Dietary Supplementation of Flaxseed (Linum usitatissimum L.) Alters Ovarian Functions of Xylene-Exposed Mice.

The aim of the performed study was to examine the ability of xylene, flaxseed, and their combinations to affect morphological and endocrine indexes of murine ovaries. The 72 indexes of secondary and tertiary follicular cells, oocytes, corpora lutea, and ovarian stroma have been quantified: diameter, markers of proliferation PCNA and apoptosis caspase 3, receptors to FSH, oxytocin, estrogen (alpha and beta), and progesterone. In addition, concentrations of the ovarian hormones progesterone, estradiol, and IGF-I in the blood, as well as their production by isolated ovaries cultured with and without gonadotropins (FSH + LH mixture), were determined using histological, immunohistochemical, and immunoassay analyses. The character of xylene and flaxseed effects on ovarian functions in mice depended on the stage of ovarian folliculogenesis. It was shown that flaxseed could mitigate and prevent the major (63%) effects of xylene on the ovary. In addition, the ability of gonadotropins to affect ovarian hormone release and prevent its response to xylene has been shown. The effects of these additives could be mediated by changes in the release and reception of hormones. These observations suggest that flaxseed and possibly gonadotropins could be natural protectors of a female reproductive system against the adverse effects of xylene.

Body fat affects mouse reproduction, ovarian hormone release, and response to follicular stimulating hormone.

We investigated the effects of body fat content on mouse fecundity, ovarian hormone release, and their response to follicle stimulation hormone (FSH). 4 types of females were produced: lean (group 1), normal (group 2), slightly fat (group 3), and significantly fat (group 4). The body weights, fat content, fertility rate, embryo number produced, retarded and degenerated embryo percentage, the release of progesterone (P4), testosterone (T), and insulin-like growth factor I (IGF-I) by isolated ovaries cultured with and without FSH (1.0IU/mL medium) were evaluated. A gradual increase in body weight and fat contents from groups 1 to 4 was observed. Group 2 had higher fertility rate than those from the other groups. Groups 2 and 3 had fewer retarded and degenerated embryos that those from groups 1 and 4. Embryo production rate was not different among the groups. P4 and T secretion was higher from group 4 than in those from groups 1-3; secretion of IGF-I of group 3 was less than that of groups 1, 2, and 4. FSH promoted ovarian T output in all groups and stimulated ovarian P4 release in groups 1, 3, and 4, but not in group 2. FSH did not affect IGF-I release in any group. Therefore, both malnutrition and overfeeding can affect body weight and fat content in female mice, reducing embryo quality or developmental capacity, but not fertility and embryo production. Excess weight or fat can have stimulatory effects on ovarian P4 and T, but inhibitory effects on ovarian IGF-I release. Both leanness and excess weight or fat can induce the stimulatory action of FSH on ovarian P4.

Metabolic state can define the ovarian response to environmental contaminants and medicinal plants.

Environmental contaminants and medicinal plants can affect reproductive processes. The aim of this study was to investigate the effect of maternal metabolic status on the response of mouse ovaries to the environmental contaminants benzene and xylene, as well as to extracts of the medicinal plant yucca. Ovaries isolated from normal-lean and slightly obese mice were cultured with or without 0.1% benzene or xylene for 24 h. Similarly, ovaries isolated from normal-lean, slightly obese, and significantly obese mice were cultured for 24 h with or without an extract of Yucca shidigera (YS, 10 µ g/mL). We found that the metabolic status did not influence the release of basal progesterone (P4), testosterone (T), or insulin-like growth factor I (IGF-I), but obesity influenced the effects of the environmental contaminants and YS. Benzene reduced P4 output in ovaries from obese but not normal-lean mice; it also reduced IGF-I (but not T) release from ovaries irrespective of the metabolic status. Xylene dramatically increased P4 and T (but not IGF-I) release by ovaries from normal-lean mice, but there were no changes in P4 and only small increases in T output in obese mice. YS increased P4 (but not T or IGF-I) release in normal-lean and slightly obese animal ovaries, whilst significant obesity was associated with a lack of P4 response to YS. Obesity might affect the basal ovarian release of T or IGF-I and increases the sensitivity of ovaries to the action of benzene but decreases their responsiveness to xylene and YS.

Diverse effect of different odor stimuli on behavior and Fos protein production in the olfactory system neurogenic region of adult rats.

Previously it has been demonstrated that processes of postnatal neurogenesis in the olfactory system neurogenic region-the subventricular zone (SVZ), rostral migratory stream (RMS), and olfactory bulb (OB) can be significantly altered by different factors of an environment. However, the mechanisms involved in regulation of neurogenesis by exogenous factors in the olfactory system remain unclear. The purpose of the present study was to contribute to the understanding of these mechanisms by immunohistochemical assessment of Fos protein induction in areas of adult neurogenesis. To evaluate the coordinate activation of Fos production in neurons of the olfactory system neurogenic region, a brief exposure to artificial odor (eau de Cologne) or naturalistic odor (cat odor) has been used in alert rats. Our results revealed that the effects of these odors are easily distinguishable at both the behavioral and the morphological level. Cat odor induced greater changes in anxiety level, and produced typical pattern of Fos activation in the accessory olfactory bulb (AOB), a brain region associated with defensive behavior. An important finding is, that next to distinct Fos expression in the OB and the AOB, Fos positive cells have been found also within the SVZ/RMS of the odor stimulated rats. Interestingly, Fos expression in the RMS was detected only after exposure to artificial odor stimulus. These results provide new evidence that some SVZ/RMS cells have complete prerequisites necessary for the Fos signal transduction cascade.

Akt/PKB plays role of apoptosis relay on entry into first mitosis of mouse embryo.

The cell-cycle regulators that control meiotic divisions also regulate the events that accompany the oocyte-to-zygote transition. Thus, the meiotic machinery functions as an internal pacemaker that propels the oocyte toward embryogenesis. The preimplantation embryo expresses a number of receptors that are important for initial activity of the phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt/PKB) pathway. The complete PI3K-Akt/PKB-CDK1 cascade is implicated as a key regulator of a number of cellular functions. Selective inhibition of protein kinase B (Akt/PKB) with inhibitor SH6 and cyclin-dependent kinase 1 (CDK1) with inhibitor roscovitine arrest development of the 1-cell preimplantation mouse embryo before entry into the first mitosis. The pronuclei of these inhibited embryos migrate to one another, but do not progress to pronuclei envelope breakdown and pronuclear fusion running immediately before the onset of mitosis. SH6-treated 1-cell mouse embryos showed a high occurrence of apoptosis features (nuclear fragmentation, positive terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), active caspase-3 in both cytoplasm and nucleoplasm). In the Akt/PKB-inhibited embryos, the active phosphorylated form Ser473Akt/PKB was not detected in pronuclear areas when compared with inhibitor-free controls. Although CDK1-inhibited 1-cell embryos also failed to enter into the first mitosis, the presence of apoptotic cell death features was not observed. In the roscovitine-treated embryos, Ser473Akt/PKB was detected in the pronuclei independently of CDK1 activity. We conclude that Akt/PKB plays an important role during entry of the 1-cell mouse embryo into the first mitosis, and probably functions as a relay in the cell-cycle stage. We assume that Akt/PKB is the primary target responsible for mediating anti-apoptotic signals in the 1-cell mouse embryo.

Gene expression in mouse preimplantation embryos affected by apoptotic inductor actinomycin D.

The aim of this study was to test the effect of actinomycin D on the expression of selected genes and to elucidate possible components of its apoptotic pathway in mouse embryos. Selected mRNAs and Trp53 protein were examined in blastocysts cultured for 24 h in vitro with or without the presence of a high concentration of actinomycin D. In all tested genes, the relative quantities of mRNA were significantly lower in treated blastocysts than in controls. The mRNA quantities of H2afz, Actb, Bax, Bad and Bcl2 were reduced at a similar rate, but the decreases in Bcl2l2 and Trp53 mRNA were significantly greater. Treatment with actinomycin D also changed the ratio between the mRNA levels of some pro-apoptotic and anti-apoptotic genes: the Bad/Bcl2l2 and the Bax/Bcl2l2 ratios were on average 4.39 and 2.66 times higher in the treated embryos than in the controls, respectively. Generally, treatment led to developmental arrest and significant increase in the incidence of cells with typical apoptotic features. However, its effect on Trp53 protein expression was not significant. The results suggest that mechanisms beyond the apoptotic effect of actinomycin D might include specific changes in the expression of pro-apoptotic and anti-apoptotic genes, shifting the expression ratio in favor of the pro-apoptotic ones. The results also show that the role of Trp53 is probably not crucial in this apoptotic pathway.

Apoptotic processes and DNA cytosine methylation in mouse embryos arrested at the 2-cell stage.

The present study evaluates the role of apoptotic cell death and DNA methylation reprogramming in early developmental failures occurring in embryos at the 2-cell stage. Mouse 2-cell embryos were cultured in vitro and treated with chemicals that cause developmental arrest and apoptosis (alpha-amanitin, actinomycin D, TNF-alpha). After 24 h, 48 h and 72 h culture, embryos were analysed using cell-death assays (annexin V staining, TUNEL labelling and immunodetection of active caspase-3) and genome methylation assay (immunodetection of 5-methylcytosine). The ability of embryos at the 2-cell stage to undergo apoptotic processes was very low. In arrested embryos, the presence of all evaluated features of apoptosis was recorded only after 72 h culture and their incidence was sporadical. Interestingly, the most frequently observed apoptotic sign was nuclear condensation and the timing of its appearance preceded even the phosphatidylserine flip. Both normally developing and arrested embryos displayed reduction in DNA cytosine methylation. In arrested embryos, this process was independent of cellular cleavage, was more pronounced and finished in almost complete demethylation of the embryonic genome. The timing of the demethylation overlapped with the onset of major apoptotic events. Although observed apoptotic cells showed either demethylated or methylated DNA cytosine in their nuclei, at blastocyst stage the demethylated status appeared more frequently in them.