Protective effects and mechanisms investigation of Kuntai capsule on the ovarian function of a novel model with accelerated aging ovaries.

ETHNOPHARMACOLOGICAL RELEVANCE: Kuntai capsule, a traditional Chinese medicine, has been widely used for the clinical treatment of menopausal syndrome. However, its mechanisms remain poorly understood. Considering that aging ovaries are the primary cause of menopause, this study was designed to investigate the effects and mechanisms of Kuntai capsule on ovarian function in a novel mice model with accelerated aging ovaries. MATERIALS AND METHODS: Seventy-five female C57BL/6 mice were chosen for this study. Fifteen of the mice were separated into the normal control group (NC). The remaining sixty were used to establish the novel accelerated aging ovary model by superovulation and oxidative stress and then by randomly dividing the mice into four equal groups. One group was considered the model group (Mod). The other three groups were treated with low (0.4g/kg), middle (0.8g/kg) and high (1.6g/kg) doses of Kuntai capsule intragastrically every day for 4 weeks. During the treatment, the body weight and fur condition of all mice were recorded. All the mice were forced to swim to record their exhaustive swimming time (EST), which measures their strength. Mice were then sacrificed for sampling. Ovarian reserve was evaluated using follicle counts and AMH expression. Ovarian function was evaluated using estrous cycle, sex hormone level and litter experiments. Ovarian follicles were categorized and counted to estimate ovarian reserve, and ovarian histologic sections were stained for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to detect apoptotic cells. The ultrastructure of ovarian cells was observed using transmission electron microscopy. Western blotting was used to measure expression of Bax, Bcl2, AMH and SOD2 protein. RESULTS: Compared with the NC GROUP, the Mod group clearly displayed worse fur condition and ovarian function. These situations showed some improvement after Kuntai capsule treatment. Specifically, the fur condition and the EST of the Kuntai capsule groups were superior to the fur condition and EST of the Mod group. In cases of damaged ovarian function, Kuntai capsule can regulate the estrous cycles, increase hormone secretion and fertility and significantly decrease atretic follicles. The transmission electron microscopy results revealed that Kuntai capsule rescued the ovarian ultrastructure of mice. TUNEL staining confirmed that the apoptotic cells were reduced after Kuntai capsule treatment. Western blotting revealed that Kuntai capsule can increase AMH, SOD2, and Bcl2 protein expression and decrease Bax expression. CONCLUSIONS: Kuntai capsule may improve damaged ovarian function, which may be related to its antioxidant and anti-apoptosis effects.

Supplementation of culture media with vitamin E improves mouse antral follicle maturation and embryo development from vitrified ovarian tissue.

AIM: The aim of this study was to evaluate the effects of preventive vitamin E (α-tocopherol) on antral follicle development and embryogenesis of oocytes obtained after vitrification of mouse ovarian tissue. METHODS: Female Balb/c mice were killed by cervical dislocation after the injection of pregnant mare's serum gonadotrophin (10 IU) and their ovaries were randomly divided into three groups: control or non-vitrified (n = 10), vitrification 1 (5, 10% ethylene glycol + 5, 10% dimethylsulfoxide) (n = 15), and vitrification 2 (10, 15% ethylene glycol + 10, 15% dimethylsulfoxide) (n = 15) with ascending concentration of cryoprotectants. After toxicity tests and vitrification-warming, mechanically isolated antral follicles were cultured in α-minimum essential medium, which was supplemented with or without α-tocopherol (100 µM). The follicular maturation rates and embryo development were collected and assessed. Also, the viability, morphology and ultrastructure of derived antral follicles from vitrified ovaries were analyzed. RESULTS: The morphology and ultrastructure of follicles were well preserved in the vitrified groups and α-tocopherol supplementation of culture media significantly increased the proportion of oocytes that reached metaphase II blastocyst rates compared to non-α-tocopherol supplemented media (P < 0.01). CONCLUSION: Vitamin E improves in vitro maturation rates and blastocyst rates of oocytes that are isolated from vitrified ovarian tissue.

The base medium affects ultrastructure and survival of bovine preantral follicles cultured in vitro.

The aim of this study was to determine the effectiveness of minimum essential medium alpha modification (α-MEM), tissue culture medium 199 (TCM-199), and McCoy's medium (McCoy's) on IVC of preantral follicles included in the bovine ovarian cortex (in situ). Bovine ovarian fragments were cultured in α-MEM, TCM-199, or McCoy supplemented ((+)) with glutamine, insulin, transferrin, selenium, ascorbic acid, BSA, penicillin, streptomycin, and HEPES buffer in 24-well plates, at 37 °C and 5% CO2 for 1 or 7 days. The morphology of follicles, normal, primordial and development (primary and secondary), as well as viability and morphometric variables of follicles and oocytes were assessed. The morphology and morphometry of preantral follicles were analyzed by ANOVA followed by the Tukey and Dunnett tests, and viability variables were determined by the chi-square test. The results showed that TCM-199(+) reduced significantly (P < 0.05) the percentage of morphologically normal and viable follicles after 7 days of culture compared to the control. Similar results were observed in McCoy(+), in which the percentage of viable follicles after 7 days of culture was significantly lower (P < 0.05) than in the control. However, it was similar (P > 0.05) between α-MEM(+) and TCM-199(+). Moreover, follicular diameters in McCoy(+) and TCM-199(+) were significantly smaller (P < 0.05) than in control and α-MEM(+) after 7 days of culture. In addition, the ultrastructure of preantral follicles was similar between the control and α-MEM(+) after 7 days of culture. In conclusion, α-MEM(+) showed to be the most effective medium to preserve morphology, morphometry and ultrastructure of bovine preantral follicles, ensuring their viability and growth after in situ culture.

Quantitative elemental analysis of bovine ovarian follicles using X-ray fluorescence imaging.

The X-ray Fluorescence Micro-spectroscopy (XFM) beamline at the Australian Synchrotron was used to image 97 follicle histological sections from 45 different bovine ovaries focusing on healthy antral follicles ranging from small (<4 mm) up to preovulatory sizes (>16 mm) and on antral follicles undergoing atresia. This analysis identified five elements (Cu, Fe, Zn, Se and Br) consistently present within the ovarian tissue with Fe, Zn and Se localised to specific structures. GeoPIXE v6.4g was subsequently used to extract quantitative information pertaining to the elemental concentrations surrounding each of these follicles. Statistical analysis suggested that significant elemental differences were evident between follicle groups sorted according to their health status (Fe and Br), and their size (Se). Se appeared to be the element which most greatly distinguished large antral follicles from smaller counterparts. The ability to use synchrotron radiation to measure trace element distributions in bovine follicles at such high resolutions could have a significant impact on understanding the mechanisms of follicular development. This research is intended to form a baseline study of healthy cycling ovaries which could later be extended to disease states, thereby improving our current understanding of infertility and endocrine diseases involving the ovary.

Evaluation of apoptosis in long-term culture of vitrified mouse whole ovaries.

The purpose of the present study was to investigate the development of follicles and incidence of apoptosis in vitrified neonatal mouse ovaries cultured in vitro in the presence of leukemia inhibitory factor (LIF). The vitrified and non-vitrified ovaries of 1-week-old mouse were cultured in the presence or absence of LIF for 7 days. At the beginning and at the end of culture period in each ovary of all groups of study the mean area and the development of ovarian follicles were analyzed; moreover, the incidence of apoptosis was assessed by transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method, DNA laddering and caspase-3/7 activity technique. The hormonal assay was done on the conditioned media collected during culture period. The proportion of preantral follicles and the levels of hormones increased in all cultured groups and it was significantly higher in LIF treated groups than in their control (P<0.001). The ultrastructural characteristics of cell death, DNA fragmentation and TUNEL positive signals were prominent in vitrified cultured ovaries. The level of caspase-3/7 activity was higher in vitrified cultured ovaries. LIF supplementation during 7 days of culture appeared to significantly preserve cells function and increase the follicular development of both vitrified and non-vitrified ovaries.

Interaction between melatonin and follicle-stimulating hormone promotes in vitro development of caprine preantral follicles.

The aim of this study was to investigate the effects of melatonin and follicle-stimulating hormone (FSH) on the in vitro culture of goat preantral follicles. Ovarian fragments were cultured for 7 d in α-minimum essential medium (α-MEM(+)) containing melatonin (100, 250, 500, or 1,000 pM), FSH (50 ng/mL), or a combination of the 2 hormones and further analyzed by histology and transmission electron and fluorescent microscopy. The results showed that after 7 d of culture, tissues cultured in α-MEM(+) alone or supplemented with FSH alone, melatonin (500 and 1,000 pM), or the combination of FSH and melatonin (1,000 pM) maintained percentages of normal preantral follicles similar to the fresh control. In contrast to the noncultured tissues, the percentage of developing follicles was increased under all culture conditions after 7 d (P < 0.05). The addition of 1,000 pM melatonin associated with FSH to the culture medium increased follicular and oocyte diameters compared with α-MEM(+) alone after 7 d of culture (P < 0.05). Ultrastructural and fluorescent analyses confirmed the integrity of follicles cultured with 1,000 pM of melatonin plus FSH for 7 d. In conclusion, this study demonstrated that the interaction between melatonin and FSH maintains ultrastructural integrity and stimulates further growth of cultured caprine preantral follicles.

The effects of insulin and follicle-simulating hormone (FSH) during in vitro development of ovarian goat preantral follicles and the relative mRNA expression for insulin and FSH receptors and cytochrome P450 aromatase in cultured follicles.

The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 µg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles.

Effects of ascorbic acid on in vitro culture of bovine preantral follicles.

The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine their effects on growth activation and viability of early-stage follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed (control) and the other fragments were cultured in minimum essential medium (MEM) supplemented or not with various doses of ascorbic acid. Ovarian tissue was processed for histology, transmission electron microscopy (TEM) and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Compared with control fragments, the percentage of primordial follicles was reduced (p < 0.05) and the percentage of growing follicles had increased (p < 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (p < 0.05), but not when cultures were supplemented with 25, 50 or 100 µg/ml of ascorbic acid. Ultrastructural and immunohistochemical analysis of 8 day cultured ovarian cortical fragments, however, showed the integrity and viability of follicles only when fragments were cultured in presence of 50 µg/ml of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 µg/ml not only stimulates the activation of 8 day in vitro cultured cattle primordial follicles and subsequent growth of activated follicles, but also safeguards the viability of these early-stage follicles.

Healthy early preantral follicle can be obtained in a culture of frozen-thawed human ovarian tissue of 32 weeks.

The objective of this study was to report morphological and functional evidence of a well-preserved preantral follicle recovered from human frozen-thawed ovarian tissue in a long-term culture. The tissue was originally obtained from a 26-year-old woman with breast cancer. The ovarian cortex was collected by laparoscopy and frozen/thawed and cultured for 32 weeks in minimum essential medium alpha-MEM, supplemented with insulin transferrine selenite (ITS), human serum (HS), antibiotics, follicle-stimulating hormone (FSH). and N-acetyl cysteine (NAC). Thawed tissue samples were examined by light microscopy (LM), transmission electron microscopy (TEM), and real-time RT-PCR. LM examination of cortical pieces after 32 weeks of culture showed a healthy early preantral follicle; TEM and real-time PCR confirmed its good state of preservation. The synergy in action of NAC and FSH plays an important role in follicle growth of ovarian tissue cultures. For the first time a well-preserved preantral follicle was found in a culture of frozen-thawed human ovarian tissue.

Effects of p-nonylphenol and resveratrol on body and organ weight and in vivo fertility of outbred CD-1 mice.

The aim of this study was to analyse the multigenerational effects of para-nonylphenol (NP) and resveratrol (RES) on the body weight, organ weight and reproductive fitness of outbred CD-1 mice. The data indicate that in male mice, NP had an effect on the weight of selected reproductive organs and the kidneys in the parental (P) generation males. Effects on selected reproductive organs, the liver and kidneys in the F1-generation males were also seen. In females, effects of NP on body weight and kidney weight were seen in the P generation, but no effects on any measured parameter were seen in the F1 generation. RES had no effect on body weight but did have some effect on selected male and female reproductive organs in the P generation. RES altered the spleen and liver weights of P-generation males and the kidney weight of F1-generation males. Acrosomal integrity (using a monoclonal antibody against intra-acrosomal sperm proteins) was assessed for both generations of NP- and RES-treated mice. A significant reduction in acrosomal integrity was seen in both generations of NP-treated, but not in RES-treated, mice. Fewer offspring were observed in the second litter of the F2 generation of mice treated with NP; no similar effect was seen in RES-treated mice. The litter sex ratio was not different from controls. Unlike RES, NP had a negative effect on spermatogenesis and sperm quality with a resultant impact on in vivo fertility.

Targeted disruption of the aromatase P450 gene (Cyp19) in mice and their ovarian and uterine responses to 17beta-oestradiol.

Aromatase P450 (CYP19) is an enzyme catalysing the conversion of androgens into oestrogens. We generated mice lacking aromatase activity (ArKO) by targeted disruption of Cyp19 and report the characteristic features of the ArKO ovaries and uteri as revealed by histological and biochemical analyses. ArKO females were totally infertile but there were as many developing follicles in their ovaries at 8 weeks of age as in wild-type ovaries. Nevertheless, no typical corpus luteum was observed in the ArKO ovaries. Electron microscopy revealed the presence of well-developed smooth endoplasmic reticulum, few lipid droplets and mitochondria with less organized tubular structures in the ArKO luteinized interstitial cells. These ultrastructural features were different from those of the wild-type interstitial cells, where there are many lipid droplets and mitochondria with well-developed tubular structures, characteristic of steroid-producing cells. When ArKO mice were supplemented with 17beta-oestradiol (E(2); 15 microg/mouse) every fourth day from 4 weeks of age for 1 month, increased numbers of follicles were observed in the ovaries as compared with those of untreated ArKO mice, although no typical corpus luteum was detectable. Ultrastructural analysis revealed the disappearance of the accumulated smooth endoplasmic reticulum in the luteinized interstitial cells after E(2 )supplementation. Transcripts of pro-apoptotic genes such as p53 and Bax genes were markedly elevated in the ArKO ovaries as compared with those of wild-type mice. Although E(2) supplementation did not cause suppression of the elevated expression of p53 and Bax mRNAs, it caused marked enhancement of expression levels of lactoferrin and progesterone receptor mRNAs in the uteri as well as increases in uterine wet weight. At 8 months of age, ArKO mice developed haemorrhages in the ovaries, in which follicles were nearly depleted, while age-matched wild-type females still had many ovarian follicles. Furthermore, macrophage-like cells were occasionally observed in the ArKO ovarian follicles. These results suggested that targeted disruption of Cyp19 caused anovulation and precocious depletion of ovarian follicles. Additionally, analysis of mice supplemented with E(2) demonstrated that E(2) apparently supports development of ovarian follicles, although it did not restore the defect in ovulation.

Effects of composite root extract on histological structures of graffian follicle and endometrial epithelium in albino rat.

This study attempted in vivo testing of a group of plant root extracts in composite form on the reproductive organs of the female albino rat. These roots in composite form have been used by the folk women of Assam to prevent pregnancy. Firsthand knowledge revealed that the dry powder of these roots in composite form can induce sterility in women temporarily (reversible) or permanently which is dose-dependent when taken through oral route. The study revealed that administration of ethanolic crude extract of these composite roots in a dose of 1000 mg/kg/day, consecutively for 12 days, can modulate histological changes in the structures of ovary and uterus. This dose has previously been detected as the threshold dose to induce sterility (reversible) in albino rat. The ovarian follicle showed structural disparity in thecal cells and granulosa cells, and formation of zona pellucida. In the uterus, the endometrial epithelium on the luminal surface showed pseudostratification, vacuolation of the cells, and irregular desquamation from the stroma. Infiltration of a large number of polymorphs in the endometrial stroma and necrosis of endometrial gland tissues indicated structural and functional aberrations of the uterus.