Effects of growth factors during in vitro culture of mouse and rat embryos

In vitro culture of preimplantation embryos of ICR, HT1AN/Icgn, HT1AC/Icgn and C57BL/6J-Ay mouse strains as well as in OXYS/Icgn rat strain in media containing granulocyte-macrophage colony stimulating factor (GM-CSF) or epidermal growth factor (EGF) has been studied. Both mouse and rat embryos were first frozen in a programmable freezer after a standard protocol using a mixture of glycerol and sucrose as cryoprotectants, thawed and cultured in vitro in R1ECM (rat one-cell embryo culture medium) for 24 hours (mice) and 72 hours (rats). For the in vitro culture experiments with these growth factors, 8-cell frozen-thawed mouse embryos and 2–4-cell frozen-thawed rat embryos were used. Supplementation of the culture medium with GM-CSF improved the rate of embryonic development in HT1AC/Icgn and C57BL/6J-Ay strain mice, while EGF had no effect. The reverse was true of the rats. Supplementation of the culture medium with EGF increased the percentage of deve­loping blastocysts in OXYS/Icgn rat strain, while GM-CSF had no effect. Co-culture of four-cell embryos of HT1AN/ Icgn strain mice with more advanced embryonic stages (morulas) of a different strain ICR led to the facilitation preimplantation embryo development. Experimental results presented here reveal the species-specific effects of growth factors on mouse and rat embryos and indicate that co-culture of different stages of embryo development have stimulatory effects on earlier stages

A comparison of different cryoprotectant solutions and thawing methods for cryo­preservation of embryos of mice and rats

The proper choice of cryoprotectant and thawing method affects cryopreservation efficiency. A freezing-thawing method for sparing embryonic cells was evaluated in experiments with ICR mice. Cleavage-stage embryos of ICR mice, GC rats, and OXYS rats were collected on Day 3 of pregnancy and frozen in plastic straws according to a standard protocol. Permeating (ethylene glycol and glycerol) and nonpermeating (sucrose) cryoprotectants and their combinations were compared during the freezing of ICR mouse embryos. With these mice, two thawing methods were compared: rapid (water bath, 10 s, 37 °С) and slow (40 s, room temperature; 40 s, 30 °С). Embryo viability in mice and rats was evaluated by their in vitro culturing after thawing. Our data on mice indicate that slow thawing is more suitable for sparing the integrity of embryonic cells; moreover, supplementation of the main cryoprotectant (either ethylene glycol or glycerol) with sucrose is beneficial for subsequent in vitro culture, especially in the case of glycerol. This freezing-thawing protocol (with glycerol and sucrose as cryoprotectant agents and slow thawing) was applied to rats of the GC and OXYS strains; the survival rate after cryopreservation was 68–83.3 %, and the rate of in vitro development was 64.7–66.6 %

Effect of exogenous human chorionic gonadotropin on ovulation in mice

The implementation of assisted reproductive technologies (ART), hormonal stimulation in particular, may change the quality of ovulated oocytes. The purpose of our work was to study ovulation in CD1 mice after their stimulation with human chorionic gonadotropin (hCG) and to investigate the effects of such hormonal stimulation on the pregnancy duration, fetal losses and the weight of the offspring. No significant differences were found in the total number of ovulated oocytes or in the number of immature (without a polar body) ovulated oocytes; nor were there differences between the groups in the number of oocytes with a developing polar body. However, the number of matured oocytes with a distinct polar body was significantly higher (p < 0.05) in mice stimulated with hCG (experimental group) as compared with the controls (6.2 ± 0.86 and 2.2 ± 0.97, respectively). No significant differences were observed between the experimental and control mice in the duration of pregnancy or in the numbers of term offspring, including the percentage of live and stillborn pups. However, the body weight of the offspring in the experimental group was significantly lower (p < 0.001) as compared with the controls on the fifth day after birth (3.16 ± 0.09 and 3.76 ± 0.07, respectively). Thus, exogenous hCG facilitates the development of mouse oocytes in vivo, which leads to the larger number of their mature forms at ovulation, however, the offspring born after hCG-stimulated pregnancy was characterized by a lower body weight on the fifth day after birth

Psycho-emotional stress, folliculogenesis, and reproductive technologies: clinical and experimental data

Modern life, especially in large cities, exposes people to a high level of noise, high density of population, disrupted sleeping, large amount of excessive and controversial information as well as to other negative factors; all this may cause chronic psycho-emotional stress. The latest publications often use the term “Syndrome of megalopolis”, which means disruption of sleeping, high anxiety, and altered reproductive function. Medical treatment of infertility may also be considered as a stress factor, especially when infertility lasts for years and is aggravated with emotional frustration. Long-lasting distress may worsen health in general and suppress reproductive function, in particular. The review presents the data on the effects of maternal stress on folliculogenesis, especially when assisted reproductive technologies (ARTs) are used. Clinical data are presented alongside data from laboratory animal experiments. Different maternal stress models are taken into account in respect of their inf luence on oocyte maturation and embryo development. The interfering of psycho-emotional stress and reproductive function is the focus of the review. In these situations, exogenous hormones compensate for the stress-related disruption of the hypothalamic-pituitary-gonadal axis. When ARTs are implemented, stress-induced disruption of oogenesis is realized not via a decrease in hypothalamic and pituitary hormones, but by other ways, which involve paracrine mechanisms described in this review. Based on the literature analysis, one may conclude that stress negatively affects oocyte maturation in the ovary and suppresses subsequent embryo development. The role of some ovarian paracrine factors, such as BDNF, GDF-9, HB-EGF, TNF-α, and some others has been elucidated

Long-term effects of maternal exposure to surgical stress at the earliest stage of pregnancy on blood pressure and behavior in offspring of OXYS rats

The use of some assisted reproductive technologies, in particular, embryo transfer, may cause various physiological and behavioral changes in the offspring. The purpose of our study was to study the effects of surgery (which is used for embryo transfer) done with pregnant dams on the weight, blood pressure and behavior in the open field and elevated plus­maze tests in adult offspring. Thus, long­term effects on the offspring after maternal exposure to surgical stress given to dams at the 4th day of pregnancy were studied in OXYS rats. OXYS females were mated in estrus with fertile males of the same strain. 96 hours after spermatozoa were found in vaginal smears the surgery (sham operation, imitating embryo transfer) was performed. Body weight (BW), systolic (SAP) and diastolic (DAP) arterial pressure as well as behavior in open field (OF) and elevated plus maze (EPM) tests were studied in the offspring of females exposed to surgical treatment during pregnancy (OXYS­PS) at the age of 3 mo. Untreated offspring of OXYS rats were used as controls. BW in naturally born OXYS rats did not differ from those of the OXYS­PS group. OXYS and OXYS­PS rats exhibited higher SAP (more than 150 mm Hg) and DAP; it is noteworthy that both SAP and DAP were higher in the OXYS­PS group than in the control group. The time spent in the center of arena, the area studied, the time and number of rearing were decreased in OXYS­PS rats in the OF test as compared to the OXYS controls. Moreover, OXYS­PS rats were characterized by the absence of grooming in the OF test. As was demonstrated by the EPM test, the duration and numbers of peeking out from closed arms were decreased in the OXYS­PS rats as compared to the OXYS controls. Thus, OXYS dams’ exposure to surgical stress at their early pregnancy led to such effects in the offspring as elevated SAP and DAP, decreased overall activity and increased anxiety

Effects of reproductive technologies and SPF status on some physiological and behavioral characteristics in rats with arterial hypertension (ISIAH strain)

Modern standards of Laboratory Animal Science include working with laboratory animals of high quality, in particular, with specific pathogen free (SPF) mice and rats. On the other hand, assisted reproductive technologies (ART) are widely used in modern medicine for human infertility treatment as well as for genome resource banking. In the present study, a comparison of body weight, blood pressure (BP) and behavior in the «elevated plus maze» (EPM) test was made between three groups of ISIAH (inherited stress induced arterial hypertension) rats: a group of animals that were born and raised in a conventional animal facility and two groups from an SPF animal facility (one with animals born naturally and another with animals resulting from ART). There were no changes in BP between the groups, but the behavior of ISIAH differed depending on rearing conditions. In particular, grooming time, as well as the number of defecations and the number of urinations during the test were decreased in both groups of ISIAH rats born in the SPF animal facility as compared to ISIAH rats born in the conventional animal facility. The behavior of the ISIAH rat offspring resulting from ART was different from that of the naturally born group: the EPM test revealed reduced anxiety in the former. The results of the present study indicate that the rearing conditions as well as reproductive technologies affect some behavioral characteristics in adult ISIAH rats, although they develop arterial hypertension in all the conditions used in this study

Genome resource banking in the family Felidae

Many of the extant Felidae species are endangered or vulnerable. Others being not endangered as a whole species contain endangered subspecies. Only a very few cat species, besides domestic cats, are not in the risk group. Cryopreservation of embryos and gametes is a modern approach for ex situ mammalian genetic resources conservation. Freezing of semen has been successfully applied to the domestic cat and to more than 25 wild members of this family. However, embryos/oocytes cryopreservation was successful for only a small number of felids. Domestic cat and four wild Felidae species produced offspring after cryopreservation and subsequent embryo transfer. Regarding freezing of oocytes, so far different cryopreservation methods are still being experimentally tried exclusively for domestic cat. Genome Resource Bank (GRB) containing frozen semen of Amur leopard cat, bobcat and Eurasian lynx was established at the Institute of Cytology and Genetics, Novosibirsk. As a result of this project, original methods of feline semen freezing have been developed; embryos of domestic cat have been successfully frozen as well. Approaches to freeze domestic cat’s oocytes have also been tried. During this work, we combined biological and physical methods. In particular, the process of freezing embryos and oocytes was monitored with Raman spectroscopy. Different methods of frozen-thawed spermatozoa and embryonic viability testing were used in this study, including vital staining and subsequent fluorescent and light microscopy, and heterologous in vitro fertilization

Cryopreservation of epididymal semen of domestic cat

Domestic cat (Felis silvestris catus) is used as a model species for developing effective methods of wild felids’ semen cryopreservation. The present study represents a comparison of domestic cat semen cryopreservation with two commercially available cryoprotectant agents (CPAs): CaniPlus Freeze (CPF) and SpermFreeze (SF). Semen was collected from the caudal epididymises of adult males and frozen with CPF and SF, correspondingly. The viability of frozen-thawed spermatozoa was evaluated by VitalScreen kit, staining with hematoxylin and eosin was performed for analysis of the spermatozoa morphology; both analyses were combined with the light microscopy. The viability rate of the frozenthawed semen cryopreserved with CPF and SF did not differ: 32.3±4.4 % for CPF and 43.3±4.0 % for SF. Total percentage of morphologically normal spermatozoa after freezing and thawing domestic cat semen was 26.0±2.3 % for CPF and 23.9±1.9 % for SF. In both cases, there were no differences from non-frozen semen, in the latter group the total percentage of spermatozoa with normal morphology was 29.0±4.1 %. The most frequent anomalies were the anomalies of tail, and the rarest anomalies were head defects. The percentages of spermatozoa with anomalies of the head, mid piece, tail and combined did not differ in these three groups. Taken together these results suggest that both CPAs are suitable for the purpose of domestic cat semen freezing and cryopreservation, although CPF was designed for Canidae semen cryopreservation and SF was developed for human semen freezing and so far was used exclusively in reproductive medicine. It might be concluded that these two commercially available cryoprotectant media are applicable for the purposes of domestic cat breeds’ semen cryopreservation

Effects of a high-fat diet on the lipid profile of oocytes in mice

There are evidences that obese women exhibit a detrimental oocyte quality. However, it remains unclear how this change is associated with obesity, indirectly – or directly through a change in the content and/or composition of lipids in oocytes. The aim of this work was to study effects of a high-fat diet applied to female donor mice on the amount and qualitative composition of lipids of immature and in vivo matured oocytes. A high-fat diet caused larger body weight in female mice compared with the control (p < 0.001; 44.77±1.46 and 35.22±1.57, respectively), and increased the blood levels of cholesterol (p < 0.05; 2.06±0.10 and 1.78±0.10, respectively) and triglycerides (p < 0.05; 2.13±0.23 and 1.49±0.21, respectively). At the same time, this diet does not affect the level of unsaturation of lipids in immature (0.207±0.004 in the experiment and 0.206±0.002 in the control) and matured oocytes (0.212±0.005 in the experiment and 0.211±0.003 in the control). Total lipid content increased during in vivo maturation of mouse oocytes. The amount of lipids was greater in mature oocytes in the experimental group compared to the control (p < 0.01; 8.15±0.37 and 5.83±0.14, respectively). An increase in intracellular lipid amount during oocyte maturation was revealed both after a standard diet (p < 0.05; 4.72±0.48 and 5.83±0.14, respectively) and after a fat-rich diet (p < 0.001; 3.45±0.62 and 8.15±0.37, respectively). Thus, during in vivo oocyte maturation in mice the content of intracellular lipids enhanced, the high-fat diet aggravated this dynamics of lipid increase during in vivo maturation of oocytes

Neuronal density in the brain cortex and hippocampus in Clsnt2-KO mouse strain modeling autistic spectrum disorder

Autistic spectrum disorders (ASD) represent conditions starting in childhood, which are characterized by difficulties with social interaction and communication, as well as non-typical and stereotyping models of behavior. The mechanisms and the origin of these disorders are not yet understood and thus far there is a lack of prophylactic measures for these disorders. The current study aims to estimate neuronal density in the prefrontal cortex and four hippocampal subfields, i. e. СA1, СA2, СA3, and DG in Clstn2-KO mice as a genetic model of ASD. In addition, the level of neurogenesis was measured in the DG area of the hippocampus. This mouse strain was obtained by a knockout of the calsinthenin-2 gene (Clsnt2) in C57BL/6J mice; the latter (wild type) was used as controls. To estimate neuronal density, serial sections were prepared on a cryotome for the above-mentioned brain structures with the subsequent immunohistochemical labeling and confocal microscopy; the neuronal marker (anti-NeuN) was used as the primary antibody. In addition, neurogenesis was estimated in the DG region of the hippocampus; for this purpose, a primary antibody against doublecortin (anti-DCX) was used. In all cases Goat anti-rabbit IgG was used as the secondary antibody. The density of neurons in the CA1 region of the hippocampus was lower in Clstn2-KO mice of both sexes as compared with controls. Moreover, in males of both strains, neuronal density in this region was lower as compared to females. Besides, the differences between males and females were revealed in two other hippocampal regions. In the CA2 region, a lower density of neurons was observed in males of both strains, and in the CA3 region, a lower density of neurons was also observed in males as compared to females but only in C57BL/6J mice. No difference between the studied groups was revealed in neurogenesis, nor was it in neuronal density in the prefrontal cortex or DG hippocampal region. Our new findings indicate that calsyntenin-2 regulates neuronal hippocampal density in subfield-specific manner, suggesting that the CA1 neuronal subpopulation may represent a cellular target for earlylife preventive therapy of ASD