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To ascertain whether the Kunming (KM) mouse is an available model for age-related decline in female fertility in human or not, oocytes from young (6–8 weeks), middle-aged (9 months) and aged (12 months) female mice were compared with respect to number of oocytes, frequency of in-vitro maturation (IVM) and in-vitro fertilization (IVF), and meiotic chromosome segregation and alignment. The mean number of pups born per mouse decreased significantly from the young to the middle-aged and the aged mice. The mean number of ovarian follicles, ovarian germinal vesicle oocytes and ovulated MII oocytes decreased significantly with maternal age. The rate of IVM in oocytes from young mice (73.9%) was less significantly than that in oocytes from middle-aged and aged mice (86.1% and 84.4%, respectively). Immunocytochemical analysis showed that ageing caused a significantly higher rate (49.3%) of chromosome misalignment than that (15.7%) of the young mice. The presence of premature chromatids was also significantly higher in MII oocytes of aged mice as compared with young mice (37.8 versus 8.3%). Pronuclear formation was delayed in oocytes of middle-aged and aged females (35.5 and 42.3% respectively in 5 h of IVF) as compared with young mice (88.1%). The study suggests that KM mouse exhibits an age-related decline in female fertility. Significant reduction of germinal vesicle (GV) and MII oocytes and significant increase of metaphase chromosome misalignment and premature chromatid segregation after meiotic maturation of oocytes, similar to human, presumably contribute to the decline in aged KM mice.
It is well known that c-Jun N-terminal kinase (JNK) plays pivotal roles in various mitotic events, but its function in mammalian oocyte meiosis remains unknown. In this study, we found that no specific JNK2 signal was detected in germinal vesicle stage. JNK2 was associated with the spindles especially the spindle poles and cytoplasmic microtubule organizing centers at prometaphase I, metaphase I, and metaphase II stages. JNK2 became diffusely distributed and associated with the midbody at telophase I stage. Injection of myc-tagged JNK2α1 mRNA into oocytes also revealed its localization on spindle poles. The association of JNK2 with spindle poles was further confirmed by colocalization with the centrosomal proteins, γ-tubulin and Plk1. Nocodazole treatment showed that JNK2 may interact with Plk1 to regulate the spindle assembly. Then we investigated the possible function of JNK2 by JNK2 antibody microinjection and JNK specific inhibitor SP600125 treatment. These two manipulations caused abnormal spindle formation and decreased the rate of first polar body (PB1) extrusion. In addition, inhibition of JNK2 resulted in impaired localization of Plk1. Taken together, our results suggest that JNK2 plays an important role in spindle assembly and PB1 extrusion during mouse oocyte meiotic maturation.
Protein kinase C (PKC) is a family of Ser/Thr protein kinases that can be activated by Ca2+, phospholipid and diacylglycerol. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. The present study aimed to monitor the effect of age, germinal vesicle (GV) transfer and modified nucleoplasmic ratio on the subcellular distribution profile of PKCα, an important isozyme of PKC, in mouse oocytes undergoing meiotic maturation and following egg activation. Germinal vesicle oocytes were collected from 6–8-week-old and 12-month-old mice. Germinal vesicle-reconstructed oocytes and GV oocytes with one-half or one-third of the original oocyte volume were created using micromanipulation and electrofusion. The subcellular localization of PKCα was detected by immunocytochemistry and laser confocal microscopy. Our study showed that PKCα had a similar location pattern in oocytes and early embryos from young and old mice. PKCα was localized evenly in ooplasm, with weak staining in GV at the GV stage, and present in the entire meiosis II (MII) spindle at the MII stage. In pronuclear and 2-cell embryos, PKCα was concentrated in the nucleus except for the nucleolus. After the GV oocytes were reconstructed, the resultant MII oocytes and embryos showed a similar distribution of PKCα between reconstructed and unreconstructed controls. After one-half or two-thirds of the cytoplasm was removed from the GV oocytes, PKCα still had a similar location pattern in MII oocytes and early embryos from the GV oocytes with modified nucleoplasmic ratio. Our study showed that age, GV transfer and modified nucleocytoplasmic ratio does not affect distribution of PKCα during mouse oocyte maturation, activation, and early embryonic mitosis.
Although the underdeveloped embryo, and thus morphological (MD) or morphophysiological (MPD) seed dormancy, is basal in angiosperms, it also occurs in advanced groups. A synthesis of the literature, combining phylogeny and the kind of seed dormancy in the highly evolutionarily advanced order Dipsacales, shows that MPD (or MD) occurs throughout all clades except the most advanced one, Valerina. Seeds of taxa in the Valerina clade have fully developed embryos and physiological dormancy (PD) or are non-dormant (ND); thus, PD and ND are derived conditions in Dipsacales. Assuming that types of seed dormancy have not changed since the Early Tertiary, the fossil record suggests that MPD (or MD) was present in extant genera of Dipsacales by the Palaeocene, but PD (or ND) not until the Miocene. Molecular dating indicates that the ages of dipsacalean lineages with MPD and PD are older than those indicated by the fossil evidence.
The present study examined: (i) age-induced regional changes in fatty acid composition of brain phospholipids; (ii) α-linolenic acid deficiency-induced regional depletion and recovery of DHA in the brain. DHA and arachidonic acid (AA) did not distribute evenly in the brain. In weaning and adult rats, the region with the highest DHA percentage was the cortex whereas the medulla had the lowest DHA percentage. In the aged rats, both the cortex and cerebellum were the regions with the highest DHA percentage whereas in the neonatal rats, the striatum had the greatest percentage of DHA, and the hypothalamus and hippocampus had the least percentage of DHA. Regarding AA, the hippocampus was the region that had the highest percentage whereas the medulla was the region with the lowest percentage except for the neonatal rats, whose cerebellum, hypothalamus, striatum and midbrain had AA percentage lower than hippocampus and cortex. DHA was not proportionally depleted in various regions of brain when the rats were maintained on an n-3-deficient diet for two generations. The results demonstrated that the cortex, hippocampus, striatum, cerebellum and hypothalamus had DHA depleted by >71 %, whereas the midbrain and medulla had only 64 and 57 % DHA depleted, respectively. The most important observation was that the diet reversal for 12 weeks resulted in complete DHA recovery in all regions except for the medulla where the recovery was only 62 %. It was concluded that the location of DHA, n-3 deficiency-induced DHA depletion and reversibility of DHA deficiency across the brain were region-specific.
Conjugated linolenic acid (CLN) refers to a group of octadecatrienoic acid isomers that have three double bonds in conjugation. Both pomegranate and tung seed oils are rich in CLN but the major isomer in the former is cis9,trans11,cis13 while in the latter it is cis9,trans11,trans13. The present study examined the effects of CLN, isolated from either pomegranate seed oil or tung seed oil, and α-linolenic acid (LN), isolated from flaxseed oil, on serum cholesterol levels in male hamsters (body weight 105 g; age 10 weeks) fed a 0.1 % cholesterol and 10 % lard diet, for a period of 6 weeks. All hamsters were allowed free access to food and fluid. The blood samples were taken by bleeding from the retro-orbital sinus into a heparinized capillary tube under light ether anaesthesia after overnight fasting at weeks 0, 2, 4 and 6. It was found that supplementation of CLN at levels of 12.2–12.7 g/kg diet exhibited no significant effect on serum cholesterol level while LN at a similar level of supplementation had serum cholesterol reduced by 17–21 % compared with the control diet containing no LN and CLN. Supplementation of CLN and LN significantly decreased hepatic cholesterol but no effect was observed on heart and kidney cholesterol levels. It was concluded that LN possessed hypocholesterolaemic activity while CLN had no effect on blood cholesterol, at least in hamsters.
Canned and bottled tea drinks contain not only green tea epicatechins (GTE), namely (−)-epigallocatechin gallate (EGCG), (−)-epicatechin gallate (ECG), (−)-epigallocatechin (EGC) and (−)-epicatechin (EC), but also four GTE epimers, namely (−)-gallocatechin gallate (GCG), (−)-catechin gallate (CG), (−)-gallocatechin (GC) and (−)-catechin (C). In the present study we examined the antioxidant activity and bioavailability of these epimers compared with their corresponding precursors. The epimerisation reaction was induced by autoclaving GTE extract derived from longjing green tea at 120°C for 20 min. Isolation and purification of each GTE and epimer were accomplished by various column chromatographic and semi-preparative HPLC techniques. The antioxidant activity of each epimer with its corresponding GTE precursor was conducted in the three in vitro systems, namely human LDL oxidation, ferric reducing–antioxidant power (FRAP), and anti-2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical assays. The results of all three assays demonstrated that CG had similar antioxidant activity with its precursor ECG, while GC was less potent as an antioxidant than its precursor EGC. Regarding EGCG and GCG, the antioxidant potency was similar for both LDL oxidation and DPPH free radical assays, but GCG was statistically less effective than EGCG in the FRAP assay. For EC and C, the latter had less anti-free radical activity in the DPPH assay, but in LDL oxidation and FRAP assays the antioxidant activity was similar. Oral and intravenous dosing of GTE–epimer mixture led to increase in total plasma antioxidant capacity in rats. In general, both epicatechins and epimers had low bioavailability (0·08–0·31) and most of the observed differences between epicatechins and their corresponding epimers were small, even if they were statistically significant in some cases. It was concluded that the epimerisation reaction occurring in manufacturing canned and bottled tea drinks would not significantly affect antioxidant activity and bioavailability of total tea polyphenols.
The present study was designed to compare the conjugated linoleic acid (CLA) isomeric distribution pattern in the liver of suckling rats in relation to those in the milk and maternal diet. Silver-ion HPLC was used to separate individual CLA isomers. It was found that the isomeric distribution pattern in the milk was very similar to that in the maternal dietary fat. However, the CLA isomeric distribution patterns in the liver phospholipids (PL) and triacylglycerols were different from those in the diet and milk. In the liver PL, total cis/trans isomers accounted for 63·6–63·9 % of total CLA, which was in contrast to the values of 88·1–89·1 % in the milk and diet. In the liver PL, total trans/trans isomers were 20·6–20·8 % of the total CLA isomers whereas they were only 2·6–3·7 % in the milk and diet. It is concluded that trans/trans-CLA were preferentially incorporated into the liver whereas for the incorporation of cis/trans-CLA there was partial discrimination.
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