Stable reference gene selection for Ophiocordyceps sinensis gene expression studies under different developmental stages and light-induced conditions.

The molecular mechanism of Chinese cordyceps formation has received a substantial amount of attention because of its usage as traditional Chinese medicine. The formation process of Chinese cordyceps includes two parts: asexual proliferation (Ophiocordyceps sinensis proliferates in the hemolymph of Thitarodes armoricanus larvae) and sexual development (formation and development of fruiting bodies). Therefore, validation of reference genes under different development stages and experimental conditions is crucial for RT-qPCR analysis. However, there is no report on stable reference genes at the development stage of O. sinensis fruiting body. In this study, 10 candidate reference genes, Actin, Cox5, Tef1, Ubi, 18s, Gpd, Rpb1, Try, Tub1 and Tub2, were selected and calculated their expression stability using four methods: geNorm, NormFinder, BestKeeper, and Comparative △Ct. After comprehensive analysis of the results of these four methods with RefFinder, we determined that the most stable reference genes during asexual reproduction of O. sinensis were Tef1 and Tub1, while the most stable reference genes during fruiting body development were Tyr and Cox5, and the most stable reference genes under light-induced conditions were Tyr and Tef1. Our study provides a guidance for reference genes selections at different proliferation processes with light stress of O. sinensis, and represents a foundation for studying the molecular mechanism of Chinese cordyceps formation.

α-Lipoic acid eliminates dioxin-induced offspring sexual immaturity by improving abnormalities in folic acid metabolism.

Maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes developmental and reproductive disorders in pups due to the attenuated luteinizing hormone (LH) production during the perinatal stage; however, the administration of α-lipoic acid (LA) to TCDD-exposed pregnant rats reversed the attenuated LH production. Therefore, reproductive disorders in pups are expected to be ameliorated with LA supplementation. To address this issue, pregnant rats orally received low dose TCDD at gestational day 15 (GD15) and proceeded to parturition. The control received a corn oil vehicle. To examine the preventive effects of LA, supplementation with LA was provided until postnatal day 21. In this study, we demonstrated that maternal administration of LA restored the sexually dimorphic behavior of male and female offspring. TCDD-induced LA insufficiency is likely a direct cause of TCDD reproductive toxicity. In the analysis to clarify the mechanism of the decrease in LA, we found evidence suggesting that TCDD inhibits the synthesis and increases the utilization of S-adenosylmethionine (SAM), a cofactor for LA synthesis, resulting in a decrease in the SAM level. Furthermore, folate metabolism, which is involved in SAM synthesis, is disrupted by TCDD, which may adversely affect infant growth. Maternal supplementation of LA restored SAM to its original level in the fetal hypothalamus; in turn, SAM ameliorated abnormal folate consumption and suppressed aryl hydrocarbon receptor activation induced by TCDD. The study demonstrates that the application of LA could prevent and recover next-generation dioxin reproductive toxicity, which provides the potential to establish effective protective measures against dioxin toxicity.

Listening to Individuals with Differences in Sex Development or Intersex and Their Families: "Not Doing Surgery Does Not Mean Doing Nothing".

Informed decision-making and considerations of the child's best interest offer a starting place for building informed and lifelong discussions that promote the long-term interests and the well-being of individuals experiencing differences in sex development (DSD) or intersex traits. Parents require sufficient information and support to understand what "doing something and doing nothing" really means when learning about nonsurgical options. This may take the form of health literacy support, asking how parents are given access to meaningful and understandable information, as well as psychosocial support and psychological care. Timely psychological assessment and interventions that support informed decision-making actions are an essential aspect of holistic care for children and youth with DSD and their parents. Without actionable tools or approaches, parents cannot make informed decisions about their child's health and, as such, health literacy is a key attribute to aid decision making for both parents and children. As individuals with DSD become increasingly adept at building resourcefulness and gathering and applying knowledge about their bodies, limiting irreversible surgeries in childhood can afford wider life choices. To this end, an educated and informed comprehensive and helpful multidisciplinary group understands and embodies, as a whole team, the need for compassionate, emotionally supportive, and validating care in all interactions with parents of children and individuals with DSD. The paper draws on the primary author's experiences working with the charity, dsdfamilies, concluding with actionable approaches that include supporting personal knowledge through health literacy, examining team-based psychological care, and psychosocial approaches across the lifespan.

[Effects of electroacupuncture on sexual development and ovarian estrogen receptor ß expression in female adolescent obese rats].

OBJECTIVE: To observe the effect of electroacupuncture on sexual development and ovarian estrogen receptor ß(ER-ß) expression in female adolescent obese rats induced by high-fat diet, so as to explore its underlying mechanisms of improving adolescent obesity. METHODS: Female SD rats (age of 21 days) were randomly divided into control, model and acupuncture groups, with 6 rats in each group. The obese model was established by feeding high-fat diet for 6 weeks. Rats of the acupuncture group received electroacupuncture(2 Hz, 0.5-1.2 mA)stimulation at bilateral "Sanyinjiao"(SP6), "Fenglong"(ST40) and "Zusanli"(ST36) for 30 min, once a day for 14 days. The body mass and abdominal circumference of rats were measured before and after treatment. The contents of serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) were detected by ELISA. The number of corpus luteum and follicle were observed by HE staining. The expression levels of ER-ß mRNA and protein in ovary were detected by fluorescence quantitative PCR and Western blot, respectively. RESULTS: Compared with the control group, the body mass and abdominal circumference, the contents of serum FSH and E2, and the expression levels of ER-ß mRNA and protein in ovary were significantly increased (P<0.05)in the model group, while the number of mature follicles and corpus luteum increased significantly. Compared with the model group, the body mass and abdominal circumference, the contents of serum FSH and E2, and the expression levels of ER-ß mRNA and protein in ovary were significantly decreased (P<0.05) in the acupuncture group, while the number of mature follicles and corpus luteum decreased significantly. CONCLUSION: Electroacupuncture can effectively improve the levels of sex hormone and the development of ovary, down-regulate the expression levels of ER-ß mRNA and protein in ovary, so as to regulate the process of sexual development of female adolescent obese rats induced by high-fat diet.

Cohort profile: pathways to care among people with disorders of sex development (DSD).

PURPOSE: The 'DSD Pathways' study was initiated to assess health status and patterns of care among people enrolled in large integrated healthcare systems and diagnosed with conditions comprising the broad category of disorders (differences) of sex development (DSD). The objectives of this communication are to describe methods of cohort ascertainment for two specific DSD conditions-classic congenital adrenal hyperplasia with 46,XX karyotype (46,XX CAH) and complete androgen insensitivity syndrome (CAIS). PARTICIPANTS: Using electronic health records we developed an algorithm that combined diagnostic codes, clinical notes, laboratory data and pharmacy records to assign each cohort candidate a 'strength-of-evidence' score supporting the diagnosis of interest. A sample of cohort candidates underwent a review of the full medical record to determine the score cutoffs for final cohort validation. FINDINGS TO DATE: Among 5404 classic 46,XX CAH cohort candidates the strength-of-evidence scores ranged between 0 and 10. Based on sample validation, the eligibility cut-off for full review was set at the strength-of-evidence score of ≥7 among children under the age of 8 years and ≥8 among older cohort candidates. The final validation of all cohort candidates who met the cut-off criteria identified 115 persons with classic 46,XX CAH. The strength-of-evidence scores among 648 CAIS cohort candidates ranged from 2 to 10. There were no confirmed CAIS cases among cohort candidates with scores <6. The in-depth medical record review for candidates with scores ≥6 identified 61 confirmed cases of CAIS. FUTURE PLANS: As the first cohort of this type, the DSD Pathways study is well-positioned to fill existing knowledge gaps related to management and outcomes in this heterogeneous population. Analyses will examine diagnostic and referral patterns, adherence to care recommendations and physical and mental health morbidities examined through comparisons of DSD and reference populations and analyses of health status across DSD categories.

Development and evaluation of a patient education programme for children, adolescents, and young adults with differences of sex development (DSD) and their parents: study protocol of Empower-DSD.

BACKGROUND: Differences in sexual development (DSD) are rare diseases, which affect the chromosomal, anatomical or gonadal sex differentiation. Although patient education is recommended as essential in a holistic care approach, standardised programmes are still lacking. The present protocol describes the aims, study design and methods of the Empower-DSD project, which developed an age-adapted multidisciplinary education programme to improve the diagnosis-specific knowledge, skills and empowerment of patients and their parents. METHODS: The new patient education programme was developed for children, adolescents and young adults with congenital adrenal hyperplasia, Turner syndrome, Klinefelter syndrome or XX-/or XY-DSD and their parents. The quantitative and qualitative evaluation methods include standardised questionnaires, semi-structured interviews, and participatory observation. The main outcomes (assessed three and six months after the end of the programme) are health-related quality of life, disease burden, coping, and diagnosis-specific knowledge. The qualitative evaluation examines individual expectations and perceptions of the programme. The results of the quantitative and qualitative evaluation will be triangulated. DISCUSSION: The study Empower-DSD was designed to reduce knowledge gaps regarding the feasibility, acceptance and effects of standardised patient education programmes for children and youth with DSD and their parents. A modular structured patient education programme with four generic and three diagnosis-specific modules based on the ModuS concept previously established for other chronic diseases was developed. The topics, learning objectives and recommended teaching methods are summarised in the structured curricula, one for each diagnosis and age group. At five study centres, 56 trainers were qualified for the implementation of the training programmes. A total of 336 subjects have been already enrolled in the study. The recruitment will go on until August 2022, the last follow-up survey is scheduled for February 2023. The results will help improve multidisciplinary and integrated care for children and youth with DSD and their families. TRIAL REGISTRATION: German Clinical Trials Register, DRKS00023096 . Registered 8 October 2020 - Retrospectively registered.

The CfMcm1 Regulates Pathogenicity, Conidium Germination, and Sexual Development in Colletotrichum fructicola.

Glomerella leaf spot (GLS), caused by Colletotrichum fructicola, is a severe disease worldwide on apple, causing defoliation, leaf and fruit spot, and substantial yield loss. However, little is known about its molecular mechanisms of pathogenesis. Previous transcriptome analysis revealed that a transcription factor, CfMcm1, was induced during leaf infection. In the present work, expression pattern analysis verified that the CfMcm1 gene was strongly expressed in conidia and early infection. Phenotypic analysis revealed that the gene deletion mutant ΔCfMcm1 lost pathogenicity to apple leaves by inhibiting conidial germination and appressorium formation. In addition to appressorium-mediated pathogenicity, ΔCfMcm1 colonization and hyphal extension in wounded apple fruit was also reduced, and conidial germination mode and conidial color were altered. ΔCfMcm1 displayed impairment of cell wall integrity and response to stress caused by oxidation, osmosis, and an acid environment. Furthermore, the deletion mutant produced fewer and smaller perithecia and no ascospores. In contrast, melanin deposition in mycelia of ΔCfMcm1 was strengthened. Further comparative transcriptome and quantitative PCR analysis revealed that CfMcm1 modulated expression of genes related to conidial development (CfERG5A, CfERG5B, CfHik5, and CfAbaA), appressorium formation (CfCBP1 and CfCHS7), pectin degradation (CfPelA and CfPelB), sexual development (CfMYB, CfFork, CfHMG, and CfMAT1-2-1), and melanin biosynthesis (CfCmr1, CfPKS1, CfT4HR1, CfTHR1, and CfSCD1). Our results demonstrated that CfMcm1 is a pivotal regulator possessing multiple functions in pathogenicity, asexual and sexual reproduction, and melanin biosynthesis.

Sex influences in the preventive effects of peripubertal supplementation with N-3 polyunsaturated fatty acids in mice exposed to the two-hit model of schizophrenia.

Schizophrenia is a devastating neurodevelopmental disorder. The animal model based on perinatal immune activation, as first-hit, combined with peripubertal stress, as a second hit, has gained evidence in recent years. Omega-3 polyunsaturated fatty acids (n3-PUFAs) is being a promise for schizophrenia prevention. Nevertheless, the influence of sex in schizophrenia neurobiology and prevention has been neglected. Thus, the present study evaluates the preventive effects of n3-PUFAs in both sexes' mice submitted to the two-hit model and the participation of oxidative changes in this mechanism. The two-hit consisted of polyI:C administration from postnatal days (PNs) 5-7, and unpredictable stress from PNs35-43. n3-PUFAs were administered from PNs30-60. Prepulse inhibition of the startle reflex (PPI), social interaction, and Y-maze tests were conducted between PNs70-72 to evaluate positive-, negative-, and cognitive-like schizophrenia symptoms. We assessed brain oxidative changes in brain areas and plasma. Both sexes' two-hit mice presented deficits in PPI, social interaction, and working memory that were prevented by n3-PUFAs. In two-hit females, n3-PUFAs prevented increments in nitrite levels in the prefrontal cortex (PFC), hippocampus, striatum, and plasma TBARS levels. In two-hit males, n3-PUFAs prevented the increase in TBARS in the PFC, hippocampus, and striatum. Notably, male mice that received only n3-PUFAs without hit exposure presented impairments in working memory and social interaction. These results add further preclinical evidence for n3-PUFAs as an accessible and effective alternative in preventing behavioral and oxidative changes related to schizophrenia but call attention to the need for precaution in this indication due to hit- and sex-sensitive issues.

Effects of Prepubertal Exposure to Aroclor-1221 on Reproductive Development and Transcriptional Gene Expression in Female Rats.

Polychlorinated biphenyls (PCBs), as persistent organic pollutants, are environmental endocrine-disrupting chemicals (EDCs). We aim to investigate the effects of prepubertal exposure to PCBs on the reproductive development and expression and regulation of related genes in rats. Female rats were treated with Aroclor-1221 (A-1221) (4 mg/kg/day, 0.4 mg/kg/day) or castor oil daily from postnatal day (PND) 28 for 2 weeks by gavage. Morphological, histological, hormonal, and biochemical parameters were studied. Lower weight and relative weight of hypothalamus, earlier puberty onset, a longer length of the estrous cycle, lower serum estradiol and progesterone levels, accelerated ovarian folliculogenesis, and higher apoptotic index in the ovary were found. The in vitro fertilization study showed a lower fertilization rate and cleavage rate. The genetic study revealed higher expression of Kiss-1 mRNA and lower expression of GnRH mRNA in the hypothalamus and higher expression of AMH mRNA and lower expression of C-myc mRNA in the ovary. These confirmed the reproductive damage of A-1221 in rats.

Chronic effects of maternal tobacco-smoke exposure and/or α-lipoic acid treatment on reproductive parameters in female rat offspring.

Prenatal tobacco-smoke exposure negatively affects the reproductive functions of female offspring and oxidative stress plays a major role at this point. Alpha-lipoic acid (ALA), well known as a biological antioxidant, has been used as a nutritional supplement and as a therapeutic agent in the treatment of certain complications during pregnancy. We aimed to investigate the effects of maternal tobacco-smoke exposure and/or ALA administration on puberty onset, sexual behavior, gonadotrophin levels, apoptosis-related genes, apoptotic cell numbers and oxidative stress markers in the adult female rat offspring. Sprague-Dawley rats were divided into four groups; control, tobacco smoke (TS), TS+ALA and ALA groups. Animals were exposed to TS and/or ALA for 8 weeks before pregnancy and throughout pregnancy. All treatments ended with birth and later newborn female rats were selected for each experimental group. The experiment ended at postnatal day 74-77. Maternal tobacco smoke advanced the onset of puberty in the female offspring of the TS group (p < 0.05). In all treatment groups; the mean number of anogenital investigations and lordosis quality scores showed a decline, serum luteinizing hormone levels significantly increased (p < 0.05) and several histopathological changes in ovaries were observed compared to the control group. In addition, an increase in apoptotic marker levels and apoptotic cell numbers was detected in the ovaries of all treatment groups. Decreased TAS and increased TOS levels were detected in all treatment groups compared to control. These findings suggested that maternal tobacco smoke and/or ALA administration may be leading to the impaired reproductive health of female offspring. Abbreviations: ALA: alpha-lipoic acid; LH: luteinizing hormone; FSH: follicle-stimulating hormone; TAS: total antioxidant status; TOS: total oxidant status; Apaf1: apoptotic protease-activating factor 1; Casp3: caspase 3; Casp9: caspase 9; CF: cyst follicles; 4-HNE: 4-Hidroxynonenal; 8-OHdG: 8-hydroxydeoxyguanosine; TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine-biotin nick end labeling; ROS: reactive oxygen species; GnRHR: gonadotropin-releasing hormone receptor; HPG: hypothalamic-pituitary-gonadal; AMPK: AMP-activated protein kinase; ELISA: enzyme-linked immunosorbent assay; cDNA: complementary DNA; qPCR: quantitative real-time PCR; FC: follicular cysts; PF: primary follicle; SF: secondary follicle; GF: graafian follicle; CL: corpus luteum; DF: degenerated follicle; AF: atretic follicle.

Caffeine as a promotor of sexual development in sterile Queensland fruit fly males.

Sterile insect technique (SIT) is an environmentally benign pest management technique that involves releasing millions of sterile insects to suppress reproduction of pest populations. Many fruit flies, including Queensland fruit fly (Bactrocera tryoni Froggatt, 'Q-fly'), have long adult maturation periods such that pre-maturation mortality can greatly reduce abundance of sexually active sterile males and impede SIT efficacy. Q-fly is the most difficult and costly challenge to market access for Australia's horticulture industries, and has been targeted for intensive use of SIT program. We here demonstrate potential of pre-release caffeine supplements as a novel means to accelerate sexual maturation in male Q-fly. In mating trials, analytical caffeine was very effective at accelerating sexual maturation, while no positive effects of caffeine-containing instant coffee or guarana supplements were detected. In parallel, development of testes and ejaculatory apodemes was accelerated in males provided analytical caffeine but not instant coffee or guarana. High doses of guarana and instant coffee reduced longevity while even the highest doses of analytical caffeine did not affect longevity. Pre-release caffeine supplements promote sexual maturation in Q-flies, and similar benefits are expected in other fruit flies having long adult maturation periods.

Opposite effects of high- and low-dose di-(2-ethylhexyl) phthalate (DEHP) exposure on puberty onset, oestrous cycle regularity and hypothalamic kisspeptin expression in female rats.

Di-(2-ethylhexyl) phthalate (DEHP) is ubiquitous in the environment and has been proposed to lead to reproductive disruption. In this study, we systematically investigated the effects of different doses of DEHP exposure on female hypothalamic-pituitary-gonadal axis development. Female Sprague-Dawley rats were gavaged with vehicle (corn oil) or DEHP (5 or 500mgkg-1 day-1) during postnatal Days (PNDs) 22-28 or PNDs 22-70. Results demonstrated that the low and high doses of DEHP exerted opposite effects on puberty onset, circulating luteinising hormone, serum oestradiol and progesterone levels, with the low dose (5mgkg-1) promoting and the high dose (500mgkg-1) inhibiting these parameters. Significant dose-related differences were also found in the D500 group with longer oestrous cycle duration, lower ovarian/bodyweight ratio, fewer corpus lutea and more abnormal ovarian stromal tissue in comparison with the oil or D5 groups. Molecular data showed that the hypothalamic Kiss1 mRNA expression in the anteroventral periventricular but not in the arcuate nucleus significantly decreased in the D500 rats and increased in the D5 rats relative to the rats in the oil group. These findings suggested that the kisspeptin system is a potential target for DEHP to disrupt reproductive development and function.

Supplementation of various zinc sources modify sexual development and testicular IGF family gene expression in pre-pubertal male Japanese quail.

Zinc plays an important role in the regulation of insulin-like growth factor-I (IGF-I). IGF system, in turn, has a key role in the development and functions of the reproductive organs. This research was performed to investigate the effects of different sources of zinc on IGF-I gene expression and testicular development in pre-pubertal male Japanese quail. A total of 512 unsexed day-old Japanese quail chicks were randomly divided into 16 groups (4 dietary treatments × 4 replicates) and kept for 35 days. The control group diet was not supplemented with zinc whereas the diets of three groups were supplemented with 25 mg kg-1 zinc oxide (ZnO), zinc oxide nanoparticle (ZnON), and zinc-methionine (Zn-Met). On days 28 and 35, one birds from each subgroup were weighed, bled, and euthanized to evaluate gonado-somatic index (GSI), testicular histology, serum testosterone concentration, cloacal gland index (CGI), and the testicular IGF family gene expression. The results showed that GSI was higher in ZnON (2.307) than control (1.619) on day 35 (P < .05). Germinal epithelium thickness was higher in ZnON (78.88 µm) and Zn-Met (79.73 µm) than control (67.73 µm) on day 35 (P < .05). On day 35, the testosterone concentration was lowest in the control (5.830 ng/ml, P < .05). The CGI of 35-day-old birds was higher in Zn-Met (411.28) than the control (307.59, P < .05). IGF-IR mRNA expression was highest in Zn-Met group on day 28. Therefore, supplementation of diet with Zn-methionine is superior to other sources of zinc for diet supplementation in immature Japanese quail.

Molecular correlates of hypothalamic development in songbird ontogeny in comparison with the telencephalon.

Development of the songbird brain provides an excellent experimental model for understanding the regulation of sex differences in ontogeny. Considering the regulatory role of the hypothalamus in endocrine, in particular reproductive, physiology, we measured the structural (volume) and molecular correlates of hypothalamic development during ontogeny of male and female zebra finches. We quantified by relative quantitative polymerase chain reaction (rqPCR) the expression of 14 genes related to thyroid and steroid hormones actions as well as 12 genes related to brain plasticity at four specific time points during ontogeny and compared these expression patterns with the expression of the same genes as detected by transcriptomics in the telencephalon. These two different methodological approaches detected specific changes with age and demonstrated that in a substantial number of cases changes observed in both brain regions are nearly identical. Other genes however had a tissue-specific developmental pattern. Sex differences or interactions of sex by age were detected in the expression of a subset of genes, more in hypothalamus than telencephalon. These results correlate with multiple known aspects of the developmental and reproductive physiology but also raise a number of new functional questions.

Intestinal delta-6-desaturase activity determines host range for Toxoplasma sexual reproduction.

Many eukaryotic microbes have complex life cycles that include both sexual and asexual phases with strict species specificity. Whereas the asexual cycle of the protistan parasite Toxoplasma gondii can occur in any warm-blooded mammal, the sexual cycle is restricted to the feline intestine. The molecular determinants that identify cats as the definitive host for T. gondii are unknown. Here, we defined the mechanism of species specificity for T. gondii sexual development and break the species barrier to allow the sexual cycle to occur in mice. We determined that T. gondii sexual development occurs when cultured feline intestinal epithelial cells are supplemented with linoleic acid. Felines are the only mammals that lack delta-6-desaturase activity in their intestines, which is required for linoleic acid metabolism, resulting in systemic excess of linoleic acid. We found that inhibition of murine delta-6-desaturase and supplementation of their diet with linoleic acid allowed T. gondii sexual development in mice. This mechanism of species specificity is the first defined for a parasite sexual cycle. This work highlights how host diet and metabolism shape coevolution with microbes. The key to unlocking the species boundaries for other eukaryotic microbes may also rely on the lipid composition of their environments as we see increasing evidence for the importance of host lipid metabolism during parasitic lifecycles. Pregnant women are advised against handling cat litter, as maternal infection with T. gondii can be transmitted to the fetus with potentially lethal outcomes. Knowing the molecular components that create a conducive environment for T. gondii sexual reproduction will allow for development of therapeutics that prevent shedding of T. gondii parasites. Finally, given the current reliance on companion animals to study T. gondii sexual development, this work will allow the T. gondii field to use of alternative models in future studies.

Disturbance in biosynthesis of arachidonic acid impairs the sexual development of the onion blight pathogen Stemphylium eturmiunum.

The formation of sexual fruiting bodies for plant pathogenic fungi is a key strategy to propagate their progenies upon environmental stresses. Stemphylium eturmiunum is an opportunistic plant pathogen fungus causing blight in onion. This self-fertilizing filamentous ascomycete persists in the soil by forming pseudothecia, the sexual fruiting body which helps the fungus survive in harsh environments. However, the regulatory mechanism of pseudothecial formation remains unknown. To uncover the mechanism for pseudothecial formation so as to find a practical measure to control the propagation of this onion pathogen, we tentatively used DNA methyltransferase inhibitor 5-azacytidine (5-AC) to treat S. eturmiunum. 5-AC treatment silenced the gene-encoding monoacylglycerol lipase (magl) concomitant with the presence of the inheritable fluffy phenotype and defectiveness in pseudothecial development. Moreover, the silence of magl also resulted in a reduction of arachidonic acid (AA) formation from 27 ± 3.1 µg/g to 9.5 ± 1.5 µg/g. To correlate the biosynthesis of AA and pseudothecial formation, we created magl knockdown and overexpression strains. Knockdown of magl reduced AA to 11 ± 2.4 µg/g, which subsequently disabled pseudothecial formation. In parallel, overexpression of magl increased AA to 37 ± 3.4 µg/g, which also impaired pseudothecial formation. Furthermore, exogenous addition of AA to the culture of magl-silenced or magl knockdown strains rescued the pseudothecial formation but failed in the gpr1 knockdown strain of S. eturmiunum, which implicates the involvement of AA in signal transduction via a putative G protein-coupled receptor 1. Thus, AA at a cellular level of 27 ± 3.1 µg/g is essential for sexual development of S. eturmiunum. Disturbance in the biosynthesis of AA by up- and down-regulating the expression of magl disables the pseudothecial development. The specific requirement for AA in pseudothecial development by S. eturmiunum provides a hint to curb this onion pathogen: to impede pseudothecial formation by application of AA.

Characterization of the dynamic change of microRNA expression in mice hypothalamus during the time of female puberty.

Puberty onset is a milestone in sexual development. A tumor suppress gene (TSG) network had been reported to be involved in the regulation of female puberty onset. The observations in rodents and primates showed a potential link between microRNAs and puberty onset. To figure out what miRNAs play roles in this important biological process, profilings of microRNAs in the hypothalamus of female mice from three different pubertal stages, juvenile [postnatal day (P10)], early pubertal (P25) and pubertal (P30) were performed on the Affymetrix GeneChip miRNA 3.0 Arrays, the cerebral cortex (CTX) was used as a control tissue. 20 miRNAs were shown to be differentially expressed in hypothalamus (fold change > 1.5, P < 0.05), but not in CTX during the transition from juvenile to pubertal. Four of them were validated by real-time quantitative RT-PCR (qRT-PCR) method. 1018 genes were predicted as the targets of these miRNAs. Further bioinformatics analysis suggested that these target genes were involved in many important signaling pathways, especially in the cancer related pathways. We also found that about 90% of these target genes were expressed in the hypothalamus, as well as in the immortalized GnRH-producing GT1-7 cells, which provided additional evidence that these miRNAs could be female puberty onset related. Here we present a novel comprehensive data set of miRNA gene expression during the puberty onset; and it provides an important recourse for the future functional characterization of individual miRNAs and their targets in mouse hypothalamus and in GT1-7 cells.

Potential Interference of Oil Vehicles on Genital Tubercle Development during the Fetal Period in ICR Mice.

Corn oil, sesame oil, and 10% ethanol in corn oil are commonly used as dosing vehicles in toxicology studies. Since these vegetable oils contain bioactive compounds, it is important for toxicology studies to characterize the toxicities of the dosing vehicles themselves. It has been recently proposed that the width of the genital tubercle (GT), the dorsal-ventral length (D-V length) of the GT, and urethral tube closure in mouse fetuses can be used as novel markers for monitoring sexual development in mice. However, how these parameters are influenced by the dosing vehicles themselves remains unclear. Therefore, we evaluated the effects of corn oil, sesame oil, and 10% ethanol in corn oil on GT width, D-V length, and GT morphology in ICR mice. Our results showed that all three vehicles influenced GT width and D-V length, but not GT morphology, suggesting that the effects of dosing vehicles themselves might need to be considered when GT width or D-V length is used as a parameter to evaluate the effects of chemicals on GT development.

Does long-term androgen deficiency lead to metabolic syndrome in middle-aged rats?

Evidence from clinical observational studies and animal experiments suggests that hypogonadism is associated with the metabolic syndrome. In most of the experiments, androgen deficiency is induced by gonadectomy in the adulthood and relatively short-term effects of hypogonadism on metabolic parameters are usually observed. The purpose of this study was to evaluate the metabolic effects of long-term androgen deficiency starting before puberty in middle-aged male rats. The components of the metabolic syndrome were examined in male, female and gonadectomized male rats at the age of 18months. Sex differences were observed in plasma testosterone, cholesterol, high-density lipoproteins and also in body weight and in glycemia dynamics during oral glucose tolerance test. Gonadectomy and long-term hypogonadism did not affect most of the analyzed metabolic parameters such as blood pressure, glycemia, plasma insulin and uric acid. The only exception was the significantly higher liver enzymes in plasma and triacylglycerol in liver found in gonadectomized males. Except low-density lipoprotein, neither treatment of middle-aged males and females with letrozole, nor supplementation of estradiol as the metabolite of testosterone in gonadectomized male rats changed any of the observed metabolic parameters. Our results suggest that long-term hypogonadism started before puberty does not induce metabolic syndrome in middle-aged male rats, but may affect the liver. Sex differences in metabolic parameters in middle-aged rats are not mediated by testosterone. Whether hypogonadism predispose to metabolic syndrome in combination with other risk factors needs further clarification.

Adolescent binge-pattern alcohol exposure alters genome-wide DNA methylation patterns in the hypothalamus of alcohol-naïve male offspring.

Teenage binge drinking is a major health concern in the United States, with 21% of teenagers reporting binge-pattern drinking behavior in the previous 30 days. Recently, our lab showed that alcohol-naïve offspring of rats exposed to alcohol during adolescence exhibited altered gene expression profiles in the hypothalamus, a brain region involved in stress regulation. We employed Enhanced Reduced Representation Bisulfite Sequencing as an unbiased approach to test the hypothesis that parental exposure to binge-pattern alcohol during adolescence alters DNA methylation profiles in their alcohol-naïve offspring. Wistar rats were administered a repeated binge-ethanol exposure paradigm during early (postnatal day (PND) 37-44) and late (PND 67-74) adolescent development. Animals were mated 24 h after the last ethanol dose and subsequent offspring were produced. Analysis of male PND7 offspring revealed that offspring of alcohol-exposed parents exhibited differential DNA methylation patterns in the hypothalamus. The differentially methylated cytosines (DMCs) were distinct between offspring depending on which parent was exposed to ethanol. Moreover, novel DMCs were observed when both parents were exposed to ethanol and many DMCs from single parent ethanol exposure were not recapitulated with dual parent exposure. We also measured mRNA expression of several differentially methylated genes and some, but not all, showed correlative changes in expression. Importantly, methylation was not a direct predictor of expression levels, underscoring the complexity of transcriptional regulation. Overall, we demonstrate that adolescent binge ethanol exposure causes altered genome-wide DNA methylation patterns in the hypothalamus of alcohol-naïve offspring.