The effect of diethylstilbestrol on inducing abdominal cryptorchidism and relevant genetic expression in rats

Objective To study the effect of diaethylstilbestrol on the transabdomial phase of testicular descent in the rat.Methods Fifty E 13.5(embryonic day 13.5)pregnant female SD rats were randomlv divided into five groups that received a subcutaneous injection of DMSO,2.5、5、10、20 mg/kg DES respectively.Male offspring were killed at E 19.5,and then fetal mortality,the degree of transabdominal testicular ascent(DTA)was determined by a stereomicroscope.The tuRNA expressions of INSL3、SF-1 in the testis and HOXA10 in the gubernaculums were determined by RT-PCR.The expression of INSL3 protein was determined by western blot.Results Higher fetal mortality,larger DTA were induced by DES dose-dependently(P＜0.01).DES down-regulated the expression of INSL-3 and SF-1 in a dose-effect manner(P＜0.01).Compared with the DMSO group,the expression of HOXA10 mRNA in the group of 2.5 mg/kg was not significant difference(P＞0.05),the expression of HOXA10 mRNA in other groups were down-regulated statistically(P＜0.05).Conclusions DES inhibited transabdominal testicular descent dose-dependently via dose-dependent down-regulating the expression of INSL3,which was induced by dose-dependent down-regulating the expression of SF1.HOXA10 may play no role in low-dosage DES induced abdominal cryptorchidism,but down-regulated HOXA10 mRNA was involved in high-dosage DES induced ones. Key words: Cryptorchidism; Diaethylstilbestrol; Rats

Objective To investigate the expression levels and the clinical significance of INSL3 and LGR8 in patients with unilateral cryptorchidism.Methods The expression levels of INSL3 and LGR8 were evaluated using RTRCR in gubernaculum and perididymis in 42 unilateral cryptorchidism (high position 4 cases; middle 31 cases; low 7 cases).Controls were taken from 40perididymis in 40 hydrocele patients and 40 hernia sacs in female indirect inguinal hernia patients.Results The expression levels of INSL3 and LGR8 in perididymis tissues and gubernaculums of different type of unilateral cryptorchidism showed no significant difference (P＞0.05).The expression levels of INSL3 and LGR8 in gubernaculums were lower than those in perididymis tissues (P＜0.05) ; and the expressions of INSL3 and LGR8 in perididymis tissues in unilateral cryptorchidism were lower than those in hydrocele testis (P＜0.05).The expressions of INSL3 and LGR8 in hemia sac of female indirect inguinal hernia were lower than those in perididymis of hydrocele testis (P＜0.05).Conclusions The expression levels of INSL3 and LGR8 in perididymis tissues and gubernaculums of different type of unilateral cryptorchidism have no significant difference.It would imply that INSL3 and LGR8 might have a role in different stages of testicular descent.Lower levels of INSL3 and LGR8 may be associated with altered gubernacular development and cryptorchidism. Key words: Insulin like factor-3; Receptors,G-protein-coupled; Cryptorchidism

Recently, it has been shown that targeted inactivation of the Insl3 gene in male mice results in cryptorchidism. The Insl3 gene encodes insulin-like factor 3 (Insl3), which is expressed in fetal Leydig cells. The testicular factor Insl3 appears to play an important role in the transabdominal phase of testis descent, which involves development of the gubernaculum. Other studies have demonstrated that in utero exposure to diethylstilbestrol (DES), a synthetic estrogen, can lead to cryptorchidism both in humans and in animal models. The present study was undertaken to investigate whether prenatal DES-exposure might interfere with testicular Insl3 mRNA expression. Furthermore, the effect of DES on steroidogenic factor 1 (SF-1) mRNA expression level was determined, since it has been shown that SF-1 plays an essential role in transcriptional activation of the Insl3 gene promoter. Timed pregnant mice were treated with DES (100 microg/kg body weight) or vehicle alone on days E9 (gestational day 9) through E17. Control and DES-exposed mouse fetuses were collected at E16, E17 and E18, when transabdominal testis descent is taking place. Lack of gubernaculum development in DES-exposed animals was confirmed by histological analyses at E17. Expression of Insl3 and SF-1 mRNAs was studied in testes of control and DES-exposed fetuses at E16 and E18 by RNase protection assay. Prenatal DES-exposure resulted in a three-fold decrease in Insl3 mRNA expression level (P<0.005), at both E16 and E18. In contrast, DES treatment had no effect on the expression of SF-1 mRNA. These results support our hypothesis that DES may interfere with gubernaculum development by altering Insl3 mRNA expression, providing a possible mechanism by which DES may cause cryptorchidism.

Objective To investigate the impact of RNA interference silencing HOXA10 gene on proliferation and apoptosis of K562 cell strain of human chronic myeloid leukemia (CML) by construction of eukaryotic expression vector targeting HOXA10.Methods Based on short hairpin RNA(shRNA) oligo that was designed and compounded by the selected specific small interference RNA (siRNA) targeting HOXA 10,the eukaryotic expression vector of pGPHI-GFP-Neo-HOXA10 was successfully constructed and sequenced,and the cationic liposome was then used to transfect K562 cells.There were cell control group (equivalent cells and culture medium only),negative control group (negative control plasmid transfected by liposome)and experimental group (pGPHI- GFP- Neo- HOXA10 transfected by liposome).The HOXA10 mRNA expression was detected by RT-PCR at 24 h after transfection,cell proliferation by MTT at 24,48 and 72 h for cell inhibition ratio,and the apoptosis by flow cytometry at 48 h in all the groups.Results The vector pGPHI-GFP-Neo-HOXA10 was successfully constructed and used to transfect K562 cells.The transfection vector in experimental group could effectively decrease the expression level of HOXA10 mRNA [ (38.86±4.49)％ vs (88.52±9.24)％,(86.75±7.38)％,P＜0.05] as compared with that in cell control and negative control group,with no difference between negative control and cell control group (P＞0.05).Compared with negative control group,the experimental group had a significant decrease in cell proliferation capacity but an increase in cell inhibition ratio at 24,48 and 72 h after transfection [ (39.92±0.74)％ vs (7.98±5.52)％;(55.62±1.18)％ vs (8.27±3.45)％; (66.30±1.26)％ vs (8.63±3.58)％; all P＜0.05].Moreover,the apoptosis rate was significantly increased in experimental group as compared with that in control and negative control groups [ (22.29± 1.67)％ vs (9.82±0.69)％,(10.14±0.96)％,P＜0.05],however,no difference was found between negative control and cell control group (P＞0.05).Conclusion Since eukaryotic expression vector pGPHI-GFP-Neo-HOXA10 can effectively silence the expression of HOXA10 in K562 cells,pGPHI-GFP-Neo-HOXA10 may significantly inhibit proliferation of K562 cell and induce its apoptosis. Key words: RNA,small interfering; K562 cells; Genes,homeobox; Cell proliferation; Apoptosis

Estradiol represses Insulin-like 3 expression and promoter activity in MA-10 Leydig cells

Leukemia is the most common malignant disease in children with high incidence and mortality rates, and a poor treatment effect. The aim of the present study was to examine the changes in the expression of homeobox (Hox) A5 gene and its relationship with cell cycle and apoptosis through the intervention of human K562 myeloid leukemia cell line by all-trans retinoic acid (ATRA), to analyze the role of HOXA5 in the pathogenesis and development process of myeloid leukemia. The optimal concentration of ATRA to be used with K562 cells was determined using a cell counting kit-8 (CCK-8). After 24, 72 and 48 h following treatment of K562 cells with 10 µmol/l ATRA, cell cycle events and apoptosis were measured using flow cytometry. HOXA5 mRNA and protein expression in K562 cells was assessed by RT-PCR and western blot analysis, and the relationship between HOXA5 expression and cell cycle and apoptosis was analyzed. The HOXA5 mRNA and protein expression levels were increased following treatment with ATRA in K562 cells. Apoptosis was increased significantly. The cell cycle was inhibited in G0/G1 phase. Cell proliferation was also inhibited. HOXA5 mRNA and protein expression rates positively correlated with cell apoptosis and the increased percentage and cell cycle of the G0/G1 phase. However, HOXA5 negatively correlated with the reduced percentage of S stage. In conclusion, the expression of HOXA5 in cells was increased following treatment with ATRA in K562 cells, in a time-dependent manner. Additionally, ATRA may inhibit the proliferation of K562 cells and promote apoptosis by upregulating the HOXA5 mRNA and protein expression.

Insulin-like 3 (INSL3) is a small peptide produced by testicular Leydig cells throughout embryonic and postnatal life and by theca and luteal cells of the adult ovary. During fetal life, INSL3 regulates testicular descent in males, whereas in adults, it acts as an antiapoptotic factor for germ cells in males and as a follicle selection and survival factor in females. Despite its considerable roles in the reproductive system, the mechanisms that regulate Insl3 expression remain poorly understood. There is accumulating evidence suggesting that androgens might regulate Insl3 expression in Leydig cells, but transcriptional data are still lacking. We now report that testosterone does increase Insl3 mRNA levels in a Leydig cell line and primary Leydig cells. We also show that testosterone activates the activity of the Insl3 promoter from different species. In addition, the testosterone-stimulating effects on Insl3 mRNA levels and promoter activity require the androgen receptor. We have mapped the testosterone-responsive element to the proximal Insl3 promoter region. This region, however, lacks a consensus androgen response element, suggesting an indirect mechanism of action. Finally we show that mono-(2-ethylhexyl) phthalate, a widely distributed endocrine disruptor with antiandrogenic activity previously shown to inhibit Insl3 expression in vivo, represses Insl3 transcription, at least in part, by antagonizing testosterone/androgen receptor action. All together our data provide important new insights into the regulation of Insl3 transcription in Leydig cells and the mode of action of phthalates.

The insulin-like hormone INSL-3, also named relaxin-like factor (RLF) or Leydig-derived insulin-like peptide (LEY-IL), is expressed in various reproductive tissues and is regarded a marker of differentiation in human testicular Leydig cells. Recently, we have identified differential expression of human INSL-3 in neoplastic Leydig cells and mammary epithelial cells suggesting an involvement of INSL-3 in tumor biology. Here we have investigated the expression of INSL-3 in human thyroid carcinoma cell lines and in the human thyroid gland which has been shown to express transcripts for the G protein coupled INSL-3 receptor LGR8. When we determined the expression of INSL-3 in eight human thyroid carcinoma cell lines, a novel INSL-3 splice variant containing a 95 bp out-of-frame insertion at the beginning of exon II of the INSL-3 gene was discovered. Treatment of the human anaplastic thyroid carcinoma cell line 8505C with diethylstilbestrol (DES) caused a significant dose-dependent transcriptional down-regulation of INSL-3 and a marked up-regulation of LGR8. Employing in situ hybridization to detect INSL-3 transcripts and specific rabbit antisera against the INSL-3 proteins, both INSL-3 isoforms were detected in patients with Graves' disease (n=10), follicular carcinomas (FTC; n=12), papillary carcinomas (PTC; n=9) and undifferentiated anaplastic carcinomas (UTC; n=15). By contrast, thyrocytes of all 15 benign goiter tissues studied were devoid of both INSL-3 isoforms, mRNA and protein. Our data indicate that INSL-3 hormone is up-regulated in hyperplastic and neoplastic human thyrocytes suggesting that the INSL-3 isoforms may serve as additional markers for hyperplastic and neoplastic human thyrocytes. In the anaplastic thyroid carcinoma cell line 8505C, the regulation of both INSL-3 and LGR8 by estrogen may be the first indication of a novel hormonally responsive, auto-/paracrine INSL-3 LGR8 ligand receptor system active in human thyroid carcinoma cells.

Nuclear receptor subfamily 4 group A member1 (NR4A1), an orphan nuclear receptor, is involved in the transcriptional regulation of thecal cell androgen biosynthesis and paracrine factor insulin-like 3 (INSL3) expression. Androgens are known to play an important regulatory role in ovarian follicle growth. Using a chronically androgenized rat model, a preantral follicle culture model and virus-mediated gene delivery, we examined the role and regulation of NR4A1 in the androgenic control of preantral follicular growth. In the present study, Ki67 staining was increased in preantral follicles on ovarian sections from 5α-dihydrotestosterone (DHT)-treated rats. Preantral follicles from DHT-treated rats cultured for 4 d exhibited increased growth and up-regulation of mRNA abundance of G(1)/S-specific cyclin-D2 (Ccnd2) and FSH receptor (Fshr). Similarly, DHT (1 μm) increased preantral follicular growth and Ccnd2 and Fshr mRNA abundance in vitro. The NR4A1 expression was high in theca cells and was down-regulated by DHT in vivo and in vitro. Forced expression of NR4A1 augmented preantral follicular growth, androstenedione production, and Insl3 expression in vitro. Inhibiting the action of androgen (with androgen receptor antagonist flutamide) or INSL3 (with INSL3 receptor antagonist INSL3 B-chain) reduced NR4A1-induced preantral follicular growth. Furthermore, NR4A1 overexpression enhanced DHT-induced preantral follicular growth, a response attenuated by inhibiting INSL3. In conclusion, DHT promotes preantral follicular growth and attenuates thecal NR4A1 expression in vivo and in vitro. Our findings are consistent with the notion that NR4A1 serves as an important point of negative feedback to minimize the excessive preantral follicle growth in hyperandrogenism.

In utero exposure to mono- n-butyl phthalate impairs insulin-like factor 3 gene expression and the transabdominal phase of testicular descent in fetal rats

Failure of the testes to descend into the scrotum (cryptorchidism) is one of the most common birth defects in humans. In utero exposure to estrogens, such as 17beta-estradiol (E2) or the synthetic estrogen diethylstilbestrol (DES), down-regulates insulin-like 3 (Insl3) expression in embryonic Leydig cells, which in turn results in cryptorchidism in mice. To identify the molecular mechanism whereby xenoestrogens block Insl3 gene transcription, we performed a microarray analysis of wild-type or estrogen receptor (ER) alpha-mutant testes exposed in utero to pharmacological doses of E2 or DES. Six and 31 genes were respectively down-regulated and up-regulated by estrogen exposure (> or =4-fold). All six genes down-regulated by estrogen exposure, including Insl3 and the steroidogenic genes steroidogenic acute regulatory protein and cytochrome P450 17alpha-hydroxylase/17,20-lyase, were done so by an ERalpha-dependent mechanism. In contrast, up-regulation was mediated either by ERalpha for 12 genes or by an independent mechanism for the 19 remaining genes. Finally, we show that Insl3 gene expression and testicular descent were not affected by in utero exposure to E2 or DES in ERalpha mutant mice, whereas absence of ERbeta did not influence the effect of these estrogens. Collectively, these data demonstrate that xenoestrogens inhibit the endocrine functions of fetal Leydig cells through an ERalpha-dependent mechanism.

To explore the effects of HOXA10 gene expression in undescended and descended testis. Twenty-four male Sprague Dawley rats were surgically prepared for unilateral cryptorchidism model. Their undescended and descended testes were removed at days 7 (infancy period, group B7) and 60 (sexual maturity period, group B60) post-operation. And contralateral descended testis served as controls (group A7, group A60). The expression of HOXA10 with histologic changes in experimental cryptorchidism were detected with one-step real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Compared with the control group, the levels of HOXA10 gene for groups B7 and B60 decreased markedly to 0.78 ± 0.11 (P < 0.01) and 0.56 ± 0.03 (P < 0.01) respectively.However the levels of HOXA10 gene and protein in group B60 were significantly lower than those in group B7 (P < 0.01). There is a lower expression of HOXA10 in undescended testes than in normally developed counterparts. And the expression of HOXA10 decreases with the elapsing time of cryptorchidism.

Objective To investigate the methylation of HOXA4 in breast cancer patients and its correlation with clinicopathological characteristics. Methods NEBNext® Ultra™ RNA Library Prep Kit for Illumina® was used for gene expression microarray and the screening of abnormally expressed genes in breast cancer tissues and adjacent breast tissues from 9 breast cancer patients (enrolled by a random number table)in Bao’an Maternal and Child Health Hospital in 2014. Illumina Infinium HD Methylation450K Assay was used for DNA methylation microarray and detection of differentially methylated genes in breast cancer. Then the differentially expressed genes and methylated genes in breast cancer were further explored based on The Cancer Genome Atlas (TCGA). The genes with significant hypermethylation and low expression were selected. Combined with bioinformatics, HOXA4 was identified as a candidate gene, with the potential for the detection of early breast cancer. The methylation and mRNA expression of HOXA4 gene in breast cancer tissues and adjacent breast tissues from 86 breast cancer patients (enrolled by a random number table)in Bao’an Maternal and Child Health Hospital from 2014 to 2017 were detected by pyrophosphoric acid sequencing and RT-PCR. And the correlation between HOXA4 mehtylation and the clinicopathological characteristics was also analyzed by the Fisher exact test. Cox proportional hazards model was used for univariate and multivariate analysis of risk factors. The breast cancer MCF-7 cells were treated with 0, 0.5, 1, 5, 10, 20 μmol/L methylation inhibitor RG108 for 5 days, then HOXA4 mRNA expression was detected. Results The gene expression microarray showed that 1 680 upregulated genes and 1 249 downregulated genes were determined in breast cancer tissue. The overall methylation levels in different regions of breast cancer tissues were significantly higher than those in adjacent tissues (All P<0.001). In 86 breast cancer patients, the methylation rate of HOXA4 gene was 95% (82/86) in breast cancer tissue(52 samples with low methylation and 30 with hypermethylation), 57% (49/86) in the corresponding adjacent tissues (49 samples with low methylation and none with hypermethylation) (χ2=4.779, P=0.029). The expression of HOXA4 mRNA in HOXA4 methylation group was significantly lower than that in non-methylation group (P=0.031). HOXA4 methylation was correlated with lymph node metastasis(P=0.039) and ER negative (P=0.017). Univariate analysis showed that TNM stage, histological grade, lymph node metastasis and HOXA4 methylation were risk factors for DFS (RR=4.008, 95%CI=1.296-12.393, P=0.016; RR=10.111, 95%CI=2.607-39.217, P=0.001; RR=4.588, 95%CI=1.201-17.523, P=0.026; RR=1.051, 95%CI=1.007-1.098, P=0.024). Multivariate analysis showed that histological grade was an independent prognostic factor for DFS in breast cancer patients (RR=14.461, 95%CI=2.429-86.100, P=0.003). After treatment with different concentrations of RG108, the expression of HOXA4 mRNA in MCF-7 cells was significantly different among six groups (χ2=4.472, P=0.029). Conclusion The methylation of HOXA4 plays an important role in the occurrence and development of breast cancer, which may serve as a novel molecular biological marker for clinical diagnosis of breast cancer. Key words: Breast neoplasms; Gene, HOXA4; Methylation

Endocrine Society classified patients with serum 25-hydroxyvitamin D [25(OH)D] levels below 20 ng/ml as deficiency, 20-30 ng/ml as insufficiency, and >30 ng/ml as replete. This study was planned to investigate the relationship between serum vitamin D level and homeobox 10 mRNA expression in women with polycystic ovary syndrome (PCOS). Thirty women with PCOS who failed the first IVF/ICSI attempt and were decided to have endometrial injury before second attempt were included in the study. Before the endometrial injury, the serum vitamin D levels of the women were measured, and they were divided into three equal groups as proposed by the Endocrine Society. Group 1 consisted of vitamin D deficient women (<20 ng/mL), Group 2 consisted of vitamin D insufficient women (20-30 ng/mL), and Group 3 consisted of vitamin D replete women (>30 ng/mL). Women in each group were injured with a Pipelle cannula during mid-luteal phase. Endometrial samples collected during injury were analyzed for HOXA10 mRNA expression by RT-PCR and correlated with serum vitamin D level. When analyzing the results according to different vitamin D thresholds, as proposed by the Endocrine Society, HOXA10 mRNA expression was comparable between vitamin D deficient and vitamin D insufficient women. The HOXA10 mRNA expression of vitamin D replete women(Group 3) was found to be higher than both vitamin D deficient (Group 1) and vitamin D insufficient women (Group 2). HOXA10 mRNA expression of the women in Group 3 was 3.3-fold higher than Group 1 and 2.6-fold higher than Group 2. HOXA10 mRNA expression was correlated to the levels of vitamin D in the Group 3 (r=0.655, p=0.02). There was no significant correlation between serum vitamin D levels and endometrial HOXA10 mRNA expression of women in both Group 1 (r=0.343, p=0.06) and Group 2 (r=0.456, p=0.08). Endometrium of women with PCOS with sufficient serum vitamin D levels express significantly higher HOXA10 mRNA than patients with low serum vitamin D levels.

Objectives: The downregulation of anti-adhesive regulatory proteins and upregulation of adhesive genes are critical for the receptive endometrium. This study was designed to determine whether switching between the anti-adhesive podocalyxin (PDX) and adhesive HOXA10 receptivity modulator occurs in the endometrium of women with recurrent implantation failure (RIF). Methods: Twenty-four patients with RIF who could not conceive for three or more cycles despite good-quality embryo transfer constituted the study group. Twenty-four patients with unexplained infertility (UEI) matched for age, BMI, and infertility duration were included in the control group. Twenty women scheduled to undergo intrauterine device (IUD) placement for birth control were included in the comparative group. Endometrial tissue was collected from patients with RIF and UEI during egg retrieval after ovarian stimulation using the GnRH antagonist protocol. In the fertile group, endometrial tissues were collected during IUD insertion. HOXA10 mRNA expression was analyzed by qRT-PCR and PDX protein expression was analyzed by ELISA. The relative expression of endometrial HOXA10 mRNA was calculated using the 2-ΔΔCt equation. Results: The relative expression of HOXA10 mRNA in the RIF group was significantly lower than that in the UEI group (p < 0.001). HOXA10 mRNA expression in the fertile group was significantly higher than that in the RIF group and was similar to that in the UEI group. PDX expression in the RIF group was significantly higher than that in the UEI group (p < 0.001). PDX expression in the fertile group was significantly lower than in the RIF and UEI groups. A negative correlation was detected between the anti-adhesive PDX protein and adhesive HOXA10 (r = -0.797, p < 0.001). Although there was a positive correlation between endometrial thickness recorded on the day of hCG administration and HOXA10 (r = 0.590, p < 0.01), endometrial thickness was negatively correlated with PDX (r = -0.529, p < 0.01). Conclusions: The failed physiological downregulation of the anti-adhesive PDX protein in patients with RIF prevented the upregulation of adhesive HOXA10 mRNA.