CD-1 mice females recognize male reproductive success via volatile organic compounds in urine.

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.

Body composition as an indicator of metabolic changes in mice obtained by in vitro fertilization.

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.

Evaluation of the α-casein (CSN1S1) locus as a potential target for a site-specific transgene integration.

Transgenic animals are an important tool in biotechnology, including the production of recombinant proteins in the milk. Traditionally, expression constructs are based on hybrid vectors bearing mammary gland specific regulatory elements from the α-casein (Csn1s1), ß-casein (Csn2), whey acidic protein (WAP), or ß-lactoglobulin (BLG) genes. Overexpression from the randomly integrated vectors typically provides high levels of expression, but has drawbacks due to unpredictable genome localization. CRISPR-Cas9 targeted transgene integration into the endogenous casein locus could alleviate the need for extensive animal screening to achieve high and reproducible expression levels. We decided to evaluate such a "precise" integration approach, placing the human granulocyte-macrophage colony-stimulating factor (hGMCSF) gene under control of the mouse endogenous alpha-S1-casein (Csn1s1) promoter. We designed two types of transgene integrations: a knock-in in the second exon of the Csn1s1 (INS-GM) and a full-size Csn1s1 replacement with hGMCSF (REP-GM) which was never tested before. The INS-GM approach demonstrated low transgene expression and milk protein levels (0.4% of Csn2 transcripts; 2-11 µg/ml hGMCSF). This was probably caused by the absence of the 3'-polyadenylation signal in the hGMCSF transgene. REP-GM animals displayed high transgene expression, reaching and slightly exceeding the level of the endogenous Csn1s1 (30-40% of Csn2 transcripts), but yielded less hGMCSF protein than expected (0.2-0.5 mg/ml vs 25 mg/ml of Csn1s1), indicating that translation of the protein is not optimal. Homozygous inserts leading to the Csn1s1 knock-out did not have any long standing effects on the animals' health. Thus, in our experimental design, site-specific transgene integration into the casein locus did not provide any significant advantage over the overexpression approach.

Generation of donor organs in chimeric animals via blastocyst complementation.

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.

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Assisted reproductive technologies (ART) increasingly occupy the study of human reproduction. In addition, in developed countries they contribute to breeding of more than 50 % of cattle. In the management of collections of genetic lines of laboratory animals, these technologies are obligatory components of cryopreservation and rederivation. ART procedures include the development of early embryos outside the mother's body and the high probability of incomplete synchronization of the physiological state of the surrogate mother and transplanted embryos. Since all this occurs at the stage of the highest susceptibility of embryos to epigenetic reprogramming, the full cycle of ART and its individual components can lead to stable phenotypic changes in the offspring. Their reality is confirmed by studies of the morphological and functional characteristics of sexually mature offspring of CD1 outbred mice, obtained using different variants of early embryo transplantation. Comparative studies of body mass and body composition, basal glucose level and response to glucose load (glucose-tolerance test - GTT) have been done on sexually mature males and females. Animals were separated in 4 groups according to the variant of embryo transplantation: group (control) - natural mating; group (2cl-bl) - incubation of 2-cell up to blastocysts; group (2cl-2cl) - removal and transplantation of the 2-cell embryo without incubation; group (Bl-bl) removal and transplantation of the blastocysts without incubation. All embryos were transplanted to recipient females of the same line. It was found that sexually mature offspring obtained with all variants of transplantations had a higher relative fat content and, correspondingly, lower lean mass compared to the control. This effect was more pronounced in females than in males. Unlike body compositions, embryo transplantations had a greater effect on basal glucose concentration and GTT in males than in females. In this case, the offspring of the 2cl-2cl and 2cl-bl groups were characterized by a higher tolerance to glucose load (GTT) compared with the control and the Bl-bl group. Stable deviations of body compositions and glucose homeostasis indices detected in experimental groups of progenies indicate the phenotypic significance of the embryo transplantations per se.

TNFα is responsible for the canonical offspring number-size trade-off.

There is a canonical life-history trade-off between quantity and quality of offspring, but molecular determinants for this are unknown. Here, we show that knockout of tumor necrosis factor (TNF-KO) in mice switched a relation between the number and size of developing embryos from expectedly negative to unexpectedly positive. Depletion of TNFα imbalanced humoral and trophic maintenance of embryo growth during gestation with respect to the litter size. The levels of embryotrophic GM-CSF cytokine and placental efficiency attained positive correlations with the number and size of embryos in TNF-KO females. Thus, TNFα oversees mother's resource allocations to balance embryo growth with the number of offspring. Consequently, this suggests an intricate link between the number-size trade-off and immunity given a pivotal role of TNFα in immune homeostasis.

Nasal aerodynamics protects brain and lung from inhaled dust in subterranean diggers, Ellobius talpinus.

Inhalation of air-dispersed sub-micrometre and nano-sized particles presents a risk factor for animal and human health. Here, we show that nasal aerodynamics plays a pivotal role in the protection of the subterranean mole vole Ellobius talpinus from an increased exposure to nano-aerosols. Quantitative simulation of particle flow has shown that their deposition on the total surface of the nasal cavity is higher in the mole vole than in a terrestrial rodent Mus musculus (mouse), but lower on the olfactory epithelium. In agreement with simulation results, we found a reduced accumulation of manganese in olfactory bulbs of mole voles in comparison with mice after the inhalation of nano-sized MnCl2 aerosols. We ruled out the possibility that this reduction is owing to a lower transportation from epithelium to brain in the mole vole as intranasal instillations of MnCl2 solution and hydrated nanoparticles of manganese oxide MnO · (H2O)x revealed similar uptake rates for both species. Together, we conclude that nasal geometry contributes to the protection of brain and lung from accumulation of air-dispersed particles in mole voles.

Association of polymorphism harbored by tumor necrosis factor alpha gene and sex of calf with lactation performance in cattle.

In a majority of mammals, male infants have heavier body mass and grow faster than female infants. Accordingly, male offspring nursing requires a much greater maternal energy contribution to lactation. It is possible that the maternal-fetal immunoendocrine dialog plays an important role in female preparation for lactation during pregnancy. Immune system genes are an integral part of gene regulatory networks in lactation and tumor necrosis factor alpha (TNFα) is a proinflammatory cytokine that also plays an important role in normal mammary gland development. The aim of this study was to evaluate the influence of the sex of calf and/or the -824A/G polymorphism in the promoter region of TNFα gene on milk performance traits in Black Pied cattle over the course of lactation. We also studied the allele frequency differences of -824A/G variants across several cattle breeds, which were bred in different climatic conditions. The G allele frequency decreased gradually over the course of lactation events in the Black Pied dairy cattle because of a higher culling rate of cows with the G/G genotype (p<0.001). In contrast to the genotypes A/A and A/G, cows with G/G genotype showed significant variability of milk and milk fat yield subject to sex of delivered calf. Milk yield and milk fat yield were significantly higher in the case of birth of a bull calf than with a heifer calf (p<0.03). The G allele frequency varies from 48% to 58% in Grey Ukrainian and Black Pied cattle to 77% in aboriginal Yakut cattle. Our results suggest that the TNFα -824A/G gene polymorphism may have an influence on the reproductive efforts of cows over the course of lactation events depending on the sex of progeny. Allocation of resources according to sex of the calf allows optimizing the energy cost of lactation. This may be a probable reason for high G allele frequency in Yakut cattle breeding in extreme environmental conditions. Similarly, the dramatic fall in milk production after birth of a heifer calf increases the probability of culling for the cows with the G/G genotype in animal husbandry.

Influence of genetic dissimilarity of mother and fetus on progesterone concentrations in pregnant mice and adaptive features of offspring.

Concentrations of progesterone in blood plasma and tissue were studied in pregnant mice of strains BALB/cLac and C57BL/6J. Both intra- and interstrain mating and embryo transplantations were used as models of homo- and heterotopic pregnancy. On day 4 of heterotopic pregnancy, plasma progesterone concentrations of females of both strains were higher than those in females of both strains undergoing homotopic pregnancy. In addition, tissue progesterone content of hybrid embryos was higher than that of purebred embryos. Adrenocortical responses to social conflict as indicators of stress resistance were studied in progeny aged 2-3 months. There was a minimal increase in plasma glucocorticoid concentrations in heterotopic transplantants after 15 min pair-matching tests and 30 min crowding compared with those of other progeny, including purebred male mice of BALB/cLac and C57BL/6J strains, homotransplantants and reciprocal hybrids. Thus, genetic dissimilarity of mother and fetuses plays an important role in progesterone provision during pregnancy and also modifies development of progeny.