Kruppel-like factor 5 (Klf5) in fetal-maternal tissue during periimplantation and effects of ovarian steroid hormone antagonist on its expression during uterine receptivity of albino mice

Background Embryo implantation is a tightly regulated sequence of events regulated by ovarian steroids, estrogen and progesterone, and their downstream targets. Ovarian steroids regulate most of the genes involved in embryo implantation and pregnancy. However, some factors are not regulated by ovarian steroids, estrogen, progesterone, or both. Kruppel-like factor 5 (Klf5) is an example of an ovarian steroid–independent factor having a role in cellular proliferation, differentiation. The detailed expression profile of Klf5 during uterine receptivity and periimplantation has not been studied till now. In the present research work, an attempt was made to investigate the expression pattern of Klf5 in mice fetal-maternal tissue during periimplantation (day 4–day 8). The expressional and functional independence of Klf5 on the ovarian steroids was studied using estrogen and progesterone antagonist. The study was carried out in female Swiss albino mice of LACA strain during the periimplantation period. KLF5 was localized in the fetal-maternal tissues using the immunofluorescence technique in paraffin-embedded tissues. Ovarian steroid antagonists were administered subcutaneously from day 1 to day 3 of gestation, and the uterus was collected on the morning of day 4. Klf5 protein and mRNA levels were studied by western blot and quantitative real-time PCR (qPCR), respectively. Results KLF5 was localized in the embryo, uterine luminal epithelium, glandular epithelium, and proliferating stromal cells during periimplantation. In ovarian steroid antagonist–treated groups, KLF5 was localized in the luminal and glandular epithelium and stroma. Western blot and qPCR confirmed translation and transcription of KLF5 during the experimental period. The KLF5 protein level significantly increased on day 6, day 7, and day 8 when compared with day 4 (P < 0.05). The mRNA level of Klf5 increased significantly on day 7 and day 8 when compared with day 4 (P < 0.05). In ovarian steroid antagonist–treated groups, protein and mRNA corresponding to Klf5 were observed. From this finding, it can be assumed that Klf5 may be a steroid-independent factor expressed during uterine receptivity. Conclusion Spatiotemporal KLF5 expression in fetal-maternal tissue was observed during the experimental period. The results suggest that Klf5 is an ovarian steroid–independent factor that may play a pivotal role in implantation, decidualization, and embryogenesis.

The Arabidopsis MATERNAL EFFECT EMBRYO ARREST45 protein modulates maternal auxin biosynthesis and controls seed size by inducingAINTEGUMENTA

Seed size is a major factor determining crop yields that is controlled through the coordinated development of maternal and zygotic tissues. Here, we identified Arabidopsis MATERNAL EFFECT EMBRYO ARREST45 (MEE45) as a B3 transcription factor that controls cell proliferation and maternally regulates seed size through its transcriptional activation of AINTEGUMENTA (ANT) and its downstream control of auxin biosynthesis in the ovule integument. After characterizing reduced seed and organ size phenotypes in mee45 mutants and finding that overexpression of MEE45 causes oversized seeds, we discovered that the MEE45 protein can bind to the promoter region of the ANT locus and positively regulate its transcription. ANT in-turn activates the expression of auxin biosynthetic genes (e.g. YUCCA4) in the ovule integument. Our results thus illustrate mechanisms underlying maternal tissue-mediated regulation of seed size and suggest that MEE45 and its downstream components can be harnessed to develop higher-yielding crop varieties.

APETALA2 functions as a temporal factor together with BLADE-ON-PETIOLE2 and MADS29 to control flower and grain development in barley

Cereal grain develops from fertilised florets. Alterations in floret and grain development greatly influence grain yield and quality. Despite this, little is known about the underlying genetic control of these processes, especially in key temperate cereals such as barley and wheat. Using a combination of near-isogenic mutant comparisons, gene editing and genetic analyses, we reveal that HvAPETALA2 (HvAP2) controls floret organ identity, floret boundaries, and maternal tissue differentiation and elimination during grain development. These new roles of HvAP2 correlate with changes in grain size and HvAP2-dependent expression of specific HvMADS-box genes, including the B-sister gene, HvMADS29. Consistent with this, gene editing demonstrates that HvMADS29 shares roles with HvAP2 in maternal tissue differentiation. We also discovered that a gain-of-function HvAP2 allele masks changes in floret organ identity and grain size due to loss of barley LAXATUM.A/ BLADE-ON-PETIOLE2 (HvBOP2) gene function. Taken together, we reveal novel, pleiotropic roles and regulatory interactions for an APETALA2-like gene controlling floret and grain development in a temperate cereal.

Single-cell analysis of prostaglandin E2-induced decidual cell differentiation: does extracellular 8-Br-cAMP cause artifacts?

Development of the uterine decidua, the transient maternal tissue contacting the fetus during extended gestation, is the hallmark of reproduction in many placental mammals. Differentiation of decidual stromal cells is known to be induced by stimuli that activate the nuclear progesterone receptor and the cyclic AMP/protein kinase A (cAMP/PKA) pathways. The nature of the stimulus upstream of PKA has not been clearly defined, although a number of candidates have been proposed. To bypass this uncertainty for in vitro experiments, direct addition of membrane-permeable cAMP along with progestin has been the prevailing method. Phylogenetic inference suggests that the inflammatory eicosanoid prostaglandin E2 (PGE2) was the stimulus that ancestrally induced decidualization. Accordingly, we developed a protocol to decidualize human endometrial stromal fibroblasts using progestin and PGE2 and analyzed the response in comparison with a cAMP-based protocol. Transcriptomic comparison reveals a common activation of core decidual cell genes between both treatments, and a set of senescence-related genes exaggerated under cAMP treatment. Single-cell transcriptomic analysis of PGE2-mediated decidualization revealed a major transcriptomic transition between an early activated cell state and a differentiated decidual state, but notably did not identify a developmental trajectory representing a distinct senescent decidual state as reported in recent literature. Furthermore, investigation of the signal transduction process underlying PGE2-mediated decidualization showed that it depends upon progestin-dependent induction of PGE2 receptor 2 (PTGER2 aka EP2) and PKA, the kinase activated by PTGER2. This progesterone-dependent induction of PTGER2 is absent in the opossum, a species incapable of decidualization. Together, these findings suggest that the origin of the decidual cell type involved the evolution of progesterone-dependent activation of the PGE2/EP2/PKA axis. We propose the use of PGE2 for in vitro decidualization studies as a potentially more physiological model than 8-Br-cAMP.

Single nucleus analysis of Arabidopsis seeds reveals new cell types and imprinting dynamics

Seeds are the basis of agriculture, yet their full transcriptional complexity has remained unknown. Here, we employ single-nucleus RNA-sequencing to characterize developing Arabidopsis thaliana seeds, with a focus on endosperm. Endosperm, the site of gene imprinting in plants, mediates the relationship between the maternal parent and embryo. We identify new cell types in the chalazal endosperm region, which interfaces with maternal tissue for nutrient unloading. We further demonstrate that the extent of parental bias of maternally expressed imprinted genes varies with cell cycle phase, and that imprinting of paternally expressed imprinted genes is strongest in chalazal endosperm. These data indicate imprinting in endosperm is heterogeneous and suggest that parental conflict, which is proposed to drive the evolution of imprinting, is fiercest at the boundary between filial and maternal tissues.

Super-Sweet Purple Sweetcorn: Breaking the Genetic Link

Purple-pericarp supersweet sweetcorn currently does not exist as a horticultural product. Purple pericarp comprises the outer layers of the kernel, with the purple pigment being produced by anthocyanin. Unlike the aleurone layer which can also be pigmented, the pericarp is maternal tissue. Although standard purple sweetcorn based on mutations such as sugary1 (su1) and sugary enhancer (se1) are in existence, the development of purple supersweet sweetcorn based on the widely used shrunken2 (sh2) gene mutation is much more challenging. This is because there is an extremely close genetic linkage between the supersweet shrunken-2 mutation and the anthocyanin biosynthesis gene, anthocyaninless-1 (a1). As distance between these two genes is only 0.1 cM, the development of purple supersweet sweetcorn depends on breaking this close genetic link, which occurs at a very low frequency of 1 in 1000 meiotic crossovers. To make this possible, we crossed a white supersweet variety (a1a1sh2sh2) with a purple-pericarp Peruvian maize (A1A1Sh2Sh2) to obtain an initial heterozygous hybrid (A1a1Sh2sh2). The hybrid seed was sown and subsequently self-pollinated to produce seed segregating for the double recessive homozygote, sh2sh2 (1 in 4). These kernels present a visually distinctive phenotype, characterised by the seed’s shrunken appearance. Approximately 2760 sh2sh2 seeds were separated and resown. Due to the low frequency of linkage breakage, the majority of these plants (~99.9%) produced supersweet white cobs (a1a1sh2sh2). Three plants (0.1%) however, produced supersweet purple cobs (A1a1sh2sh2), due to a single low-frequency linkage break. These cobs will form the basis for a purple-pericarp supersweet sweetcorn breeding program.

Promoting Roles of Embryonic Signals in Embryo Implantation and Placentation in Cooperation with Endocrine and Immune Systems

Embryo implantation in the uterus is an essential process for successful pregnancy in mammals. In general, the endocrine system induces sufficient embryo receptivity in the endometrium, where adhesion-promoting molecules increase and adhesion-inhibitory molecules decrease. Although the precise mechanisms remain unknown, it is widely accepted that maternal–embryo communications, including embryonic signals, improve the receptive ability of the sex steroid hormone-primed endometrium. The embryo may utilize repulsive forces produced by an Eph–ephrin system for its timely attachment to and subsequent invasion through the endometrial epithelial layer. Importantly, the embryonic signals are considered to act on maternal immune cells to induce immune tolerance. They also elicit local inflammation that promotes endometrial differentiation and maternal tissue remodeling during embryo implantation and placentation. Additional clarification of the immune control mechanisms by embryonic signals, such as human chorionic gonadotropin, pre-implantation factor, zona pellucida degradation products, and laeverin, will aid in the further development of immunotherapy to minimize implantation failure in the future.

Effects of diet type on nutrient utilization and energy balance in drylot heifers1

Feeding cattle in intensified settings allows cow-calf producers to decrease their reliance on grazed forage and utilize alternative feedstuffs. During times of intense management, diet type may alter energy utilization. Fourteen pregnant MARC III heifers (405 ± 44 kg BW) were used in a 180 d experiment to determine effects of diet type on nutrient and energy utilization. Heifers were randomly assigned to one of two treatments, a forage diet (FOR; 2.10 Mcal metabolizable energy [ME]/kg; 95.75% forage) or a concentrate diet (CONC; 2.94 Mcal ME/kg; 71% concentrate), and individually fed to meet maintenance energy requirements (0.135 Mcal ME/kg BW0.75). The CONC diet contained dry-rolled corn, corn stalks (10.16 cm grind size), soybean meal, corn silage (approximately 45% corn grain; stored in a plastic bag), dicalcium phosphate, urea, and a premix pellet; FOR contained alfalfa hay (harvested at mid-bloom), corn silage, dicalcium phosphate, and a premix pellet. Measurements of energy intake and digestibility were measured over a 4-d period on days 116, 172, and 235 of gestation. Using portable headbox calorimeters, measurements of O2, CO2, and CH4 gases were collected over a period of 24 h. Data were analyzed in a completely randomized design with diet as fixed effect. Dry matter and organic matter digestibility were greater for CONC than FOR (P &lt; 0.01). Intake of gross energy (GE) and digestible energy (DE) were greater for FOR (P &lt; 0.01), but by design, ME intake was not different between treatments (P = 0.26). Energy lost as methane (% of GE intake) was not different between treatments (P = 0.49). The ratio of ME to DE was greater for CONC (86.8 vs. 82.8; P = 0.01) than FOR. Heat production relative to ME was not different between treatments (P = 0.85). Maternal tissue energy did not differ and was 1.2 Mcal/d for CONC and 0.9 Mcal/d for FOR (P = 0.73). Greater nitrogen (N) consumption was observed for FOR (192.2 g/d) than CONC (134.0 g/d; P &lt; 0.01), and retained N was greater for FOR than CONC (P &lt; 0.01) on days 116 and 235 of gestation. Neither concentrate-based or forage-based diets affected body condition score (P = 0.26). Heifers fed concentrate-based diets retained more energy in part because they had larger calves, but this energy was not recovered in maternal tissue.

Serum and tissue pregnanes and pregnenes after dexamethasone treatment of cows in late gestation

Dexamethasone (DEX) initiates parturition by inducing progesterone withdrawal and affecting placental steroidogenesis, but the effects of DEX in fetal and maternal tissue steroid synthetic capacity remains poorly investigated. Blood was collected from cows at 270 days of gestation before DEX or saline (SAL) treatment, and blood and tissues were collected at slaughter 38 h later. Steroid concentrations were determined by liquid chromatography tandem mass spectrometry to detect multiple steroids including 5α-reduced pregnane metabolites of progesterone. The activities of 3β-hydroxysteroid dehydrogenase (3βHSD) in cotyledonary and luteal microsomes and mitochondria and cotyledonary microsomal 5α-reductase were assessed. Quantitative PCR was used to further assess transcripts encoding enzymes and factors supporting steroidogenesis in cotyledonary and luteal tissues. Serum progesterone, pregnenolone, 5α-dihydroprogesterone (DHP) and allopregnanolone (3αDHP) concentrations (all <5 ng/mL before treatment) decreased in cows after DEX. However, the 20α-hydroxylated metabolite of DHP, 20αDHP, was higher before treatment (≈100 ng/mL) than at slaughter but not affected by DEX. Serum, cotyledonary and luteal progesterone was lower in DEX- than SAL-treated cows. Progesterone was >100-fold higher in luteal than cotyledonary tissues, and serum and luteal concentrations were highly correlated in DEX-treated cows. 3βHSD activity was >5-fold higher in luteal than cotyledonary tissue, microsomes had more 3βHSD than mitochondria in luteal tissue but equal in cotyledonary sub-cellular fractions. DEX did not affect either luteal or cotyledonary 3βHSD activity but luteal steroidogenic enzyme transcripts were lower in DEX-treated cows. DEX induced functional luteal regression and progesterone withdrawal before any changes in placental pregnene/pregnane synthesis and/or metabolism were detectable.

Improved FPGA controlled artificial vascular system for plethysmographic measurements

The fetal oxygen saturation is an important parameter to determine the health status of a fetus, which is until now mostly acquired invasively. The transabdominal, fetal pulse oximetry is a promising approach to measure this non-invasively and continuously. The fetal pulse curve has to be extracted from the mixed signal of mother and fetus to determine its oxygen saturation. For this purpose efficient algorithms are necessary, which have to be evaluated under constant and reproducable test conditions. This paper presents the improved version of a phantom which can generate artificial pulse waves in a synthetic tissue phantom. The tissue phantom consists of several layers that mimic the different optical properties of the fetal and maternal tissue layers. Additionally an artificial vascular system and a dome, which mimics the bending of the belly of a pregnant woman, are incorporated. To obtain data on the pulse waves, several measurement methods are included, to help understand the behavior of the signals gained from the pulse waves. Besides pressure sensors and a transmissive method we integrated a capacitive approach, that makes use of the so called “Pin Oscillator” method. Apart from the enhancements in the tissue phantom and the measurements, we also improved the used blood substitute, which reproduces the different absorption characteristics of fetal and maternal blood. The results show that the phantom can generate pulse waves similar to the natural ones. Furthermore, the phantom represents a reference that can be used to evaluate the algorithms for transabdominal, fetal pulse oximetry.