Mikhail Pavlovich Moshkin

In vitro fertilization (IVF) exposes embryos to environmental stressors that can disrupt early development and confer long-term health risks, though the mechanisms remain poorly understood. Here, we tested the hypothesis that reducing incubation temperature during the first zygotic cleavage would promote long-term developmental stability in IVF-conceived offspring. Using a mouse model, we compared the long-term effects of standard (37 °C) versus reduced (35 °C) IVF culture temperature on energy balance, circadian rhythms, sleep architecture, and brain histone modifications. Although offspring from both IVF groups exhibited increased body mass without notable effects on glucose metabolism, significant disruptions in circadian rhythms and sleep–wake patterns were detected. The 37 °C group exhibited altered amplitudes in oxygen consumption rhythms and respiratory exchange ratios, as well as pronounced alterations in sleep–wake patterns, including reduced sleep duration and increased nighttime activity. The 35 °C group displayed intermediate phenotypes, substantiating the importance of optimizing embryo incubation parameters. These metabolic and behavioral changes were paralleled by altered histone modifications in the cerebral cortex of IVF offspring, suggesting an epigenetic basis for circadian misalignment. Our results identify disrupted circadian rhythm and sleep architecture as a novel mechanism contributing to metabolic dysfunction in IVF-conceived offspring. The partial mitigation of these effects through reduced culture temperature underscores the importance of optimizing IVF protocols to minimize long-term epigenetic and metabolic risks.

Zygotic genome activation (ZGA) represents one of the most vulnerable periods to environmental perturbations. The objective of this study was to investigate the formation of stress granules in mouse embryos in response to temperature reduction during ZGA, preimplantation embryo mortality, and long-term phenotypic outcomes. These outcomes included the evaluation of expression noise in bilateral right/left limbs of offspring as an indicator of developmental instability, behavioral deviation, hippocampal volume, and metabolomics profiling in adult offspring. Exposure to hypothermia during ZGA was associated with an increased number and inter-blastomere variability of stress granules, extended duration of the second embryonic division, and elevated embryonic mortality during the second and third cleavage stages. The embryonic response to hypothermic stress correlated with phenotypic traits indicative of increased pathology risk. Expression noise, serving as an indicator of developmental instability, was reduced in adult offspring derived from two-cell embryos incubated at 35 °C compared to those at 37 °C, while showing no significant difference relative to the control group. These results suggest that embryos surviving hypothermic exposure (35 °C) possess enhanced resilience to the adverse effects commonly associated with embryo transfer procedures. Furthermore, increased hippocampal volume and augmented auditory startle reflex observed in offspring that endured hypothermia during ZGA imply reduced risks of cognitive-related pathologies and reduced risks of pathologies associated with cognitive functions.

At the early developmental stage, embryos are susceptible to environmental factors, which modulate development trajectories. In our study, we examined how different incubation temperatures (35 °C, 37 °C, and 39 °C) in vitro during the first embryonic cleavage affect the morphology, cell division rate, and DNA methylation in two-, four-, and eight-cell embryos and the viability of these two-cell embryos transferred to recipient females. Embryos kept at 35 °C for the first 24 h after in vitro fertilization in two- and four-cell embryos at 37 °C showed enhanced variability in the size of blastomeres and DNA 5mC level among blastomeres, as compared to the groups kept at 37 °C and 39 °C. This was associated with the highest rate of embryo death in four- and eight-cell embryos and the highest viability of newborns. In contrast, incubation at 39 °C did not significantly impact developmental dynamics and viability in vitro but led to a notably higher rate of gestation failure compared to other groups. The indicators of the 37 °C group fell within an intermediate range. Therefore, we conclude that a decrease in temperature during zygotic genome activation (ZGA) highlights the adaptive potential of embryos during their initial cleavages, while an increase in temperature does not show clear effects on their fate.

Introduction. Beside the excretion of metabolic wastes, the lymphatic system in CNS play a crucial role in the regulation of intracranial pressure that is vital for the organism. One of the possible pathways of cerebrospinal fluid (CSF) drainage is its flow through the foramen of the cribriform plate (CP) into the nasal cavity. Despite the significant contribution of the nasal tract to the overall dynamics of the liquor, not much is known about the mechanisms of this process and how it is regulated. Due to its influence on the tone of blood vessels and peristalsis of lymphatic vessels, nitric oxide (NO) is a powerful modulator of liquor outflow, but its effects on nasal CSF outflow have not been studied yet. Aim and Methods. Using diffusion-weighted magnetic resonance imaging (DW MRI), we characterized the changes in CSF outflow through the CP of C57Bl/6 mice influenced by intranasal application of NO synthesis modulators. Results. In our study, using DW MRI and computer tomography (CT) mapping of the CP, we detected significant CSF outflow through its large dorsal and ventral foramen located along the nasal septum. At the same time, the CSF flow rate through the dorsal orifices of the CP was the highest. In addition, we showed that intranasal introduction of NO donor after 30 min leads to a significant decrease of water diffusion through the CP whereas application of a nonspecific NO synthase inhibitor into the nasal cavity enhances nasal outflow. The effects of the NO modulators did not have any significant spatial patterns; CSF outflow was significantly altered across all CP orifices regardless of their size or localization. Conclusion. The obtained results demonstrate the potential possibility of noninvasive local regulation of liquor dynamics, which may be used in the development of new approaches to the therapy of intracranial hypertension of various etiologies and methods of CNS detoxification.

Sexual selection is considered as one of the leading factors of evolutionary development. In the conditions of incessant competition, specialized methods of attracting individuals of the opposite sex as well as criteria for assessing the quality of a sexual partner have been formed. In order for animals to rely on signaling from sexual partners, the signal must reflect the morpho-physiological status of animals. A high reproductive efficiency of male mice is a good advantage for mate selection and thus must be somehow demonstrated to potential mates. The aim of our study was to find out if male mice could demonstrate their reproductive efficiency through urine volatile organic compounds. The experiment implies cohabiting one male with two mature females for 6 days. The reproductive success of the male was assessed by the presence or absence of pregnant females. At the same time, naive females, who did not participate in reproduction, assessed the urine of the successful males as more attractive, which was expressed in shorter Latency time of sniffs in the Olfactory test. Using a rapid headspace GC/MS analysis, we have found volatile organic compounds (VOCs) in male urine that correlated with female behavior. It turned out that these substances are derivatives of mouse pheromone 6-hydroxy-6-methyl-3-heptanone. The amplitude of peaks corresponding to this pheromone correlated with the testosterone level in blood and the weight of preputial glands. The amplitude of peaks increased in males after mating with whom the females turned out to be pregnant. It is important to note that body weight, weight of testes, weight of seminal vesicles, weight of preputial glands, and plasma testosterone level alone are not reliable indicators of male reproductive success. Thus, the content of the pheromone 6-hydroxy-6-methyl-3-heptanone in the urine of males can serve as a good predictor of the quality of the male as a sexual partner for female CD-1 mice.

To identify body systems subject to epigenetic transformation during in vitro fertilization (IVF), comparative morphological and functional studies were performed on sexually mature offspring of outbred CD1 mice, specific-pathogen-free (SPF), obtained by IVF (experiment) and natural conception (control). The studies included assessment of age-related changes in body weight and composition, energy intake and expenditure, and glucose homeostasis. To level the effects caused by the different number of newborns in the control and in the experiment, the size of the fed litters was halved in the control females. Males obtained using the IVF procedure were superior in body weight compared to control males in all age groups. As was shown by analysis of variance with experiment/control factors, gender, age (7, 10 and 20 weeks), the IVF procedure had a statistically significant and unidirectional effect on body composition. At the same time, IVF offspring outperformed control individuals in relative fat content, but were behind in terms of lean mass. The effect of the interaction of factors was not statistically significant. IVF offspring of both sexes had higher fat to lean mass ratios (FLR). Since adipose tissue contributes significantly less to total energy intake compared to muscle, the main component of lean mass, it is not surprising that at the same level of IVF locomotor activity offspring consumed less food than controls. When converted to one gram of body weight, this difference reached 19 %. One of the consequences of reduced utilization of IVF energy substrates by offspring is a decrease in their tolerance to glucose loading. The integral criterion for the effectiveness of restoring the initial glucose level is the area under the curve (AUC), the value of which was 2.5 (males) and 3.2 (females) times higher in IVF offspring compared to the corresponding control. Thus, the totality of our original and literature data shows an increase in the risk of metabolic disorders in IVF offspring, which is confirmed by epidemiological studies of a relatively young cohort of people born using assisted reproductive technologies.

Developmental instability (DI) is thought to be inversely related to a capacity of an organism to buffer its development against random genetic and environmental perturbations. DI is represented by a trait’s inter- and intra-individual variabilities. The inter-individual variability (inversely referred to as canalization) indicates the capability of organisms to reproduce a trait from individual to individual. The intra-individual variability reflects an organism’s capability to stabilize a trait internally under the same conditions, and, for symmetric traits, it is expressed as fluctuating asymmetry (FA). When representing a trait as a random variable conditioned on environmental fluctuations, it is clear that, in statistical terms, the DI partitions into “extrinsic” (canalization) and “intrinsic” (FA) components of a trait’s variance/noise. We established a simple statistical framework to dissect both parts of a symmetric trait variance/noise using a PCA (principal component analysis) projection of the left/right measurements on eigenvectors followed by GAMLSS (generalized additive models for location scale and shape) modeling of eigenvalues. The first eigenvalue represents “extrinsic” and the second—“intrinsic” DI components. We applied this framework to investigate the impact of mother–fetus major histocompatibility complex (MHC)-mediated immune cross-talk on gene expression noise and developmental stability. We showed that “intrinsic” gene noise for the entire transcriptional landscape could be estimated from a small subset of randomly selected genes. Using a diagnostic set of genes, we found that allogeneic MHC combinations tended to decrease “extrinsic” and “intrinsic” gene noise in C57BL/6J embryos developing in the surrogate NOD-SCID and BALB/c mothers. The “intrinsic” gene noise was negatively correlated with growth (embryonic mass) and the levels of placental growth factor (PLGF), but not vascular endothelial growth factor (VEGF). However, it was positively associated with phenotypic growth instability and noise in PLGF. In mammals, the mother–fetus MHC interaction plays a significant role in development, contributing to the fitness of the offspring. Our results demonstrate that a positive impact of distant MHC combinations on embryonic growth could be mediated by the reduction of “intrinsic” gene noise followed by the developmental stabilization of growth.

A paradoxical reduction in anxiety levels in chronic predator stress paradigm (PS) in Sprague–Dawley rats has recently been shown in previous works. In this paper, we studied the possible neurobiological mechanism of this phenomenon. We segregated PS-exposed Sprague–Dawley rats into the high- and low-anxiety phenotypes. The long-lasting effects of PS on corticosterone levels, blood flow speed in the carotid arteries, diffusion coefficient, and 1H nuclear magnetic resonance spectra in the hippocampus were compared in the high-anxiety and low-anxiety rats. In addition, we evaluated the gene BDNF expression in the hippocampus which is considered to be a main factor of neuroplasticity. We demonstrated that in low-anxiety rats, the corticosterone level was decreased and carotid blood flow speed was increased. Moreover, in the hippocampus of low-anxiety rats compared to the control group and high-anxiety rats, the following changes were observed: (a) a decrease in N-acetyl aspartate levels with a simultaneous increase in phosphoryl ethanol amine levels; (b) an increase in lipid peroxidation levels; (c) a decrease in apparent diffusion coefficient value; (d) an increase in BDNF gene expression. Based on these findings, we proposed that stress-induced anxiety reduction is associated with the elevation of BDNF gene expression directly. Low corticosterone levels and a rise in carotid blood flow speed might facilitate BDNF gene expression. Meanwhile, the decrease in apparent diffusion coefficient value and decrease in N-acetyl aspartate levels, as well as an increase in the lipid peroxidation levels, in the hippocampus possibly reflected destructive changes in the hippocampus. We suggested that in Sprague–Dawley rats, these morphological alterations might be considered as an impetus for further increase in neuroplasticity in the hippocampus.

The latest vaccination campaign has actualized the potential impact of antigenic stimuli on reproductive functions. To address this, we mimicked vaccination’s effects by administering keyhole limpet hemocyanin (KLH ) to CD1 male mice and used their sperm for in vitro fertilization (IVF). Two-cell embryos after IVF with spermatozoa from control (C) or KLH-treated (Im) male mice were transferred to surrogate mothers mated with vasectomized control (C) or KLH-treated (Im) male mice, resulting in four experimental groups: C–C, Im–C, C–Im, and Im–Im. The pre-implantation losses were significantly lower in the Im–C group than in the C–Im group. At the same time, the resorption rates reduced markedly in the C–Im compared to the Im–C group. Embryo and placenta weights were significantly higher in the Im–Im group. Although the GM-CSF levels were lower in the amniotic fluid of the gestating surrogate mothers in the Im–Im group, they were strongly correlated with embryo mass. The number–size trade-off was only significant in the Im–Im group. This suggests a positive, cooperative effect of spermatozoa and seminal fluid from immune-primed males on embryo growth and the optimal distribution of surrogate mother maternal resources despite the negative impact of males’ antigenic challenge on the IVF success rate.

The lack of organs for transplantation is an important problem in medicine today. The growth of organs in chimeric animals may be the solution of this. The proposed technology is the interspecific blastocyst complementation method in combination with genomic editing for obtaining “free niches” and pluripotent stem cell production methods. The CRISPR/Cas9 method allows the so-called “free niches” to be obtained for blastocyst complementation. The technologies of producing induced pluripotent stem cells give us the opportunity to obtain human donor cells capable of populating a “free niche”. Taken together, these technologies allow interspecific blastocyst complementation between humans and other animals, which makes it possible in the future to grow human organs for transplantations inside chimeric animals. However, in practice, in order to achieve successful interspecific blastocyst complementation, it is necessary to solve a number of problems: to improve methods for producing “chimeric competent” cells, to overcome specific interspecific barriers, to select compatible cell developmental stages for injection and the corresponding developmental stage of the host embryo, to prevent apoptosis of donor cells and to achieve effective proliferation of the human donor cells in the host animal. Also, it is very important to analyze the ethical aspects related to developing technologies of chimeric organisms with the participation of human cells. Today, many researchers are trying to solve these problems and also to establish new approaches in the creation of interspecific chimeric organisms in order to grow human organs for transplantation. In the present review we described the historical stages of the development of the blastocyst complementation method, examined in detail the technologies that underlie modern blastocyst complementation, and analyzed current progress that gives us the possibility to grow human organs in chimeric animals. We also considered the barriers and issues preventing the successful implementation of interspecific blastocyst complementation in practice, and discussed the further development of this method.