (FSH HP: 88.8%, HMG: 80.6%; P = 0.009). Furthermore, in the FSH HP group, significantly fewer oocytes with dark cytoplasm were observed (FSH HP: 14.4%, HMG: 22.4%; P = 0.02). Fertilization, cleavage and pregnancy rates (FSH HP 38%, HMG: 34% per retrieval) were comparable in both groups. Based on the results obtained, it can be concluded that the short-term FSH HP treatment protocol synchronizes oocyte maturation better than comparable stimulation with HM

Abnormal chromosomal arrangements in human oocytes

Ninety-one human oocytes, lacking signs of fertilization 50 h after insemination in vitro, were investigated cytogenetically to assess the frequency and type of chromosomal abnormalities. Chromosome spreading permitted adequate karyotyping in 55 oocytes. Non-determined numerical aberrations occurred with the following frequencies: hypohaploidy, 10.9% (6/55), hyperhapJoidy, 14.5% (8/55) and hyperdiploidy, 3.6% (2/55). Total aneuploidy occurred with a frequency of 29.1% and was observed in oocytes from 30 patients. No correlation was found between specific chromosomal aberrations and type of infertility, stimulation treatment or gonadotrophin levels. On the other hand, the frequency of aneuploidy was significantly higher (P 35 years of age. Two chromosomal complements (3.6%) had structural rearrangements; one oocyte had both structural and numerical chromosomal abnormalities and the other had differently condensed regions on the long arms of three chromosomes from group C. The overall frequency of chromosomal aberrations was 32.7%. Only two samples contained an additional set of polar body chromosomes. Thirteen oocytes presented sperm chromosomes in an arrested stage of premature chromosome condensation of the G1, phase and four oocytes showed asynchronous condensation of pronuclear chromosomes. Finally, it was concluded that the high proportion of chromosomal aberrations observed in human oocytes may contribute significantly to abnormal embryonic development in vitr

Identification of nitric oxide synthase in human and bovine oviduct

Nitric oxide synthase (NOS) is responsible for the biological production of nitric oxide (NO) in several organs. NOS activity has also been localized in the reproductive tract, although direct evidence for its presence in the human or bovine oviduct is still lacking. In the present study, four different techniques were used to identify the presence of NOS activity in human (n = 11) and bovine (n = 9) oviduct: (i) conversion of [3H]-L-arginine to [3H]-L-citrulline; (ii) production of nitrite/nitrate (NO2/NO3; stable NO metabolites); (iii) identification of NADPH-diaphorase activity; and (iv) immunostaining with antiserum to endothelial NOS. Cytosolic extracts from human ampullary segments of the Fallopian tube, obtained from post-partum patients (n = 4), converted [3H]-L-arginine to [3H]-L-citrulline (21.0 ± 8.8 fmol/mg protein/min). This conversion rate was significantly (P <0.05) reduced in the presence of either EDTA or N-monomethyl-L-arginine monoacetate (L-NMMA), an inhibitor of NOS activity. When bovine (n = 3) ampullary segments were incubated for 36 h in Hanks' balanced salt solution, the concentration of NO2/NO3 in the medium was increased (P <0.05) if segments were pretreated with lipopolysaccharide (LPS; an inducer of inducible NOS), but not after treatment with LPS + L-NMMA. Additionally, epithelial cells cultured from ampullary segments showed positive staining both for NADPH-diaphorase activity and with antiserum to endothelial NOS. The results of the present study provide direct evidence for the presence of both the Ca2+ -dependent constitutive form of NOS, as well as the inducible form of NOS activity in human and bovine oviduct. Since the oviduct plays a key role in the reproductive process, it is possible that the two forms of NOS may be involved in the physiological regulation of oviduct functio

Swine manure management by hydrothermal carbonization: comparative study of batch and continuous operation

Hydrothermal carbonization (HTC) is considered a promising technology for biomass waste management without pre-drying. This study explores the potential for swine manure management by comparing batch and continuous processes, emphasizing the benefits of the continuous mode, particularly for its potential full-scale application. The continuous process at low temperature (180 ◦C) resulted in a hydrochar with a lower degree of carbonization compared to the batch process, but similar characteristics were found in both hydrochars at higher operating temperatures (230–250 ◦C), such as C content (~ 52 wt%), fixed carbon (~ 24 wt%) and higher calorific value (21 MJ kg− 1 ). Thermogravimetric and combustion analyses showed that hydrochars exhibited characteristics suitable as solid biofuels for industrial use. The process water showed a high content of organic matter as soluble chemical oxygen demand (7–22 g L− 1 ) and total organic carbon (4–10 g L− 1 ), although a high amount of refractory species such as N- and O-containing long aromatic compounds were detected in the process water from the batch process, while the process water from the continuous process presented more easily biodegradable compounds such as acids and alcohols, among others. The longer time required to reach operating temperature in the case of the batch system (longer heating time to reach operating temperature) resulted in lower H/C and O/C ratios compared to hydrochar from the continuous process. This indicates that the dehydration and decarboxylation reactions of the feedstock play a more important role in the batch process. This study shows the efficiency of the continuous process to obtain carbonaceous materials suitable for use as biofuel, providing a solution for swine manure managementAuthors greatly appreciate funding from Spanish MCIN/AEI/ 10.13039/501100011033 and European Union "NextGenerationEU/ PRTR" (TED2021-130287B-I00, PDC 2021-120755-I00, and PID 2022- 138632OB-I00) and Grupo Kerbest Company. R.P. Ipiales acknowledges the financial support from the Community of Madrid (IND2019/AMB17092) and Arquimea Agrotech Compan

Stretching the Rules: Monocentric Chromosomes with Multiple Centromere Domains

The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3-5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel ``meta-polycentric'' functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function

Wnt/β-Catenin-Signaling Modulates Megakaryopoiesis at the Megakaryocyte-Erythrocyte Progenitor Stage in the Hematopoietic System

The bone marrow (BM) hematopoietic system (HS) gives rise to blood cells originating from hematopoietic stem cells (HSCs), including megakaryocytes (MKs) and red blood cells (erythrocytes; RBCs). Many steps of the cell-fate decision remain to be elucidated, being important for cancer treatment. To explore the role of Wnt/β-catenin for MK and RBC differentiation, we activated β-catenin signaling in platelet-derived growth factor b (Pdgfb)-expressing cells of the HS using a Cre-lox approach (Ctnnb1BM-GOF). FACS analysis revealed that Pdgfb is mainly expressed by megakaryocytic progenitors (MKPs), MKs and platelets. Recombination resulted in a lethal phenotype in mutants (Ctnnb1BM-GOFwt/fl, Ctnnb1BM-GOFfl/fl) 3 weeks after tamoxifen injection, showing an increase in MKs in the BM and spleen, but no pronounced anemia despite reduced erythrocyte counts. BM transplantation (BMT) of Ctnnb1BM-GOF BM into lethally irradiated wildtype recipients (BMT-Ctnnb1BM-GOF) confirmed the megakaryocytic, but not the lethal phenotype. CFU-MK assays in vitro with BM cells of Ctnnb1BM-GOF mice supported MK skewing at the expense of erythroid colonies. Molecularly, the runt-related transcription factor 1 (RUNX1) mRNA, known to suppress erythropoiesis, was upregulated in Ctnnb1BM-GOF BM cells. In conclusion, β-catenin activation plays a key role in cell-fate decision favoring MK development at the expense of erythroid production

In Depth Characterization of Repetitive DNA in 23 Plant Genomes Reveals Sources of Genome Size Variation in the Legume Tribe Fabeae

The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55-83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encountered. Differential accumulation of repetitive DNA was found to account for 85% of the genome size differences between the species, and most (57%) of this variation was found to be driven by a single lineage of Ty3/gypsy LTR-retrotransposons, the Ogre elements. Although the amounts of several other lineages of LTR-retrotransposons and the total amount of satellite DNA were also positively correlated with genome size, their contributions to genome size variation were much smaller (up to 6%). Repeat analysis within a phylogenetic framework also revealed profound differences in the extent of sequence conservation between different repeat types across Fabeae. In addition to these findings, the study has provided a proof of concept for the approach combining recent developments in sequencing and bioinformatics to perform comparative analyses of repetitive DNAs in a large number of non-model species without the need to assemble their genomes

Experimental evidence for splicing of intron-containing transcripts of plant LTR retrotransposon Ogre

Ogre elements are a distinct group of plant Ty3/gypsy-like retrotransposons characterized by several specific features, one of which is a separation of the gag-pol region into two non-overlapping open reading frames: ORF2 coding for Gag-Pro, and ORF3 coding for RT/RH-INT proteins. Previous characterization of Ogre elements from several plant species revealed that part of their transcripts lacks the region between ORF2 and ORF3, carrying one uninterrupted ORF instead. In this work, we investigated a hypothesis that this region represents an intron that is spliced out from part of the Ogre transcripts as a means for preferential production of ORF2-encoded proteins over those encoded by the complete ORF2–ORF3 region. The experiments involved analysis of transcription patterns of well-defined Ogre populations in a model plant Medicago truncatula and examination of transcripts carrying dissected pea Ogre intron expressed within a coding sequence of chimeric reporter gene. Both experimental approaches proved that the region between ORF2 and ORF3 is spliced from Ogre transcripts and showed that this process is only partial, probably due to weak splice signals. This is one of very few known cases of spliced LTR retrotransposons and the only one where splicing does not involve parts of the element’s coding sequences, thus resembling intron splicing found in most cellular genes

Independent, Rapid and Targeted Loss of Highly Repetitive DNA in Natural and Synthetic Allopolyploids of Nicotiana tabacum

Allopolyploidy (interspecific hybridisation and polyploidy) has played a significant role in the evolutionary history of angiosperms and can result in genomic, epigenetic and transcriptomic perturbations. We examine the immediate effects of allopolyploidy on repetitive DNA by comparing the genomes of synthetic and natural Nicotiana tabacum with diploid progenitors N. tomentosiformis (paternal progenitor) and N. sylvestris (maternal progenitor). Using next generation sequencing, a recently developed graph-based repeat identification pipeline, Southern blot and fluorescence in situ hybridisation (FISH) we characterise two highly repetitive DNA sequences (NicCL3 and NicCL7/30). Analysis of two independent high-throughput DNA sequencing datasets indicates NicCL3 forms 1.6–1.9% of the genome in N. tomentosiformis, sequences that occur in multiple, discontinuous tandem arrays scattered over several chromosomes. Abundance estimates, based on sequencing depth, indicate NicCL3 is almost absent in N. sylvestris and has been dramatically reduced in copy number in the allopolyploid N. tabacum. Surprisingly elimination of NicCL3 is repeated in some synthetic lines of N. tabacum in their forth generation. The retroelement NicCL7/30, which occurs interspersed with NicCL3, is also under-represented but to a much lesser degree, revealing targeted elimination of the latter. Analysis of paired-end sequencing data indicates the tandem component of NicCL3 has been preferentially removed in natural N. tabacum, increasing the proportion of the dispersed component. This occurs across multiple blocks of discontinuous repeats and based on the distribution of nucleotide similarity among NicCL3 units, was concurrent with rounds of sequence homogenisation