The effect of icariin on autoimmune premature ovarian insufficiency via modulation of Nrf2/HO-1/Sirt1 pathway in mice

Primary ovarian insufficiency (POI) is a common gynecological disease. Autoimmunity is a common cause of POI. Icariin (ICA) plays a therapeutic role in many autoimmune diseases. This study aims to investigate the effect of ICA on autoimmune POI mice and its effect on immune regulation. Sixty-three female BALB/c mice were randomized into three groups (control, POI, POI + ICA). POI and POI + ICA group were hypodermically injected with zona pellucida three peptides (pZP3) to induce autoimmune POI. Then the POI + ICA group was gavaged with ICA. A vaginal smear was to observe estrous cycles, hematoxylin-eosin staining was to count follicles. Enzyme-linked immunosorbent analysis determined serum FSH, LH, AMH, and anti-zona pellucida antibody (AZPAb) levels. In addition, flow cytometry detected the expression of Th1 cells and Treg cells, and Western blot was used to detect the expression of Nuclear factor E2 related factor 2(Nrf2), heme oxygenase-1 (HO-1), and Sirtuin-1 (Sirt1) proteins. pZP3 treatment decreased serum AMH levels and increased FSH, LH, and AZPAb levels. Additionally, decreases in the number of healthy follicles at all stages and an increase in the number of atretic follicles. Abnormal ovarian structure and an arrested estrous cycle were also noted. However, ICA rescued POI through up-regulating Nrf2, HO-1, and Sirt1 expressions and up-regulating Treg expressions. ICA treatment improved the structure of the injured ovarian and its function in autoimmune POI mice. The mechanism is achieved by increasing the expression of Nrf2/HO-1/Sirt1 pathway in the ovary and increasing Treg cells' expression.     

Brca1 Mutations Enhance Mouse Reproductive Functions by Increasing Responsiveness to Male-Derived Scent

We compared the gene expression profiles of ovarian granulosa cells harboring either mutant or wild type Brca1 to follow up on our earlier observation that absence of a functional Brca1 in these important regulators of menstrual/estrous cycle progression leads to prolongation of the pre-ovulatory phase of the estrous cycle and to increased basal levels of circulating estradiol. Here we show that ovarian granulosa cells from mice carrying a conditional Brca1 gene knockout express substantially higher levels of olfactory receptor mRNA than granulosa cells from wild type littermates. This led us to hypothesize that reproductive functions in mutant female mice might be more sensitive to male-derived scent than in wild type female mice. Indeed, it is well established that isolation from males leads to complete cessation of mouse estrous cycle activity while exposure to olfactory receptor ligands present in male urine leads to resumption of such activity. We found that Brca1 ^-/- female mice rendered anovulatory by unisexual isolation resumed ovulatory activity more rapidly than their wild type littermates when exposed to bedding from cages where males had been housed. The prime mediator of this increased responsiveness appears to be the ovary and not olfactory neurons. This conclusion is supported by the fact that wild type mice in which endogenous ovaries had been replaced by Brca1-deficient ovarian transplants responded to male-derived scent more robustly than mutant mice in which ovaries had been replaced by wild type ovarian transplants. Our findings not only have important implications for our understanding of the influence of olfactory signals on reproductive functions, but also provide insights into mechanisms whereby genetic risk factors for breast and extra uterine Müllerian carcinomas may influence menstrual activity in human, which is itself an independent risk factor for these cancers.     

CPEB3 deficiency in mice affect ovarian follicle development and causes premature ovarian insufficiency

Premature ovarian insufficiency (POI) is a heterogeneous and multifactorial disorder. In recent years, there has been an increasing interest in research on the pathogenesis and treatment of POI, owing to the implementation of the second-child policy in China. Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that can bind to specific RNA sequences. CPEB3 can bind to and affect the expression, cellular location, and stability of target RNAs. Cpeb3 is highly expressed in the ovary; however, its functions remain unknown. In this study, Cpeb3-mutant mice were used to characterize the physiological functions of CPEB3. Cpeb3-mutant female mice manifested signs of gradual loss of ovarian follicles, ovarian follicle development arrest, increased follicle atresia, and subfertility with a phenotype analogous to POI in women. Further analysis showed that granulosa cell proliferation was inhibited and apoptosis was markedly increased in Cpeb3-mutant ovaries. In addition, the expression of Gdf9, a potential target of CPEB3, was decreased in Cpeb3-mutant ovaries and oocytes. Altogether, these results reveal that CPEB3 is essential for ovarian follicle development and female fertility as it regulates the expression of Gdf9 in oocytes, disruption of which leads to impaired ovarian follicle development and POI.Subject terms: RNA-binding proteins, Infertility     

Laser capture microdissection of gonads from juvenile zebrafish

Background

Until recently, the limit of spatial resolution of ultrasound systems has prevented characterization of structures <1 mm. Hence, the study of ovarian follicular development in rodents has been based on one-time histological examination of excised tissues; i.e., longitudinal study of day-to-day ovarian changes has not been possible in mice and rats. The objective was to establish an ultrasonographic approach to study follicular and luteal dynamics in mice and rats.

Methods

Experiment 1 was a pilot study to develop methods of immobilization (physical restraint vs. general anesthesia) and determine technical factors affecting ovarian images using ultrasound bio-microscopy in rats vs. mice. The hair coat was removed over the thoraco-lumber area using depilation cream, and a highly viscous acoustic gel was applied while the animals were maintained in sternal recumbency. In Experiment 2, changes in ovarian structures during the estrous cycle were monitored by twice daily ultrasonography in 10 mice for 2 estrous cycles.

Results

Ovarian images were not distinct in rats due to attenuation of ultrasound waves. Physical restraint, without general anesthesia, was insufficient for immobilization in mice. By placing the transducer face over the dorsal flank, the kidney was visualized initially as a point of reference. A routine of moving the transducer a few millimetres caudo-laterally from the kidney was established to quickly and consistently localize the ovaries; the total time to scan both ovaries in a mouse was about 10 minutes. By comparing vaginal cytology with non-anesthetized controls, repeated exposure to anesthesia did not affect the estrous cycle. Temporal changes in the number of follicles in 3 different size categories support the hypothesis that follicles ≥ 20 microns develop in a wave-like fashion.

Conclusion

The mouse is a suitable model for the study of ovarian dynamics using transcutaneous ultrasound bio-microscopy. Repeated general anesthesia for examination had no apparent effect on the estrous cycle, and preliminary results revealed a wave-like pattern of ovarian follicle development in mice.     

Crybb2 deficiency impairs fertility in female mice

Beta-B2-crystallin (CRYBB2), encoded by Crybb2 gene, is a major protein in the mammalian eye lens that plays an important role in maintaining the transparency of the ocular lens. However, CRYBB2 also plays important roles in many extra-lenticular tissues and organs such as the retina, brain and testis. Our previous studies demonstrated that male Crybb2 deficient (Crybb2^−/−) mice have reduced fertility compared with wild-type (WT) mice, while female Crybb2^−/− mice exhibited reduced ovary weights and shorter estrous cycle percentages. Here we specifically investigated the role of CRYBB2 in the female reproductive system. Our studies revealed that ovaries from female Crybb2^−/− mice exhibited significantly reduced numbers of primordial, secondary and pre-ovulatory follicles when compared with WT mice, while the rate of atretic follicles was also increased. Additionally, fewer eggs were collected from the oviduct of Crybb2^−/− female mice after superovulation. Estrogen levels were higher in the metestrus and diestrus cycles of female Crybb2^−/− mice, while progesterone levels were lower in diestrus cycles. Furthermore, the expression of survival and cell cycle genes, Bcl-2, Cdk4 and Ccnd2, were significantly decreased in granulosa cells isolated from female Crybb2^−/− mice, consistent with the predominant expression of CRYBB2 in ovarian granulosa cells. Our results reveal a critical role for CRYBB2 in female fertility and specific effects on the proliferation and survival status of ovarian granulosa cells.     

Loss of abnormal spindle-like,microcephaly-associated (Aspm) disrupts female folliculogenesis in mice during maturation and aging

The abnormal spindle-like, microcephaly-associated (ASPM) gene is a causative gene of autosomal recessive primary microcephaly (MCPH) 5 in humans, which is characterized by a reduction in brain volume. It was previously reported that truncated Aspm proteins in transgenic mice caused major defects in the germline, a severe reduction in ovary weight and the number of follicles accompanied by reduced fertility. However; it remains unknown whether a loss of Aspm induces abnormal ovarian function, resulting in female infertility. In order to assess the ovary function, we examined vaginal smear cytology from the age of 7 weeks to 100 weeks in CAG-mediated Cre-loxP conditional Aspm^-/- knockout mice and control female mice. In addition, we evaluated the ovarian size, fibrosis ratio and the number of follicles (primordial, primary, secondary, antral and atretic follicles) in mice from 15 weeks to 100 weeks old by image analyses. Mann-Whitney U-test was used for statistical analysis. The size of the ovary was significantly reduced in Aspm knockout mice at 15–20 weeks, 40–50 weeks and 70–80 weeks old compared with the control mice. Furthermore, at all stages, we found a severe decrease in the number of developing follicles at 10–15 weeks, 40–50 weeks and 70–80 weeks old, accompanied by disrupted cyclic changes of vaginal cytology and an aberrant upregulation of Foxo3, Kitl, and Lhcgr in Aspm knockout female. These results suggested that Aspm might play an important role in the folliculogenesis and estrous cyclicity of the postnatal ovary during maturation and aging.     

Waves of follicle development during the estrous cycle in sheep

The pattern of ovarian follicle development in maiden cyclic lambs was characterized using the definition of a follicle wave as the changes in the number of follicles among the days of the estrous cycle, as originally defined in cattle by Rajakoski in 1960. We also examined the steroid content relationships among follicles on Days 5 (Wave 1) and 14 (Waves 2 and 3) of the estrous cycle. In Experiment 1, the ovaries of 20 cyclic lambs (40 to 45 kg) were examined daily using transrectal ultrasonography for 1 or 2 estrous cycles (n = 31 cycles). The number of small (2 and 3 mm in diameter), medium (4 and 5 mm) and large (> or = 6 mm) follicles were aligned with the beginning and end of the average length estrous cycle and then compared among days. Identified follicles were defined as those that grew to > or = 4 mm and remained at > or = 3 mm for > or = 3 d. The number of identified follicles emerging (retrospectively identified at 2 or 3 mm) per ewe per day was also aligned with the average length estrous cycle. In Experiment 2, ewe lambs were ovariectomized on Day 5 (n = 6) or 14 (n = 5) of the estrous cycle, then follicle diameters and follicular fluid concentrations of estradiol and progesterone were compared among follicles. Data were analyzed by repeated measures ANOVA and compared among days using Fisher's LSD. In Experiment 1, either 2 (n = 10 cycles), 3 (n = 20 cycles) or 4 (n = 1 cycle) periods of emergence of identified follicles occurred during individual cycles, with estrous cycle lengths of 15.6 +/- 1.6, 16.1 +/- 1.1 and 17 d respectively. In animals with 2 or 3 periods of emergence of identified follicles, the total number of small, medium and large follicles differed (P < 0.05) among days of the estrous cycle showing a wave-like pattern. In Experiment 2, a single follicle collected on each of Days 5 and 14 of the cycle (6.2 +/- 0.2 and 3.9 +/- 0.2 mm in diameter) had a higher (P < 0.05) concentration of follicular fluid estradiol (36.2 +/- 4.4 and 50.9 +/- 21.6 ng/mL) than other follicles collected on the same day (next largest follicle: 4.3 +/- 0.3 and 3.5 +/- 0.4 mm; 4.3 +/- 0.9 and 18.2 +/- 6.7 ng/mL estradiol). The results showed that 1) there was a synchronous emergence of follicles associated with fluctuations in the number and size of follicles during the estrous cycle; 2) within a wave there was a hierarchy among follicles for diameter and steroid content; 3) ovarian follicle growth in ewe lambs occurred in 2 or 3 organized waves during the estrous cycle.     

Clinical analysis of human umbilical cord mesenchymal stem cell allotransplantation in patients with premature ovarian insufficiency

ObjectivePremature ovarian insufficiency (POI) is a refractory disease that seriously affects female fertility. Growing body of evidence has indicated mesenchymal stem cells (MSCs) as promising resources in regenerative medicine. In this study, we treated POI patients with umbilical cord‐derived MSCs (UCMSCs) and then investigated the restoration of ovarian function and clinical outcomes through follow‐ups.Materials and methodsSixty‐one patients diagnosed with POI participated in this study. UCMSCs were isolated and cultured according to GMP standards, and then transplanted to the patients’ ovary by orthotopic injection under the guidance of vaginal ultrasound. We monitored side effects, vital signs and changes in clinical and collected haematological and imaging parameters during the follow‐ups.ResultsAll patients showed normal clinical behaviour without serious side effects or complications relevant to the treatment. Transplantation of UCMSCs rescued the ovarian function of POI patients, as indicated by increased follicular development and improved egg collection. POI patients who experienced shorter amenorrhoea durations (<1 year) seemed to obtain mature follicles more easily after stem cell therapy, and patients with better ovarian conditions (pre‐operative antral follicles) were more likely to derive the better outcomes by UCMSC injection. Four successful clinical deliveries were obtained from POI patients after UCMSC transplantation, and all of these babies are developed normally.ConclusionsThe clinical trial result sugggests a possible therapy for POI by UCMSC transplantation.     

Adoptive transfers of CD4+CD25+ Tregs partially alleviate mouse premature ovarian insufficiency

This study was designed to investigate the protective effect of CD4⁺CD25⁺ regulatory T cells (Tregs) against zona pellucida glycoprotein 3 peptide (pZP3) immunization‐induced premature ovarian insufficiency (POI) in mice. A mouse POI model was induced by two subcutaneous injections of pZP3 (50 nmol/L). Mice in the pZP3‐Treg group were intraperitoneally injected with 5 × 10⁵ CD4⁺CD25⁺ Tregs after the POI model was established. Sex hormone levels, follicle numbers, apoptotic events, and the Akt/FOXO3a signaling pathway molecules in the ovaries were assessed. Compared with control group, the weight of ovaries in both pZP3 group and pZP3‐Treg group was decreased and no difference was found between them. The number of follicles in the Treg transferred mice, like in pZP3 group, was significantly reduced compared to the control group, but showed a modest improvement when compared the pZP3 group alone. Significantly lower serum concentrations of follicle‐stimulating hormone, luteinizing hormone, and anti‐zona pellucida antibodies (AZPAbs) were found, while the concentrations of estradiol and anti‐Mullerian hormone increased. In mechanism, Treg cell transfer to ZP3 treated mice restored the levels of Caspase3 to control levels, and partially restored Bax, however, had no effect on Bcl‐2. Moreover, Treg cell transfer to ZP3 treated mice partially restored the levels of Akt and FOXO3a, and partially restored the ratios of p‐Akt/Akt and p‐FOXO3a/FOXO3a. In conclusion, Treg cells improved some aspects of ZP3‐induced POI which may be mediate by suppressing ovarian cells apoptosis and involving the Akt/FOXO3a signaling pathway. Therefore, Treg cells may be protective against autoimmune POI.     

Salubrinal enhances eIF2α phosphorylation and improves fertility in a mouse model of Classic Galactosemia

Loss of galactose-1 phosphate uridylyltransferase (GALT) activity in humans results in Classic Galactosemia, and the GalT-deficient (GalT^?/?) mouse mimics the patient condition. GalT^?/? ovaries display elevated endoplasmic reticulum (ER) stress marker, BiP, and downregulated canonical phosphatidylinositol 3-kinase (Pi3k)/protein kinase B (Akt) growth/pro-survival signaling. Numbers of primordial follicles are reduced in the mutants, recapitulating the accelerated ovarian aging seen in human patients. We previously found that oral administration of the compound Salubrinal (an eIF2α phosphatase inhibitor), resulted in reduction of ovarian BiP expression, rescued Pi3k/Akt signaling, and a doubling of primordial follicles in GalT^?/? adults. Here, we further characterized galactosemic stress in GalT^?/? mice versus wild-type (WT) controls, and examined whether Salubrinal treatment improved broader reproductive parameters. We assessed the expression levels of factors of the unfolded protein response (UPR), and found that BiP, phospho-Perk, and phospho-eIF2α were all elevated in GalT^?/? ovaries. However, neither IKK activation (NFκB pathway) nor alternative Xbp1 splicing downstream of ER membrane protein Ire1α activation was induced, suggesting an Xbp1-independent UPR in galactosemic stress. Moreover, Salubrinal treatment significantly increased the number of ovulated eggs in mutant animals after gonadotrophic superovulation. Salubrinal treatment also normalized estrus cycle stage lengths and resulted in significantly larger litter sizes than vehicle-treated mutants. Overall, we show that Salubrinal protects against galactosemia-induced primordial follicle loss in a fashion that includes suppressing the de-phosphorylation of eIF2α, and that intervention in this way significantly improves and extends ovarian function, fertility, and fecundity.     

Influence of the estrous cycle on clock gene expression in reproductive tissues: Effects of fluctuating ovarian steroid hormone levels

Circadian rhythms in physiology and behavior are known to be influenced by the estrous cycle in female rodents. The clock genes responsible for the generation of circadian oscillations are widely expressed both within the central nervous system and peripheral tissues, including those that comprise the reproductive system. To address whether the estrous cycle affects rhythms of clock gene expression in peripheral tissues, we first examined rhythms of clock gene expression (Per1, Per2, Bmal1) in reproductive (uterus, ovary) and non-reproductive (liver) tissues of cycling rats using quantitative real-time PCR (in vivo) and luminescent recording methods to measure circadian rhythms of PER2 expression in tissue explant cultures from cycling PER2::LUCIFERASE (PER2::LUC) knockin mice (ex vivo). We found significant estrous variations of clock gene expression in all three tissues in vivo, and in the uterus ex vivo. We also found that exogenous application of estrogen and progesterone altered rhythms of PER2::LUC expression in the uterus. In addition, we measured the effects of ovarian steroids on clock gene expression in a human breast cancer cell line (MCF-7 cells) as a model for endocrine cells that contain both the steroid hormone receptors and clock genes. We found that progesterone, but not estrogen, acutely up-regulated Per1, Per2, and Bmal1 expression in MCF-7 cells. Together, our findings demonstrate that the timing of the circadian clock in reproductive tissues is influenced by the estrous cycle and suggest that fluctuating steroid hormone levels may be responsible, in part, through direct effects on the timing of clock gene expression.     

The effect of the absence or presence of a corpus luteum on the ovarian follicular population and serum oestradiol concentrations during the estrous cycle in Sanjabi ewes

The aim of this study was to determine if the presence or absence of a corpus luteum (CL) during estrous synchronization in ewes can affect the ovarian follicular population and the serum oestradiol concentrations. The estrous cycles of 197 Sanjabi ewes were synchronized using a 12-day treatment with intravaginal progestagen sponges (Chronogest^®). Estrus was detected in 144 ewes, 27–39 h after sponge removal. Blood samples were taken daily from day 2 and continued for 19 days and analyzed for serum oestradiol concentration. Nine ewes were slaughtered on each experimental day (days 1–16 after estrus) for ovary collection. The ovaries per ewe were classified as those without, or with one or two CL's, for each slaughter day. Visible follicles on the surface of the ovaries were classified, based on their diameter, into (i) very small (<2 mm), (ii) small (2–3.4 mm), (iii) medium (3.5–5 mm) and (iv) large (>5 mm) categories, and the respective numbers recorded. Results indicated, the number of ovarian follicles to decrease (P < 0.01) from days 1 to 5 of the cycle and showed a significant increase on day 7. Numbers were high again on day 11 and decreased (P < 0.01) on day 16 of the estrous cycle. The serum oestradiol concentrations were significantly higher (P < 0.001) in the double than in the single ovulating animals (one or two CL's, respectively) on days 2–0. However serum levels were also significantly higher (P < 0.001) in single, than twin ovulating animals on days 4–5 and 12–16 of the estrous cycle. There were no significant differences in the total number of very small follicles between animals without and those with two CL's. The number of small, medium and large follicles in ewes, with or without a CL on the ovary was significantly higher (P < 0.01) than ewes with two ovulations at certain stages of the estrous cycle. The present study provides evidence of differences in the follicular ovarian population in ovaries without CL's and double ovulations. The existence of an intraovarian effect of the CL numbers on follicular population is demonstrated.     

Cryopreservation of ovarian tissues temporarily suppresses the proliferation of granulosa cells in mouse preantral follicles

Cryopreservation of ovarian tissue has been reported to delay the development of preantral follicles during in vitro culture, but the mechanism causing this impairment has not been brought to light. In order to elucidate the underlying mechanism of delayed follicular development, we evaluated the effects of cryopreservation on the proliferation of granulosa cells during culture of mouse preantral follicles, as a sufficient population of granulosa cells is critical for normal follicular development. Additionally the initial cell death of granulosa cells was estimated immediately after cryopreservation. The ovarian tissues obtained from 12-day-old female mice were cryopreservation by vitrification. The granulosa cell proliferation was evaluated by measuring the PCNA expression and the expression of cell cycle regulators such as cyclin D2, CDK4, cyclin E and CDK2 in preantral follicles isolated from fresh and cryopreserved ovarian tissues that were cultured for 48 h. The viability of granulosa cells was evaluated by measuring the proportion of necrotic areas. The granulosa cell proliferation of the cryopreserved preantral follicles was decreased significantly compared to that of the fresh controls at 0 and 24 h after culture (P < 0.05), and this was increased to the control levels after 48 h of culture. The expressions of cyclin D2, Cdk 4, cyclin E and Cdk2 were also decreased in the cryopreserved ovarian tissues at 0 and 24 h after culture (P < 0.05), but they were increased to the control levels after 48 h of culture. The proportion of the necrotic area was significantly higher in cryopreserved preantral follicles compared to that of the fresh preantral follicles (P < 0.05). This suggests that cryopreservation of ovarian tissues may delay the preantral follicle development by temporary suppressing the granulosa cell proliferation through the cell cycle regulators (cyclin D2, Cdk4, cyclin E and Cdk2) and by granulosa cell death immediately after warming.     

The Influence of LepR Tyrosine Site Mutations on Mouse Ovary Development and Related Gene Expression Changes

Leptin exerts many biological functions, such as in metabolism and reproduction, through binding to and activating the leptin receptor, LepRb, which is expressed in many regions of the brain. To better understand the roles of LepR downstream signaling pathways, Y123F mice, which expressed mutant leptin receptors with phenylalanine (F) substituted for three tyrosines (Y) (Tyr985, Tyr1077 and Tyr1138), were generated. The body weight and abdominal fat deposits of Y123F homozygous mice (HOM) were higher than those of wild-type mice (WT). HOM ovaries were atrophic and the follicles developed abnormally; however, the HOM ovaries did not exhibit polycystic phenotypes. Moreover, Y123F HOM adults had no estrous cycle and the blood estrogen concentration remained stable at a low level below detection limit of 5 pg/ml. LepR expression in HOM ovaries was higher than in WT ovaries. Using cDNA Microarrays, the mRNA expressions of 41 genes were increased, and 100 were decreased in HOM vs. WT ovaries, and many signaling pathways were evaluated to be involved significantly. The expressions of 19 genes were validated by real-time quantitative PCR, most of which were consistent with the microarray results. Thus, Y123F HOM mice were suggested as a new animal model of PCOS for research that mainly emphasizes metabolic disorders and anovulation, but not the polycystic phenotype. Meanwhile, using the model, we found that JAK-STAT and hormone biosynthesis pathways were involved in the follicular development and ovulation disorders caused by LepR deficiency in ovaries, although we could not exclude indirect actions from the brain.     

Meloxicam and Buprenorphine Treatment after Ovarian Transplantation Does Not Affect Estrous Cyclicity and Follicular Integrity in Aged CBA/J Mice

Angiogenesis, the formation of new blood vessels, is important for the survival of ovarian transplants and the restoration of ovarian functions. Without angiogenesis, transplanted ovarian tissue becomes more susceptible to tissue damage and necrosis. Administration of analgesics for pain management has been shown to decrease angiogenesis, which can influence transplant success especially in aged animals. Aging and the effects of hypoxia after transplantation decrease reproductive viability of the ovarian transplant; therefore, it is important to understand the additional effects of analgesics on aged animal models. The present study investigated the effects of two analgesics, buprenorphine, an opiate, and meloxicam, a non-steroidal anti-inflammatory drug (NSAID), on the reproductive indicators related to estrous cyclicity and follicular integrity after ovarian transplantation of young ovaries into aged CBA/J mice. These aged females did not show any different reproductive responses when treated with either buprenorphine or meloxicam. No significant differences were observed in estrous cycle length, the onset of estrous cycling, the regularity of estrous cycles, and the proportion of viable follicles and total number of follicles per ovarian sample across treatment groups.     

Di (2-ethylhexyl) Phthalate Exposure Impairs Growth of Antral Follicle in Mice

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plastic additive. As an environmental endocrine disruptor, it has been shown to be harmful to the mammalian reproductive system. Previous studies indicated that DEHP inhibited the development of mouse ovarian follicles. However, the mechanisms by which DEHP affects ovarian antral follicle development during the pre-puberty stage are poorly understand. Thus, we investigated the effects of direct DEHP exposure on antral follicle growth in pre-pubescent mice by use of intraperitoneal injection. Our results demonstrated that the percentage of large antral follicles was significantly reduced when mice were exposed to 20 or 40 μg/kg DEHP every 5 days from postnatal day 0 (0 dpp) to 15 dpp. In 20 dpp, we performed microarray of these ovaries. The microarray results indicated that mRNA levels of apoptosis related genes were increased. The mRNA levels of the apoptosis and cell proliferation (negative) related genes Apoe, Agt, Glo1 and Grina were increased after DEHP exposure. DEHP induced the differential gene expression of Hsp90ab1, Rhoa, Grina and Xdh which may play an important role in this process. In addition, TUNEL staining and immunofluorescence showed that DEHP exposure significantly increased the number of TUNEL, Caspase3 and γH2AX positive ovarian somatic cells within the mouse ovaries. Flow cytometer analyses of redox-sensitive probes showed that DEHP caused the accumulation of reactive oxygen species. Moreover, the mRNA expression of ovarian somatic cell antioxidative enzymes was down-regulated both in vivo and in vitro. In conclusion, our data here demonstrated that DEHP exposure induced oxidative stress and ovarian somatic cell apoptosis, and thus may impact antral follicle enlargement during the pre-pubertal stage in mice.