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.

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.

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.

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.

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.

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.

Regulation of follicle growth and apoptosis in cultured mouse ovaries by NGF and oxygen.

Cryopreservation of ovarian tissue has been proposed for use in preserving female fertility before anticancer chemo-radiotherapy, because ovarian tissue contains a large pool of non-growing, primordial follicles. The mechanisms that regulate the exit of follicles from the pool are poorly understood. To determine optimal conditions for in vitro ovarian culture, we investigated the effects of nerve growth factor (NGF) and oxygen concentration on follicle growth and apoptosis. Oxygen concentration affected both cell proliferation and apoptosis. Under 20% oxygen, but not 1.5% or 5%, NGF decreased apoptosis in mouse ovaries by down-regulating the pro-apoptotic genes Bax and p53. In conclusion, high oxygen tension during in vitro ovarian culture promotes follicle growth and, in conjunction with NGF, suppresses apoptosis. The efficiency of this method to preserve fertility depends in part on the level of atresia. These results suggest that oxygen and NGF may be used to increase numbers of preantral follicles and mature oocytes in the culture of mammalian ovarian cortical strips.

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.

Ovarian cyst torsion coexisting with acute pyelonephritis in an eight-year-old premenarchal girl.

We report the case of a premenarchal eight-year-old girl with an adnexal torsion accompanied by acute pyelonephritis with vague, non-specific symptoms. In young girls, symptoms may be vague, with patients complaining of non-specific gastro-intestinal problems. Combination with other complications such as pyelonephritis makes the diagnosis of adnexal torsion even more difficult as in the present case. To preserve future reproductive function, early surgical intervention is required. Although the impact on menarchal development of oophorectomy or partial removal of ovarian tissue before menarche remains to be evaluated, timely recognition and treatment of adnexal torsion clearly increases the possibility of retaining the ovary. Therefore, to decrease the time between the onset of symptoms and the identification of patients at risk, sonography should be considered as a first imaging modality for evaluating children with any degree of abdominal pain accompanied by non-specific symptoms.

Clinicopathological characteristics of ovarian tumours according to menarchal status in Korean girls.

AIM: To evaluate our experience with ovarian tumours in young girls and to identify specific characteristics in relation to their menarchal status that might assist in the early diagnosis and prompt management. METHODS: Girls younger than 18 years of age who had an adnexal tumour confirmed at surgery from 1985 to 2008 were identified from the pathology database of Hanyang University Medical Centre. Clinicopathological characteristics such as patient's age, age at menarche, presenting symptoms, operative findings and final histological diagnosis were analysed. RESULTS: Twenty-four of the 90 patients operated on for an ovarian tumour were pre-menarchal and 66 were post-menarchal. Most tumours were of germ cell origin: 83% of tumours in pre-menarchal and 53% in post-menarchal girls. Most of the patients complained of abdominal pain (∼70%), although a palpable mass was identified in only 17% of pre-menarchal and 30% of post-menarchal patients. Vomiting was a major complaint in pre-menarchal patients (30%) but was uncommon in post-menarchal patients (<5%). The frequency of torsion was significantly higher in pre-menarchal (50%) than in post-menarchal (17%) patients, and more than 90% of the torsion in pre-menarchal patients occurred in tumours of germ cell origin. CONCLUSIONS: The incidence of ovarian tumour and torsion is rare, especially in pre-menarchal girls. However, we found a higher prevalence of torsion in pre-menarchal girls with an ovarian tumour. A high level of suspicion for torsion should be considered when treating pre-menarchal girls with an ovarian tumour, irrespective of the tumour size to preserve adnexal tissue.

Lactobacillus acidophilus contributes to a healthy environment for vaginal epithelial cells.

Lactobacillus species in the female genital tract are thought to act as a barrier to infection. Several studies have demonstrated that lactobacilli can adhere to vaginal epithelial cells. However, little is known about how the adherence of lactobacilli to vaginal epithelial cells affects the acidity, cell viability, or proliferation of the lactobacilli themselves or those of vaginal epithelial cells. Lactobacillus acidophilus was co-cultured with immortalized human vaginal epithelial cells (MS74 cell line), and the growth of L. acidophilus and the acidity of the culture medium were measured. MS74 cell density and viability were also assessed by counting cell numbers and observing the cell attachment state. L. acidophilus showed exponential growth for the first 6 hr until 9 hr, and the pH was maintained close to 4.0-5.0 at 24 hr after culture, consistent with previous studies. The growth curve of L. acidophilus or the pH values were relatively unaffected by co-culture with MS74 cells, confirming that L. acidophilus maintains a low pH in the presence of MS74 cells. This co-culture model could therefore potentially be used to mimic vaginal conditions for future in vitro studies. On the other hand, MS74 cells co-cultured with L. acidophilus more firmly attached to the culture plate, and a higher number of cells were present compared to cells cultured in the absence of L. acidophilus. These results indicate that L. acidophilus increases MS74 cell proliferation and viability, suggesting that lactobacilli may contribute to the healthy environment for vaginal epithelial cells.

Luteinizing hormone-stimulated pituitary adenylate cyclase-activating polypeptide system and its role in progesterone production in human luteinized granulosa cells.

The present study examined the gonadotropin regulation of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type I receptor (PAC(1)-R) expression, and its role in progesterone production in the human luteinized granulosa cells. The stimulation of both PACAP and PAC(1)-R mRNA levels by LH was detected using a competitive reverse transcription-polymerase chain reaction (RT-PCR). PACAP transcript was stimulated by LH reaching maximum levels at 12 hours in a dose dependent manner. LH treatment also stimulated PAC(1)-R mRNA levels within 24 hours. Addition of PACAP-38 (10(-7) M) as well as LH significantly stimulated progesterone production during 48 hours culture. Furthermore, co-treatment with PACAP antagonist partially inhibited LH-stimulated progesterone production. Treatment with vasoactive intestinal peptide, however, did not affect progesterone production. Taken together, the present study demonstrates that LH causes a transient stimulation of PACAP and PAC(1)-R expression and that PACAP stimulates progesterone production in the human luteinized granulosa cells, suggesting a possible role of PACAP as a local ovarian regulator in luteinization.

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.

Hormonal regulation of proprotein convertase subtilisin/kexin type 5 expression during ovarian follicle development in the rat.

The proprotein convertase subtilisin/kexin (PCSKs), a family of subtilisin-like proteases, is the processing enzymes for the activation of many hormone precursors. The present study was designed to identify the PCSK isoform expressed in the ovary and to examine its expression in gonadotropin-stimulated rat ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed an increased expression of Pcsk5 messenger RNA (mRNA) during development with the highest levels at 21 days of age. Treatment of immature rats with PMSG further increased ovarian Pcsk5 expression, and in situ hybridization analysis revealed the localization of Pcsk5 mRNA in theca-interstitial cells of follicles in different sizes. Interestingly, treatment of PMSG-primed rats with hCG resulted in a transient stimulation of ovarian Pcsk5 mRNA levels within 3-6 h. In addition to theca-interstitial cells, hCG treatment induced the expression of Pcsk5 in granulosa cells of preovulatory follicles. Pcsk1, 2 and 4 mRNAs were not detected whereas Pcsk7 mRNA was slightly expressed. Injection of a progestin antagonist RU486 or an inhibitor of 3beta-hydroxysteroid dehydrogenase epostane at 1h before hCG treatment inhibited hCG-induced Pcsk5 mRNA levels. Treatment with LH stimulated both Pcsk5 mRNA and protein levels in preovulatory follicles cultured in vitro. In addition, forskolin but not TPA stimulated Pcsk5 mRNA levels. RNase protection assay revealed that the soluble Pcsk5A variant was the predominant form stimulated by gonadotropins in the ovary. Finally, the predicted proprotein substrates cleaved by PCSK5 were analyzed in preovulatory follicles using regular expressions. The present study demonstrates PCSK5A as the gonadotropin-regulated PCSK isoform in the ovary, and its possible contribution to ovulation by processing pro-TGFbeta and matrix metalloproteinase family.

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.

Heterogeneous rRNA molecules encoded by Streptomyces coelicolor M145 genome are all expressed and assembled into ribosomes.

The Streptomyces coelicolor M145 genome harbors six copies of divergent rRNA operons that differ at ~0.2% and ~0.6% of the nucleotide positions in small subunit (SSU) and large subunit (LSU) rRNA genes, respectively. When these rRNA genes are expressed, a single cell may harbor three different kinds of SSU rRNA and five kinds of LSU rRNA. Primer extension analyses revealed that all of the heterogeneous rRNA molecules are expressed and assembled into ribosomes. This finding and the maintenance of the intragenomic variability of rRNA operons imply the existence of functional divergence of rRNA species in this developmentally complex microorganism.