Astrocytic connexin 43 deletion ameliorates SNI-induced neuropathic pain by reducing microglia activation.

Neuropathic pain (NP) is a chronic disease caused by damage to the peripheral or central nervous system. Connexin 43 (Cx43), the primary connexin expressed by astrocytes, has been reported to be significantly increased in NP. However, the roles and mechanisms of Cx43 in the development and maintenance of NP remain largely unknown, while microglia activation has been commonly regarded as a key factor of NP. In the present study, we found that Cx43 deletion significantly ameliorated spared nerve injury (SNI)-induced NP and suppressed SNI induced c-Fos expression in the spinal cord. Notably, Cx43 deletion led to much less SNI-induced microglia activation in the spinal cord. These results suggest that astrocyte Cx43 may play a significant role in regulating microglial activation and NP.

Adiponectin receptor agonist AdipoRon modulates human and mouse platelet function.

Adiponectin, an adipokine secreted by adipocytes, has anti-atherosclerotic and antithrombotic activities. AdipoRon is synthetic small molecule adiponectin receptor agonist. In this study, we investigated the effect of AdipoRon on platelet activation and thrombus formation. Washed human platelets were prepared from the peripheral blood of healthy donors. In a series of in vitro platelet functional assays, pre-treatment with AdipoRon (10, 20, 40 µg/mL) dose-dependently inhibited the aggregation, granule secretion and spreading of washed human platelets. We showed that AdipoRon (20, 40 µg/mL) significantly inhibited AMPK, Syk, PLCγ2, PI3K, Akt, p38-MAPK and ERK1/2 signalling pathways in washed human platelets. In addition, we demonstrated that the phosphorylation of CKII at Tyr255 was an important mechanism of the integrin αIIbß3-mediated platelet activation. Meanwhile, AdipoR1 deficiency impaired the inhibitory effect of AdipoRon on mouse platelets. In ferric chloride-induced carotid injury model, injection of AdipoRon (5 or 12.5 mg/kg, iv) significantly attenuated arterial thrombosis. In conclusion, AdipoRon attenuates platelet function via the AdipoR1/AMPK/CKII/PI3K/AKT signalling pathways, while exerting a protective effect against arterial thrombosis. This study offers new insights into the fields of cardiovascular disease and antiplatelet drug discovery.Schematic model of AdipoRon regulating platelet activation. (BioRender.com).

[Clinical Study on Early Extubation and Sequential Non-Invasive Respiratory Support for Children with Acute Respiratory Failure Receiving Invasive Mechanical Ventilation].

Objective: To explore the treatment outcome of the strategy of early extubation and then switching to non-invasive mechanical ventilation in children with acute respiratory failure, and the safety and feasibility of using the strategy to replace traditional methods. Methods: A total of 102 children, aged between 1 month to 14 years old, who had acute respiratory failure and were admitted to the pediatric ICU of West China Second University Hospital, Sichuan University between January 2019 and December 2020 were enrolled and randomly assigned to treatment group 1 (n=55) and treatment group 2 (n=47). In addition, 53 children who had the same condition in the 12 month prior to the beginning of the study were included in the control group. In the two treatment groups, the patients were extubated first, and then weaned off the ventilator. In group 1, when the patient met the invasive-non-invasive switching criteria, the tracheal tube was pulled out and non-invasive bi-level positive airway pressure (BiPAP) ventilation was used for respiratory support. In group 2, high-flow nasal cannula (HFNC) oxygen therapy was used for respiratory support. The traditional progressive weaning method was adopted for the control group (extubing and weaning were performed at the same time). The incidence of ventilator-associated pneumonia (VAP) during the period of tracheal intubation was compared and the mortality of the two groups was evaluated from the point when the patients were recruited. At the time of extubation in the treatment groups and extubation plus weaning in the control group, the pressure support levels, or PC above PEEP, intubation time, sequential time (between 2 treatment groups only), weaning failure rate, and the incidence of laryngeal edema and nasal pressure ulcer were compared. Results: The subjects of the study were predominantly infants (93 cases, 60%) and young children (31 cases, 20%). Among the 155 cases, 82 (53%) were male. There was no statistical difference in age distribution or gender among the groups. There was no significant difference in the clinical indicators among the three groups before tracheal intubation. At the time of extubation, the PC above PEEP in the two treatment groups was higher than that in the control group, and higher in group 1 than that of group 2, the difference being statistically significant (P<0.05). The intubation time of the two treatment groups was shorter than that of the control group, and shorter in group 1 than that of group 2 (P<0.05). The sequential time of group 2 was shorter than that of group 1 (P<0.05). The extubation failure rate and the incidence of VAP in the two treatment groups were lower than those in the control group, and there was no statistically significant difference between the two treatment groups. The incidence of nasal pressure ulcers in group 1 was higher than that in the other two groups (P<0.05). There was 1 death in treatment group 1, and no deaths in treatment group 2 or the control group. There was no significant difference in mortality or the incidence of laryngeal edema after extubation in the three groups. Conclusion: Early extubation and then switching to non-invasive mechanical ventilation can be well tolerated by the patients, and can be used in clinical practice as an effective weaning method for children with acute respiratory failure.

Simvastatin Improves Outcomes of Endotoxin-induced Coagulopathy by Regulating Intestinal Microenvironment.

OBJECTIVE: The systemic inflammatory response is regarded as the major cause of endotoxin-induced coagulopathy, which is a strong predictor of mortality in patients with severe sepsis. Simvastatin plays an important role in reducing inflammation. In addition, the gut has long been hypothesized to be the "motor" of critical illness, driving or aggravating sepsis by the increased intestinal permeability and bacterial translocation. Whether simvastatin plays a role in severe endotoxin-induced coagulopathy through the gut is unclear. METHODS: In this study, mice were administered 20 mg/kg simvastatin by gavage for 2 weeks and then intraperitoneally injected with 50 mg/kg endotoxin. Twelve h later, cytokine release, coagulation dysfunction, organ damage, and survival were assessed. Besides, the intestinal barrier, permeability, bacteria abundance, and translocation were evaluated. RESULTS: We found that the severity of endotoxin-induced coagulopathy was significantly improved in simvastatin-pretreated mice, who showed attenuated depletion of coagulation factors and platelets, decreased plasminogen activator inhibitor-1 (PAI-1) expression, reduced organ fibrin deposition, and improved survival time. Also, simvastatin reduced epithelial apoptosis and improved intestinal barrier function by upregulating antimicrobial peptides, lysozyme, and mucins. Simvastatin increased Lactobacillales counts, while the lipopolysaccharide group showed increased Desulfovibrio and Mucispirillum, which can produce harmful toxins. Finally, the decreased intestinal permeability in the simvastatin group caused reduced bacterial translocation in the organs and blood, both in terms of quantity and species. CONCLUSION: Simvastatin improves the prognosis of severe endotoxemia, and the intestinal microenvironment participates in this process.

Frequency and clinical manifestation of prenatal cytogenetic diagnosis of chromosomal polymorphisms in Northeast China.

OBJECTIVE: To retrospectively analyze the incidence of chromosomal polymorphisms in prenatal cytogenetic diagnostic cases and the effect of the clinical manifestation of these fetuses. MATERIALS AND METHODS: 490 fetuses with chromosomal polymorphisms among 9996 pregnant women who underwent prenatal cytogenetic diagnosis were included in this study and were set as group 1. Other 500 pregnant women, whose fetuses were with normal karyotypes, were randomly selected from the remaining pregnant women and set as group 2. Clinical information and outcomes and maternal serum screening results of group 1 were compared with group 2. RESULTS: The frequency of fetal chromosomal polymorphism was 4.90% (490/9996). The most common variants observed were 1/9/16 qh± (2.27%, 227/9996), followed by inv(9) (0.90%, 90/9996). 94.62% (264/279) of fetal chromosomal variants were inherited from parents. No statistical difference was found in clinical information and outcomes and maternal serum screening results between group 1 and group 2. CONCLUSION: The fetus with chromosomal polymorphism has no impact on serum markers of second trimester screening and does not play an important role for the clinical outcome of the current pregnancy either, whether it is inherited from the parents or a de novo mutation.

Indigenization of the median of markers for Down syndrome screening based on statistical analysis of medical big data.

OBJECTIVE: To indigenize the median of Down syndrome (DS) screening markers for first and second trimester, and compare the impact of the indigenized and built-in median data on the efficiency of DS screening. MATERIALS AND METHODS: Data derived from first and Second-trimester screening (FTS and STS) for DS, composed of selected pregnancies deemed to be normal, were examined in a retrospective study. Indigenization regression analysis was calculated by using five models to fit statistical the raw data. Multiple of median (MoM) values estimated by using indigenized medians were compared with those calculated by using built-in. RESULTS: This study established a regression equation which is more suitable for the median of each screening marker in the local pregnant women. The changes of median MoM of screening markers were statistically significant after indigenization. For FTS, the detection rate was 100% when the false positive rate was 5%, and the cut-off value was 1/262. On the other hand, for STS, the detection rate of the model with indigenized parameters was 77.42%, which is 16.13% higher than that of built-in parameters. CONCLUSION: For the individual specific risk of pregnancy, when the indigenized parameters was used to calculate, is more accurately and screening effectiveness has been improved. This is a great reference significance for the current prenatal screening whether indigenized data should be used.

Ultrasound-Guided Transversus Abdominis Plane Block for Cesarean Delivery: Injection Site Pain as a New Complication and Dexamethasone Reduced Incidence.

BACKGROUND: Although ultrasound-guided transversus abdominis plane block (TAPB) is widely used in multimodal analgesia after cesarean delivery (CD), the complications of TAPB during analgesia after CD have rarely been reported. METHODS: A total of 84 cases of CD were randomly assigned to either a ropivacaine group (R group) or ropivacaine + dexamethasone group (RD group) in this double-blind trial. The pain site and pain degree at rest and during activity at 2 h, 6 h, 10 h, 12 h, 14 h, 16 h, 20 h, and 24 h after maternal surgery were recorded. The consumption of opioids at 24h, postoperative nausea, vomiting, exhaustion, and other adverse reactions were recorded. RESULTS: A total of 80 patients were included in the analysis of results. A total of 19 patients developed ISP, 14 in the R group and 5 in the RD group. The incidence of ISP in the R and RD groups was 35% and 12.5%, respectively. The results described above showed that combining dexamethasone with ropivacaine reduced the incidence of ISP, and the difference was statistically significant (P<0.05). Two groups of women with positive ISP had higher values of opioid consumption than women with negative ISP, but the difference was not significant. CONCLUSION: Dexamethasone as an adjuvant for ropivacaine can effectively relieve the ISP of ultrasound-guided TAPB after CD, and can enhance the analgesic effect of ropivacaine.

SIRT1 knock-in mice preserve ovarian reserve resembling caloric restriction.

Previous studies have proposed that caloric restriction (CR) regulates many cell functions and prolongs the lifespan of an organism. Our previous studies proposed that CR also prevents follicular activation and preserves the ovarian reserve in mice by activating SIRT1. To test if SIRT1 preserves the ovarian reserve and prolongs the ovarian longevity, we generated SIRT1 knock-in mice that can overexpress SIRT1 in oocytes of the mouse. Ovaries of the mice at ages 35 days and 15 months were collected, and the follicular development and follicular reserve were examined. The vaginal opening and onset of estrus of transgenic female mice (both the homozygous and heterozygous for SIRT1 overexpression) were later than that of wild-type mice. Both the homozygous and heterozygous SIRT1-overexpressing mice had a larger and stronger reproductive capacity than wild-type mice. Moreover, 35-day-old and 15-month-old homozygous and heterozygous SIRT1-overexpressing mice also had a higher mean number and percentage of healthy follicles, fewer atretic follicles than wild-type mice, and the mean number and percentage of primordial follicles in both the homozygous and heterozygous SIRT1-overexpressing mice were higher than wild-type mice at the same age. However, the phenotypes of heterozygous and homozygous transgenic mice came no difference. Immunohistochemistry showed increased expression of SIRT1 and FOXO3a, and decreased expression of mTOR in both the homozygous and heterozygous SIRT1-overexpressing mice compared with wild-type mice. Thus, oocyte-specific SIRT1-overexpressing mice continuously activate FOXO3a and suppress mTOR and have a larger reproductive capacity, larger follicle reserve and longer ovarian lifespan.

[Role of c-Jun N-terminal kinase-mediated FOXO3a nuclear translocation in neuronal apoptosis in neonatal rats with hypoxic-ischemic brain damage].

OBJECTIVE: To explore the mechanisms of neuroprotective effects of c-Jun N-terminal kinase (JNK)/FOXO3a transcription factor signaling pathway inhibition on hypoxic-ischemic neuronal apoptosis in neonatal rats with hypoxic-ischemic brain damage (HIBD). METHODS: Sixty-four 7-day-old Sprague-Dawley rats were divided into four groups: hypoxia-ischemia (HI), sham-operated, JNK specific inhibitor AS601245-treated, and DMSO vehicle. Rats' cerebral cortexes were collected at 24 hours after HI. Western blot was used to detect the protein expression of JNK, p-JNK, FOXO3a, nuclear and cytoplasmic FOXO3a, Bim, and CC3. TUNEL staining was used to detect the apoptotic cells. RESULTS: Compared with the sham-operated group, p-JNK protein increased (P<0.01), nuclear protein of FOXO3a increased (P<0.01), cytoplasmic protein decreased (P<0.01), and pro-apoptotic proteins Bim and CC3 increased 24 hours after HI (P<0.01). Compared with the HI and DMSO vehicle groups, p-JNK protein was reduced (P<0.01), nuclear protein of FOXO3a was also reduced (P<0.01), cytoplasmic protein increased (P<0.01), and Bim and CC3 proteins decreased (P<0.01) in the AS601245-treated group 24 hours after HI. TUNEL positive cells were reduced in the AS601245-treated rats compared with the HI and DMSO vehicle groups 24 hours after HI (P<0.01). CONCLUSIONS: JNK activity increases in the neonatal rat brain with HI damage. JNK activity inhibition can inhibit FOXO3a translocation from cytoplasm to nucleus and downregulate the levels of pro-apoptotic proteins Bim and CC3, leading to the reduction of neuronal apoptosis.

Calorie restriction inhibits ovarian follicle development and follicle loss through activating SIRT1 signaling in mice.

BACKGROUND: Silent information regulator 2 related enzyme 1 (SIRT1) is one of the key factors in the mechanism of calorie restriction (CR) extending lifespan of animals. The aim of the study is to investigate if CR prolongs ovarian lifespan in mice through activating SIRT1 signaling. METHODS: In the present study, 21 female C57BL/6 mice were divided into three groups: the control (n = 7), CR (n = 7), and SRT1720 (n = 7) groups. After the 26-week treatment, the number of ovarian follicles at each stage was counted, and Western blot was performed. RESULTS: The number of surviving follicles in ovaries of the SRT1720 group was less than that of the CR group but more than that of the normal control (NC) group. The number of atretic follicles in the ovaries of the SRT1720 group was similar to that of the CR group but less than that of the NC group. The number of primordial follicles in the ovaries of the SRT1720 group was less than that of the CR group but more than that of the NC group. The numbers of primary follicles, secondary follicles, antral follicles, and corpora lutea in the SRT1720 group were similar to those in the CR group. Western blot analysis showed that the expression of SIRT1, SIRT6, FOXO3a, and NRF1 proteins was upregulated, and p53 was downregulated in both the CR group and the SRT1720 group compared to the control group. CONCLUSIONS: Our results indicate that CR inhibits the activation of primordial follicles and development of follicles at different stages, thus preserving the reserve of follicle pool (at least partly) through activating SIRT1 signaling.

Caloric restriction promotes the reserve of follicle pool in adult female rats by inhibiting the activation of mammalian target of rapamycin signaling.

Caloric restriction (CR) is known to increase the number of primordial follicles and prolong the reproductive life span. However, how CR modulates follicular development is not well understood. In the present study, we examined the effects of CR on follicular development in rats and investigated the underlying mechanism. After 10 weeks of CR or high-fat diet, ovarian follicles at different developmental stages were examined by histological analysis. Plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estrogen (ESG) were measured, and the levels of mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K), and phosphorylated p70S6K in the ovary were detected by Western blot. The results showed that the reserve of follicle pool in CR rats was increased, accompanied by decreased level of phosphorylated p70S6K in the ovary, and decreased serum LH, FSH, and ESG levels. Taken together, these results suggest that CR may suppress ovarian follicular development and enhance the follicle pool reserve by inhibiting mTOR signaling.

SIRT1 activator (SRT1720) improves the follicle reserve and prolongs the ovarian lifespan of diet-induced obesity in female mice via activating SIRT1 and suppressing mTOR signaling.

BACKGROUND: The prevalence of obesity is increasing worldwide and significantly affects fertility and reproduction in both men and women. Our recent study has shown that excess body fat accelerates ovarian follicle development and follicle loss in rats. The aim of the present study is to explore the effect of SIRT1 activator SRT1720 on the reserve of ovarian follicle pool and ovarian lifespan of obese mice and the underlying mechanism associated with SIRT1 and mTOR signaling. METHODS: Adult female Kunming mice (n = 36) were randomly divided into three groups: the normal control (NC) group (n = 8), the caloric restriction (CR) group (fed 70% food of the NC group, n = 8) and the high-fat diet (HF) group (fed a rodent chow containing 20% fat, n = 20). After 4 months, the HF mice were further randomly divided into three groups: the control high-fat diet (CHF, n = 8) group (treated every day with an intraperitoneal injection of vehicle), the SRT1720 (SRT, n = 6) group (treated every other day with an intraperitoneal injection of SRT1720 (50 mg/kg)), the SRT1720 and nicotinamide (NAM, n = 6) group (treated every other day with an intraperitoneal injection of SRT1720 (50 mg/kg) and every day with an intraperitoneal injection of nicotinamide (100 mg/kg)). After 6 weeks of treatment, ovaries were harvested for histological and Western blotting analyses. RESULTS: The body weight, ovary weight and visceral fat in the SRT group were significantly lower than those in the CHF group at the end of treatment. Histological analysis showed that the SRT mice had significantly greater number and percentage of primordial follicles, but lower number and percentage of corpora lutea and atretic follicles than the CHF mice and NAM mice. Western blot analysis demonstrated that the levels of SIRT1, SIRT6, FOXO3a and NRF-1 protein expression significantly increased in the ovaries of SRT mice, whereas those of mTORC1, p-mTOR, p-p70S6K, NFκB and p53 decreased compared to the CHF and NAM mice. CONCLUSIONS: Our study suggests that SRT1720 may improve the follicle pool reserve in HF diet-induced obese female mice via activating SIRT1 signaling and suppressing mTOR signaling, thus extending the ovarian lifespan.

Obesity accelerates ovarian follicle development and follicle loss in rats.

OBJECTIVE: Studies have shown that excess body fat negatively affects reproductive functions in females. However, whether obesity affects the ovarian follicle development and ovarian lifespan and the underlying mechanism has not been well elucidated. The aim of the present study was to investigate the association between obesity and ovarian follicle development. METHODS: Adult female Sprague-Dawley rats (n = 36) were randomly divided into three groups: the normal control (NC) group, the caloric restriction (CR) group (fed 70% food of the NC group) and the high-fat diet (HF) group. They were maintained on these regimens for 18 weeks. RESULTS: The body weight, ovary weight and visceral fat in the HF group were significantly higher than those in the NC group and the CR group at the end of treatment. Histological analysis showed that the HF rats had significantly less number and percentage of primordial follicles, but greater number and percentage of developing and atretic follicles than the NC rats and CR rats. Western blot analysis demonstrated that the level of mTORC1 and p-S6K1 proteins significantly increased in the ovaries of HF rats, whereas that of SIRT1, SIRT6, FOXO3a and NRF-1 decreased compared to the NC rats. In contrast, the expression of mTORC1 and p-S6K1 dramatically declined, while that of SIRT1, SIRT6, FOXO3a and NRF1 increased in the ovaries of CR rats. CONCLUSIONS: Our study suggests that the HF diet induced obesity may accelerate the ovarian follicle development and rate of follicle loss through activating mTOR and suppressing SIRT1 signaling, thus leading to POF, and that CR may inhibit the activation of primordial follicles, follicular development and loss, thus extending the ovarian lifespan through suppressing mTOR and activating SIRT1 signaling.

Rapamycin preserves the follicle pool reserve and prolongs the ovarian lifespan of female rats via modulating mTOR activation and sirtuin expression.

To maintain the normal length of female reproductive life, the majority of primordial follicles must be maintained in a quiescent state for later use. In this study, we aimed to study the effects of rapamycin on primordial follicle development and investigate the role of mTOR and sirtuin signaling. Rats were treated every other day with an intraperitoneal injection of rapamycin (5mg/kg) or vehicle. After 10weeks of treatment, ovaries were harvested for hematoxylin and eosin (HE) staining, and analysis by immunohistochemistry and Western blotting. HE staining showed that the number and percentage of primordial follicles in the rapamycin-treated group were twice the control group (P<0.001). Immunohistochemical analysis showed that mTOR and phosphorylated-p70S6K were extensively expressed in surviving follicles with strong staining observed in the cytoplasm of the oocyte. Western blotting showed decreased expression of phosphorylated mTOR and phosphorylated p70S6K in the rapamycin-treated group, and increased the expression of both SIRT1 and SIRT6 compared to the control group (P<0.05). Taken together, these results suggest that rapamycin may inhibit the transition from primordial to developing follicles and preserve the follicle pool reserve, thus extending the ovarian lifespan of female rats via the modulation of mTOR and sirtuin signalings.

The effects of caloric restriction and a high-fat diet on ovarian lifespan and the expression of SIRT1 and SIRT6 proteins in rats.

BACKGROUND AND AIMS: Caloric restriction (CR) extends mammals' lifespans and suppresses ovary development. Sirtuins are involved in these mechanisms. If, and to what extent CR affects ovarian lifespan and follicle development is largely unknown. We investigated the effects of moderate and severe caloric restriction compared with a high-fat dietary regimen on ovarian follicle reserves in rats. METHODS: Female Sprague-Dawley rats (n=48) randomly divided into four groups including normal control (NC), 25% caloric restriction (MCR), 45% CR (SCR) and high-fat diet (HF) were maintained on these regimens for 2 months. RESULTS: Histological analysis showed that both the 25 and 45% CR rats had a significantly higher percentage of primordial follicles and a larger number of healthy follicles than the NC rats, whereas the HF rats did not differ significantly from the NC rats. Immunohistochemical analysis revealed that SIRT1 and SIRT6 proteins were present in the nucleus and cytoplasm of the oocytes. The 25% CR diet increased the expression of both SIRT1 and SIRT6 in the ovary, whereas the 45% CR and HF diets caused a decrease in SIRT1 expression. The level of SIRT6 protein did not change with the 45% CR diet, and it appeared slightly lower in the HF than in the NC groups. CONCLUSIONS: Caloric restriction may inhibit the transition from primordial to developing follicles and extend the entire growth phase of a follicle to preserve the reserve of germ cells. SIRT1 and SIRT6 are both associated with these effects.

Caloric restriction promotes the reproductive capacity of female rats via modulating the level of insulin-like growth factor-1 (IGF-1).

The insulin-like growth factor-1 (IGF-1) plays an important role in the regulation of reproductive function. In the present study, we examined the effects of caloric restriction (CR) on the reproductive lifespan in rats and investigated the potential role of IGF-1. After 10 weeks of treatment, we determined the distribution of the ovarian follicles at various stages and measured the plasma level of IGF-1, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estrogen (ESG). Our results show that IGF-1 level was decreased after CR and correlated with the decrease in the levels of LH, FSH and ESG. Moreover, a higher percentage of primordial follicles and surviving follicles was observed in CR rats than in control rats (P<0.05). Immunohistochemical analysis showed that IGF-1 was extensively expressed in the cytoplasm of granulosa cells in the surviving follicles at different stages but not in the atretic follicles. Taken together, these results suggest that caloric restriction promotes the reproductive capacity of female rats via modulating the level of IGF-1, which then regulate pituitary gonadotrope cells to reduce the release of LH, FSH and ESG, and modulate follicular development.

Effects of plant polyphenols on ovarian follicular reserve in aging rats.

The pool of ovarian primordial follicles is established during embryonic development or at birth. During the development from primordial to primary, secondary, and antral follicles, only a small portion of follicles can mature and successfully ovulate; the others are destined to degenerate through apoptotic or atretic loss. As aging advances, females ultimately enter the cessation phase of the estrous cycle and are no longer capable of fertilization. The presumption is that if we can slow down the process of folliculogenesis or decrease follicle loss, females may have a larger ovarian follicular reserve and a longer reproductive lifespan. In our study, rats underwent intragastric administration with tea polyphenols, quercetin (meletin), genistein, or resveratrol, once a day for 4 months (from age 12 to 15 months), to test whether they have positive effects on follicular reserve or ovarian functions. The results showed that rats treated with tea polyphenols (27.8 +/- 3.2) and quercetin (36.5 +/- 4.1) had a comparable number of healthy follicles to those of controls (26.9 +/- 3.8), although significantly fewer atretic follicles were observed in the tea polyphenol group (43.4 +/- 5.9 vs 79.7 +/- 7.5; p < 0.001). Remarkably, both genistein- and resveratrol-treated rats had more healthy follicles (respectively, 42.8 +/- 3.9, p < 0.05; and 51.9 +/- 6.4, p < 0.001) and fewer atretic follicles (respectively, 58.4 +/- 8.0, p < 0.05; and 51.0 +/- 6.2, p < 0.01) than controls. These results indicate that genistein and resveratrol can increase the ovarian follicular reserve and prolong the ovarian lifespan in rats, and their positive effects may be not only due to their intervention in the transition from primordial to primary follicle, but also due to the inhibiting effect on follicular atresia.

Evidence that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary.

Previous studies have proposed that the forkhead transcription factor FOXO3a is involved in cell cycle arrest and apoptosis and that it may also repress follicular development by inducing cell cycle arrest in ovaries. We have recently demonstrated that FOXO3a induces oocyte apoptosis of neonatal rat ovaries under in vitro conditions. In the present study, we evaluated the role of FOXO3a in oocyte apoptosis under in vivo conditions. Ovaries from rats were obtained from newborns on postnatal day (PD) 1, 2, 3, and 4. TUNEL assay results showed that oocyte apoptosis occurred mainly on PD 1 and 2. Immunohistochemical staining of FOXO3a, Bim, Fas ligand (FasL), p27KIP1, caspase-8, and caspase-3 showed that they were all expressed mainly in naked oocytes on PD 1 and 2. The percentage of positive FOXO3a staining of oocytes reached peak levels in the ovaries of 2-day-old rats, which was consistent with the rate of the apoptotic profiles determined by TUNEL. The percentage between TUNEL-positive and FOXO3a-positive oocytes in the nucleus showed no statistical differences within the 4-day-old rat ovaries. Furthermore, the positive oocyte percentage of the target factors of FOXO3a (Bim, p27KIP1, and FasL) and pro-apoptotic proteins (caspase-3 and caspase-8) also reached peak levels in the ovaries of 2-day-old rats, which was similar to the rate of FOXO3a-positive oocytes. These results suggest that FOXO3a in the oocyte nucleus is involved in oocyte apoptosis; that is, FOXO3a-positive oocytes may be the apoptotic cells. To verify this, rat oocytes were subjected to TUNEL and immunofluorescent double-labeling assays. We found that TUNEL-positive cells were also FOXO3a-, Bim-, or FasL-positive. To identify the downstream target of FOXO3a, double immunofluorescent staining with antibodies to Bim and FasL was performed. We found that FOXO3a-positive cells were also Bim- and FasL-positive. We conclude that the overexpression of FOXO3a in the oocyte nucleus of neonatal rat ovaries may play an important role in the apoptosis of naked oocytes, and that Bim, FasL, and p27KIP1 are the key downstream factors of FOXO3a.

Effects of genistein on ovarian follicular development and ovarian life span in rats.

Recently, studies reported that neonatal genistein treatment inhibited breakdown of oocyte nests and increased oocyte survival, resulting in multi-oocyte survival in adult mice. However, whether the inhibition effect in ovarian follicular development exists also in other stages during ovarian development (e.g. adult or climacteric) is unknown. So far, few studies have investigated the effect of genistein in adult or pre-menopausal ovarian follicular development and follicular reserves. We investigated ovarian follicular development in 4-month and 15-month-old rats after 4 weeks and 4 months treatment with genistein in a dose of 160 mg/kg d. Genistein-treated rats obtained a higher percentage of primordial follicles by 4 months of age and a greater number of surviving follicles at 15 months of age compared to a control group (P<0.05). In addition, vaginal cytology showed that age-dependent cessation of regular estrus was delayed for 2 months in the genistein-treated group than control group. These results suggest that genistein alters rat ovarian follicular development and increases the number of surviving follicles, which may prolong ovarian reproductive life.