Age-related changes in densitometry and histomorphometry of long bone during the pubertal growth spurt in male rats.

Because experimental studies to determine the developmental toxicity of exposure to various substances in children are impossible, many studies use immature male rats. This study aimed to provide normative data for longitudinal bone growth with age during the puberty in male rats. In order to evaluate long bone growth and mineralization we examined bone size and bone density by dual-energy X-ray absorptiometry, analyzed histomorphometry of the growth plate, and serum hormone levels relevant to bone growth from postnatal day (PD)20 to PD60. The length and weight of long bones increased strongly by PD40, and no further increase was observed after PD50. On the other hand, tibial growth plate height decreased sharply after PD50 along with a reduction in the number of cells and columns, which was probably responsible for the absence of further lengthening of long bones. Parameters related to bone formation such as bone area ratio, and the thickness and number of trabeculae, also increased significantly between PD40 and PD50. Furthermore, serum levels of IGF-1 peaked at PD30 and testosterone increased rapidly on and after PD40, when IGF-1 levels were going down. These changes may participate in the parallel increase in mineral acquisition, as well as lengthening of long bones. Our findings provide comprehensive data for changes in bone density, histomorphometry of long bones, and hormone levels relevant to bone growth during the growth spurt. This will be useful for planning animal toxicological studies, particularly for deciding on the appropriate age of animals to use in given experiments.

Radioactive Iodine-induced hypothyroidism interferes with the maturation of reproductive organs during puberty in immature female rats.

Animal and human studies suggest that thyroid hormone may have critical roles in the development of the ovary. For example, thyroid deficiency disrupts the ovarian microarchitecture and menstrual cycle in neonate and adult women, respectively. Therefore, it is conceivable that thyroid deficiency might disrupt sexual maturation during the peri-pubertal period. To investigate the impact of radioactive iodine-induced thyroid deficiency on reproductive organs throughout puberty, immature female rats were given water containing radioactive iodine (0.37 MBq/g body weight) twice, on postnatal days 22 and 29. Radioactive iodine-induced hypothyroidism was revealed by low free thyroxin levels. Thyroid deficiency delayed the onset of vaginal opening, reduced ovarian weight and the number of medium-sized follicles and led to elongated uteri. However, there was no effect on the estrous cycle or absolute uterus weight. We conclude that radioactive iodine-induced thyroid deficiency delays sexual maturation and alters normal ovarian growth in peri-pubertal rats.

Luteinizing Hormone Surge-Induced Krüppel-like Factor 4 Inhibits Cyp17A1 Expression in Preovulatory Granulosa Cells.

Previous in vivo and in vitro studies have demonstrated a dramatic up-regulation of Krüppel-like factor 4 (Klf4) in rat preovulatory granulosa cells (GCs) after LH/hCG treatment and its role in regulating Cyp19A1 expression during the luteal shift in steroidogenesis. In this study, we examined whether Klf4 also mediates the LH-induced repression of Cyp17A1 expression in primary rat preovulatory GCs. In response to LH treatment of GCs in vitro, Cyp17A1 expression declined to less than half of its initial value by 1 h, remaining low for 24 h of culture. Overexpression of Klf4 decreased basal and Sf1-induced Cyp17A1 expressions and increased progesterone secretion. Reduction of endogenous Klf4 by siRNA elevated basal Cyp17A1 expression but did not affect LH-stimulated progesterone production. Overexpression of Klf4 also significantly attenuated Sf1-induced Cyp17A1 promoter activity. On the other hand, mutation of the conserved Sp1/Klf binding motif in the promoter revealed that this motif is not required for Klf4-mediated repression. Taken together, these data indicate that the Cyp17A1 gene may be one of the downstream targets of Klf4, which is induced by LH in preovulatory GCs. This information may help in identifying potential targets for preventing the molecular changes occurring in hyperandrogenic disorders.

Excretory-Secretory Products of Trichomonas vaginalis Cause Apoptosis in Mouse Sperm in Vitro.

Excretory-secretory products (ESP) of T. vaginalis have been shown to inhibit sperm motility, viability, and functional integrity, leading to a decreased fertilization rate in vitro. This study investigated whether T. vaginalis induce apoptosis and ultrastructural changes of sperm using flow cytometry and electron microscopy. Incubation of sperm with T. vaginalis ESP increased phosphatidylserine externalization and DNA fragmentation, and decreased mitochondrial membrane potential. Transmission electron microscopy of sperm incubated with ESP revealed abnormal features such as distorted heads, broken necks, and acrosomes exocytosis. This is the first report that demonstrates a direct impact of T. vaginalis ESP on sperm apoptosis and architecture in vitro.

LH-induced Transcriptional Regulation of Klf4 Expression in Granulosa Cells Occurs via the cAMP/PKA Pathway and Requires a Putative Sp1 Binding Site.

Krüppel-like factor 4 (Klf4) plays an important role in the transition from proliferation to differentiation in a wide variety of cells. Previous studies demonstrated its critical role in the luteal transition of preovulatory granulosa cells (GCs). This study used cultured rat preovulatory GCs to investigate the mechanism by which luteinizing hormone (LH) regulates Klf4 gene expression. Klf4 mRNA and protein were rapidly and transiently induced by LH treatment, reaching peak levels after 45 min and declining to basal levels by 3 h. Pretreatment with the protein synthesis inhibitor cycloheximide had no effect on LH-stimulated Klf4 expression, indicating that Klf4 is an immediate early gene in response to LH. To investigate the signaling pathway involved in LH-induced Klf4 regulation, the protein kinase A (PKA) and protein kinase C (PKC) pathways were evaluated. A-kinase agonists, but not a C-kinase agonist, mimicked LH in inducing Klf4 transcription. In addition, specific inhibitors of A-kinase abolished the stimulatory effect of LH on Klf4 expression. Truncation of a Klf4 expression construct to -715 bp (pKlf4-715/luc) had no effect on transcriptional activity, whereas deletion to -402 bp (pKlf4-402/luc) dramatically reduced it. ChIP analysis revealed in vivo binding of endogenous Sp1 to the -715/-500 bp region and maximal transcriptional responsiveness to LH required the Sp1 binding element at -698/-688 bp, which is highly conserved in mice, rats, and humans. These findings demonstrate that Klf4 is activated by LH via the cAMP/PKA pathway and a putative Sp1 binding element at -698/-688 bp is indispensable for activation and suggest that Klf4 could be a target for strategies for treating luteal phase insufficiency induced by an aberrant response to the LH surge.

The Effects of High Peripubertal Caffeine Exposure on the Adrenal Gland in Immature Male and Female Rats.

The consumption of high levels of dietary caffeine has increased in children and adolescents. Human and animal studies have shown that chronic intake of high doses of caffeine affects serum glucocorticoid levels. Given that glucocorticoids play a role in peripubertal organ growth and development, chronic high doses of caffeine during puberty might impair maturation of the adrenal glands. To evaluate any effects of caffeine exposure on growing adrenal glands, 22-day-old male (n = 30) and female Sprague Dawley rats (n = 30) were divided into three groups (n = 10/group); group 1 received tap water (control) and groups 2 and 3 received water containing 120 and 180 mg/kg/day caffeine, respectively, via gavage for 4 weeks. At the end of the experiment, adrenal glands were weighed and processed for histological analysis. Relative adrenal weights increased in both groups of caffeine-fed males and females, whereas absolute weights were decreased in the females. In the female caffeine-fed groups the adrenal cortical areas resembled irregularly arranged cords and the medullary area was significantly increased, whereas no such effects were seen in the male rats. Our results indicate that the harmful effects of caffeine on the adrenal glands of immature rats differ between females and males. Although female rats seemed to be more susceptible to damage based on the changes in the microarchitecture of the adrenal glands, caffeine affected corticosterone production in both female and male rats. In addition, increased basal adrenocorticotropic hormone levels in caffeine-fed groups may reflect decreased cortical function. Therefore, caffeine may induce an endocrine imbalance that disturbs the establishment of the hypothalamo-pituitary adrenal axis during puberty, thereby leading to abnormal stress responses.

Krüppel-like factor 4 plays a role in the luteal transition in steroidogenesis by downregulating Cyp19A1 expression.

The transition from granulosa cell (GC) to luteal cell involves a change from estrogen production to predominantly progesterone production. We analyzed the role of Krüppel-like factor 4 (Klf4), a transcriptional repressor used to generate pluripotent cells, in that transition. After luteinizing hormone (LH)/human chorionic gonadotropin treatment of preovulatory follicles, a major but transient increase in Klf4 transcript levels was detected. Therefore, we enquired whether Klf4 is involved in the rapid decline of aromatase, the key estrogen-producing enzyme, using preovulatory GCs obtained from pregnant mare serum gonadotropin-primed immature rat ovaries. Cyp19A1 expression in GCs transfected with FLAG-Klf4 or Klf4-specific siRNA was analyzed by real-time PCR and immunofluorescence staining. Cyp19A1 decreased when Klf4 was overexpressed, and Cyp19A1 and estradiol biosynthesis increased when Klf4 was knocked down. The mechanism by which Klf4 regulates Cyp19A1 expression was investigated using Cyp19A1 promoter-luciferase reporter assays and chromatin immunoprecipitation assays. The results revealed that the steroidogenic factor-1 (SF1)-binding motif, but not the specificity protein 1 (Sp1) binding element or the CACCC motif, was required for Klf4-mediated repression of Cyp19A1 promoter activity. Here we showed that Klf4 suppressed endogenous Cyp19A1 transcript and protein production, and this resulted from direct binding of Klf4 to the SF1 recognition motif in the Cyp19A1 promoter. These findings suggest that Klf4 is a physiologic regulator of Cyp19A1 expression in response to the LH surge in preovulatory GCs and that it has an essential role in the luteal transition in steroidogenesis.

Role of Klf4 in the Regulation of Apoptosis and Cell Cycle in Rat Granulosa Cells during the Periovulatory Period.

In the ovary, the luteinizing hormone (LH) surge suppresses the proliferation and induces the luteinization of preovulatory granulosa cells (GCs), which is crucial for the survival of terminally-differentiated GCs. Krüppel-like factor 4 (Klf4) has been shown to play a role in regulating the cell cycle and apoptosis in various cell types. The rapid induction of Klf4 expressions by LH was observed in preovulatory GCs. To evaluate whether Klf4 affects GC proliferation and survival, primary rat GCs were isolated from pregnant mare serum gonadotropin-primed Sprague⁻Dawley rat ovaries and transfected with a Klf4 expression vector or Klf4-specific siRNA, followed by determination of the transcript levels of apoptosis-related and cell cycle-related genes. Cell proliferation, viability, and apoptosis were analyzed by BrdU incorporation, a Cell Counting Kit-8 assay, a bioluminescence caspase 3/7 assay, and flow cytometry. LH treatment increased Klf4 mRNA expression in preovulatory GCs. Transcripts of B-cell lymphoma 2 (Bcl-2) and cell cycle promoters (Cyclin D1 and Cyclin D2) decreased, whereas those of the cell cycle inhibitor, p21, increased. Altering the expression of Klf4 by overexpression or knockdown consistently affected the expression of Bcl-2 and Cyclin D1. In agreement with this, Klf4 overexpression reduced cell viability, increased the fraction of apoptotic cells, and arrested cell cycle progression in G1 phase. We conclude that Klf4 increases the susceptibility of preovulatory GCs to apoptosis by down-regulating Bcl-2, and promotes LH-induced cell cycle exit. It appears to be a key regulator induced by the LH surge that determines the fate of GCs in preovulatory follicles during the luteal transition.

Effect of radioactive iodine-induced hypothyroidism on longitudinal bone growth during puberty in immature female rats.

Thyroid cancer in children, the most common endocrine malignancy, shows aggressive behavior and has a high recurrence rate after surgical ablation. Radioactive iodine (RAI) treatment is the most effective primary modality for medical ablation of juvenile thyroid cancer, and leads to intentional hypothyroidism. Although several negative impacts of hypothyroidism have been reported in children in response to other antithyroid agents, the combined effects of RAI exposure and hypothyroidism, on growing bones specifically, are unknown. In this study, we investigated the effect of RAI-induced hypothyroidism on the long bones during the pubertal growth spurt using immature female rats. Female Sprague-Dawley rats were randomly divided into a control group, and an RAI-treated group fed with RAI (0.37 MBq/g body weight) twice via gavage. After 4 weeks, we observed a significantly-reduced serum free thyroxine level in the RAI group. The latter group also displayed decreased body weight gain compared to the control. In addition, the lengths of long bones, such as the leg bones and vertebral column, as well as bone mineral content, were reduced in the RAI-treated animals. Our results confirm the negative impacts of RAI-induced thyroid deficiency during puberty on longitudinal bone growth and bone mineralization.

The Effects of Caffeine on the Long Bones and Testes in Immature and Young Adult Rats.

This study was to evaluate the age-dependent effects of caffeine exposure on the long bones and reproductive organs using male rats. A total of 15 immature male rats and 15 young adult male rats were allocated randomly to three groups: a control group and two groups fed caffeine with 120 and 180 mg/kg/day for 4 weeks. Exposure to caffeine at either dose significantly reduced body weight gain; a proportional reduction in muscle and fat mass in immature animals, whereas a selective reduction in fat mass with relatively preserved muscle mass in young adult animals. The long bones of immature rats exposed to caffeine were significantly shorter and lighter than those of control animals along with decreased bone minerals. However, there was no difference in the length or weight of the long bones in young adult rats exposed to caffeine. Exposure to caffeine reduced the size and absolute weight of the testes significantly in immature animals in comparison to control animals, but not in young adult animals exposed to caffeine. In contrast, the adrenal glands were significantly heavier in caffeine-fed young adult rats in comparison to control animals, but not in caffeine-fed immature rats. Our results clearly show that the negative effects of caffeine on the long bones and testes in rats are different according to the age of the rat at the time of exposure, and might therefore be caused by changes to organ sensitivity and metabolic rate at different developmental stages. Although the long bones and testes are more susceptible to caffeine during puberty, caffeine has negative effects on body fat, bone minerals and the adrenal glands when exposure occurs during young adulthood. There is a need, therefore, to educate the public the potential dangers of caffeine consumption during puberty and young adulthood.

Peri-pubertal high caffeine exposure increases ovarian estradiol production in immature rats.

Chronic caffeine consumption exerts a negligible effect on the reproductive organs of normal adult females, but it is not known whether this is also true for children and adolescents. Here, we investigated the effects of high caffeine exposure on sexual maturation and ovarian estradiol production in immature female rats. Immature female SD rats were divided into controls and caffeine groups fed 120 and 180mg/kg/day for 4 or 8 weeks. There was a significant delay in vaginal opening in the caffeine-fed groups. In addition, serum estradiol levels were elevated in the caffeine-fed animals after 2 and 4 weeks of exposure. Estradiol secretion as well as aromatase expression also increased significantly in the ovarian cells in response to caffeine. These results demonstrate that peripubertal exposure to high caffeine increases estradiol production in the ovary; this may disturb the coordinated regulation of the hypothalamo-pituitary-ovarian axis, thereby interfering with sexual maturation.

Dose- and time-related effects of caffeine on the testis in immature male rats.

We previously showed that prepubertal chronic caffeine exposure adversely affected the development of the testes in male rats. Here we investigated dose- and time-related effects of caffeine consumption on the testis throughout sexual maturation in prepubertal rats. A total of 80 male SD rats were randomly divided into four groups: controls and rats fed 20, 60, or 120 mg caffeine/kg/day, respectively, via gavage for 10, 20, 30, or 40 days. Preputial separation was monitored daily before the rats were sacrificed. Terminal blood samples were collected for hormone assay, and testes were grossly evaluated and weighed. One testis was processed for histological analysis, and the other was collected to isolate Leydig cells. Caffeine exposure significantly increased the relative weight of the testis in a dose-related manner after 30 days of exposure, whereas the absolute testis weight tended to decrease at the 120 mg dose of caffeine. The mean diameter of the seminiferous tubules and height of the germinal epithelium significantly decreased in the caffeine-fed groups after 40 days of caffeine exposure, which was accompanied by a reduced BrdU incorporation rate in germ cells. In addition, caffeine intake significantly reduced in vivo and ex vivo testosterone production in a dose-related manner. Our results demonstrate that caffeine exposure during sexual maturation alter the testicular microarchitecture and also slow germ cell proliferation even at the 20 mg dose level. Furthermore, caffeine may act directly on Leydig cells and interfere with testosterone production in a dose-related manner, consequently delaying onset of sexual maturation.

Longitudinal bone growth is impaired by direct involvement of caffeine with chondrocyte differentiation in the growth plate.

We showed previously that caffeine adversely affects longitudinal bone growth and disrupts the histomorphometry of the growth plate during the pubertal growth spurt. However, little attention has been paid to the direct effects of caffeine on chondrocytes. Here, we investigated the direct effects of caffeine on chondrocytes of the growth plate in vivo and in vitro using a rapidly growing young rat model, and determined whether they were related to the adenosine receptor signaling pathway. A total of 15 male rats (21 days old) were divided randomly into three groups: a control group and two groups fed caffeine via gavage with 120 and 180 mg kg-1  day-1 for 4 weeks. After sacrifice, the tibia processed for the analysis of the long bone growth and proliferation of chondrocytes using tetracycline and BrdU incorporation, respectively. Caffeine-fed animals showed decreases in matrix mineralization and proliferation rate of growth plate chondrocytes compared with the control. To evaluate whether caffeine directly affects chondrocyte proliferation and chondrogenic differentiation, primary rat chondrocytes were isolated from the growth plates and cultured in either the presence or absence of caffeine at concentrations of 0.1-1 mm, followed by determination of the cellular proliferation or expression profiles of cellular differentiation markers. Caffeine caused significant decreases in extracellular matrix production, mineralization, and alkaline phosphatase activity, accompanied with decreases in gene expression of the cartilage-specific matrix proteins such as aggrecan, type II collagen and type X. Our results clearly demonstrate that caffeine is capable of interfering with cartilage induction by directly inhibiting the synthetic activity and orderly expression of marker genes relevant to chondrocyte maturation. In addition, we found that the adenosine type 1 receptor signaling pathway may be partly involved in the detrimental effects of caffeine on chondrogenic differentiation, specifically matrix production and mineralization, as evidenced by attenuation of the inhibitory effects of caffeine by blockade of this receptor. Thus, our study provides novel information on the integration of caffeine and adenosine receptor signaling during chondrocyte maturation, extending our understanding of the effect of caffeine at a cellular level on chondrocytes of the growth plate.

Peripubertal Caffeine Exposure Impairs Longitudinal Bone Growth in Immature Male Rats in a Dose- and Time-Dependent Manner.

This study investigated the dose- and time-dependent effects of caffeine consumption throughout puberty in peripubertal rats. A total of 85 male SD rats were randomly divided into four groups: control and caffeine-fed groups with 20, 60, or 120 mg/kg/day through oral gavage for 10, 20, 30, or 40 days. Caffeine decreased body weight gain and food consumption in a dose- and time-dependent manner, accompanied by a reduction in muscle and body fat. In addition, it caused a shortening and lightening of leg bones and spinal column. The total height of the growth plate decreased sharply at 40 days in the controls, but not in the caffeine-fed groups, and the height of hypertrophic zone in the caffeine-fed groups was lower than in the control. Caffeine increased the height of the secondary spongiosa, whereas parameters related to bone formation, such as bone area ratio, thickness and number of trabeculae, and bone perimeter, were significantly reduced. Furthermore, serum levels of IGF-1, estradiol, and testosterone were also reduced by the dose of caffeine exposure. Our results demonstrate that caffeine consumption can dose- and time-dependently inhibit longitudinal bone growth in immature male rats, possibly by blocking the physiologic changes in body composition and hormones relevant to bone growth.

Coxsackievirus B3 infection reduces female mouse fertility.

Previously we demonstrated coxsackievirus B3 (CVB3) infection during early gestation as a cause of pregnancy loss. Here, we investigated the impacts of CVB3 infection on female mouse fertility. Coxsackievirus-adenovirus receptor (CAR) expression and CVB3 replication in the ovary were evaluated by immunohistochemistry or reverse transcription-polymerase chain reaction (RT-PCR). CAR was highly expressed in granulosa cells (GCs) and CVB3 replicated in the ovary. Histological analysis showed a significant increase in the number of atretic follicles in the ovaries of CVB3-infected mice (CVBM). Estrous cycle evaluation demonstrated that a higher number of CVBM were in proestrus compared to mock mice (CVBM vs. mock; 61.5%, 28.5%, respectively). Estradiol concentration in GC culture supernatant and serum were measured by an enzyme-linked immunosorbent assay. Baseline and stimulated levels of estradiol in GC were decreased in CVBM, consistent with significantly reduced serum levels in these animals. In addition, aromatase transcript levels in GCs from CVBM were also decreased by 40% relative to the mock. Bone mineral density evaluated by micro-computed tomography was significantly decreased in the CVBM. Moreover, the fertility rate was also significantly decreased for the CVBM compared to the mock (CVBM vs. mock; 20%, 94.7%, respectively). This study suggests that CVB3 infection could interfere with reproduction by disturbing ovarian function and cyclic changes of the uterus.

High doses of caffeine reduce in vivo osteogenic activity in prepubertal rats.

Caffeine adversely affects endochondral ossification during fetal skeletal growth, and results in increased incidence of delayed and abnormal fetal skeletal development. Chronic caffeine intake also decreases growth hormone secretion. Thus, it is conceivable that caffeine may disrupt bone growth during the peripubertal period. This study aimed to investigate the impact of high-caffeine consumption on bone growth throughout puberty. A total of 51 male rats (21 days old) were divided randomly into three groups: a control group and two groups fed caffeine via gavage with 120 and 180 mg kg(-1)  day(-1) for 4 weeks. After death, the final length and weight of leg bones were measured, and the tibia processed for histomorphometric analysis. Caffeine caused a significant decrease in body mass gain. This was accompanied with proportional decreases in lean body mass and body fat. In addition, bone mass and osteogenic activity in vivo were assessed using dual-energy X-ray absorptiometry and (18) F-NaF positron emission tomography. The results showed significant decreases of bone mass and in vivo osteogenic activity in the caffeine-fed groups. Rats fed with caffeine showed a significantly shorter and lighter tibia and femur and the vertebral column compared with controls. In addition, caffeine does not increase the width of the growth plates (GPs), it slows the rate at which the GP closes due to a slower rate of growth. These results demonstrated that caffeine altered osteogenic activity, leading to delayed peripubertal longitudinal bone growth and maturation. Given that osteogenic cells undergo dynamic changes in metabolic activity and that the pubertal growth spurt is mainly stimulated by growth hormone/insulin-like growth factor-1 and sex steroids during pubertal development, caffeine could suppress ossification by interfering with both physiological changes in hormonal secretion and osteogenic activity during this critical period. Further study will be needed to investigate the cellular/molecular mechanism by which caffeine affects osteogenesis using in vitro experimental models.

High Doses of Caffeine during the Peripubertal Period in the Rat Impair the Growth and Function of the Testis.

Prenatal caffeine exposure adversely affects the development of the reproductive organs of male rat offspring. Thus, it is conceivable that peripubertal caffeine exposure would also influence physiologic gonadal changes and function during this critical period for sexual maturation. This study investigated the impact of high doses of caffeine on the testes of prepubertal male rats. A total of 45 immature male rats were divided randomly into three groups: a control group and 2 groups fed 120 and 180 mg/kg/day of caffeine, respectively, via the stomach for 4 weeks. Caffeine caused a significant decrease in body weight gain, accompanied by proportional decreases in lean body mass and body fat. The caffeine-fed animals had smaller and lighter testes than those of the control that were accompanied by negative influences on the histologic parameters of the testes. In addition, stimulated-testosterone ex vivo production was reduced in Leydig cells retrieved from the caffeine-fed animals. Our results demonstrate that peripubertal caffeine consumption can interfere with the maturation and function of the testis, possibly by interrupting endogenous testosterone secretion and reducing the sensitivity of Leydig cells to gonadotrophic stimulation. In addition, we confirmed that pubertal administration of caffeine reduced testis growth and altered testis histomorphology.

The secretory products of Trichomonas vaginalis decrease fertilizing capacity of mice sperm in vitro.

Trichomonas vaginalis infection is one of the most prevalent sexually transmitted infections in humans and is now recognized as an important cause of infertility in men. There is little information about the effect of extracellular polymeric substances (EPS) from T. vaginalis on sperm, but previous reports do not provide a conclusive description of the functional integrity of the sperm. To investigate the impact of EPS on the fertilizing capacity of sperm, we assessed sperm motility, acrosomal status, hypo-osmotic swelling, and in vitrofertilization rate after incubating the sperm with EPS in vitrousing mice. The incubation of sperm with EPS significantly decreased sperm motility, viability, and functional integrity in a concentration and time-dependent manner. These effects on sperm quality also resulted in a decreased fertilization rate in vitro. This is the first report that demonstrates the direct negative impact of the EPS of T. vaginalis on the fertilization rate of sperm in vitro. However, further study should be performed using human sperm to determine if EPS has similar negative impact on human sperm fertilizing capacity in vitro.

Wilms' tumor suppressor gene (WT1) suppresses apoptosis by transcriptionally downregulating BAX expression in immature rat granulosa cells.

BACKGROUND: The important role of WT1 in early folliculogenesis was evident from its restricted expression pattern in immature follicles and from its involvement in transcriptional control of inhibin-α and FSH receptor. There is also considerable evidence that WT1 is a potent inhibitor of apoptotic cell death in the developing kidney and male germ cells, suggesting that it could play a role in the regulation of follicle survival. Therefore, we evaluated if WT1 involves in cell survival of granulosa cells (GCs) during the FSH-independent stage. METHODS: GCs were obtained from small preantral follicles of immature rat ovary. Bax and bcl-2 mRNA and protein levels in GCs transfected with WT1 (-KTS) or WT1 (+KTS) were analyzed by Real-time RT-PCR and immune-blotting analysis. Cell viability was measured with MTT assays and apoptosis was analyzed with caspase 3/7 activity and TUNEL assay. The mechanism by which WT1 regulates Bax expression was investigated using Bax promoter-luciferase reporter assay and ChIP assays from GCs. RESULTS: Here, we showed that WT1 (-KTS) suppressed endogenous Bax transcript and protein expression, and this inhibition resulted from direct binding of WT1 in the Bax promoter region in vivo. In addition, anti-apoptotic effects of WT1 (-KTS) were demonstrated based on MTT assays, a sensitive bioluminescence caspase 3/7 assay and TUNEL assays. On the other hand, WT1 has no role on bcl-2 expression in GCs. CONCLUSION: These findings suggest that activation of WT1 is necessary for maintenance of GC survival during early stage of follicles and WT1 can play a role in protecting apoptosis through the regulation of upstream activator (Bax), as well as through regulation of downstream effecter (caspases 3 and 7).

Cervical cytopathological findings in Korean women with Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum infections.

This is to investigate the cervical cytological abnormalities associated with Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, and Ureaplasma urealyticum infections on routine screen. A total of 714 subjects who had undergone cervical Pap smears and concomitant analyses for cervical infections were included by a retrospective search. The frequencies of reactive cellular change (RCC) and squamous epithelial abnormalities were significantly higher in Chlamydia positive subjects than in uninfected subjects (P<0.001). Of the 124 subjects tested for M. hominis, M. genitalium, and U. urealyticum, 14 (11%) were positive for M. hominis and 29 (23%) were positive for U. urealyticum. Squamous abnormalities were more frequent in subjects with Ureaplasma infections than in uninfected subjects (24% versus 8%). Taking together these findings, C. trachomatis and U. urealyticum may have a causal role in the development of cervical epithelial changes, including RCC. Thus, extra awareness is warranted in cervical screening of women with Chlamydia or Ureaplasma infections.