[Analysis of the feasibility and prognostic value of circulating tumor DNA in detecting gene mutations in small cell lung cancer].

Objective: To investigate the feasibility of circulating tumor DNA (ctDNA) in detecting small cell lung cancer (SCLC) gene mutations and its prognostic value in chemotherapy and/or radiotherapy for SCLC patients. Methods: A total of 77 SCLC patients who were admitted to the Department of Thoracic Medical Oncology and the Department of Thoracic Radiation Oncology of Zhejiang Cancer Hospital from July 2016 to November 2019 were included. There were 66 males and 11 females, with a median age of 60 years. Among them, 42 cases were in limited stage (LS) and 35 cases were in extensive stage (ES). Next-generation sequencing (NGS) of patients' plasma ctDNA was performed before treatment. The differences of mutated genes and signaling pathways between LS and ES patients were analyzed and compared. Blood-based tumor mutation burden (bTMB) was calculated according to detected somatic cell mutations. Patients were divided into the high bTMB and the low bTMB groups according to the optimal threshold calculated by R software. Log-rank tests were used to compare progression-free survival (PFS) between the high bTMB and the low bTMB groups. Results: Among the 77 patients, 76 patients had gene mutations detected in their plasma, and the positive rate of ctDNA test was 98%. Among the 76 patients, the genes with the highest mutation frequency were TP53 (89%), RB1 (70%), LRP1B (34%), CREBBP (21%), MLL3 (21%), MLL2 (16%), NOTCH1 (13%), ROS1 (13%), BRCA2 (12%), and PTPRD (12%). The most common mutated genes in LS patients were TP53 (90%), RB1 (68%), LRP1B (24%), MLL2 (22%), and BRCA2 (17%); the most common mutated genes in ES patients were TP53 (89%), RB1 (71%), LRP1B (46%), CREBBP (31%), and MLL3 (29%). The mutation rates of NOTCH1 and CREBBP genes were significantly higher in ES patients (31.4% and 22.9%) than those in LS patients (11.9% and 4.8%) (both P<0.05). Signaling pathway analysis showed that there were more NOTCH pathway gene variations in ES patients. Among LS patients, patients in the high bTMB group (≥ 6.96 mutations/Mb) had a longer PFS than that in the low bTMB group (<6.96 mutations/Mb) (P=0.033); but no such difference was noted in ES patients. Conclusion: Plasma ctDNA sequencing detected SCLC gene mutation profiles similar to those reported in previous literature, thus ctDNA could be used as a tool to study SCLC genomics; the mutation spectra of ES-SCLC and LS-SCLC were different. bTMB has potential prognostic value in LS-SCLCs treated with chemoradiotherapy.

[Meta-analysis of the effects of dexmedetomidine combined with ketamine during dressing changes in burn patients].

Objective: To systematically evaluate the safety and efficacy of dexmedetomidine combined with ketamine during dressing changes in burn patients using meta-analysis. Methods: Foreign language databases including PubMed, Cochrane Central, Embase, and Web of Science were searched with the terms of " burns, dexmedetomidine, ketamine, and dressing" , and Chinese databases including Chinese Journal Full-Text Database, Wanfang Data, and China Academic Journal Network Publishing Database were searched with the terms in Chinese version of ",,,,," to retrieve the publicly published randomized controlled trials on the application of dexmedetomidine combined with ketamine for sedation and analgesia during dressing changes in burn patients from the establishment of each database to March 2019. The outcome indexes included systolic blood pressure at 5 minutes after administration, arousal restlessness score, ketamine dosage, dressing change time, body movement/recovery time, pain score, Ramsay sedation scores at 10 minutes after the start of dressing change and 1 hour after dressing change, physician satisfaction score, neuropsychological symptoms, nausea and vomiting times, nausea and vomiting score. RevMan 5.3 and Stata 14.0 statistical software were used to conduct a meta-analysis of eligible studies. Results: A total of 396 burn patients were included in 7 articles, including 198 patients in dexmedetomidine+ ketamine group who received dexmedetomidine and ketamine for sedation and analgesia, and 198 patients in ketamine alone group who received ketamine alone for sedation and analgesia. The bias risks of the seven studies included were uncertain. Compared with those of ketamine alone group, the systolic blood pressure at 5 minutes after administration, arousal restlessness score, nausea and vomiting score of patients in dexmedetomidine+ ketamine group were significantly decreased, with standardized mean differences of -13.89, -0.84, and -0.99 (95% confidence interval=-20.89--6.89, -1.17--0.52, -1.31--0.68, P<0.01), the Ramsay sedation score at 10 minutes after the start of dressing change and that at 1 hour after dressing change were significantly increased, with standardized mean differences of 1.53 and 0.72 (95% confidence interval=1.05-2.02, 0.13-1.31, P<0.05 or P<0.01), and the number of neuropsychological symptom and number of nausea and vomiting were significantly reduced, with relative risks of 0.20 and 0.16 (95% confidence interval=0.07-0.58, 0.05-0.58, P<0.01). The patients in the two groups were similar in ketamine dosage, dressing change time, body movement/recovery time, pain score, and physician satisfaction score. There was no publication bias in dressing change time or ketamine dosage (P>0.05), while the other indexes might have publication bias (P<0.05). Conclusions: Compared with ketamine alone, combination of dexmedetomidine and ketamine during dressing changes in burn patients can reduce the occurrence of restlessness, nausea and vomiting, neuropsychological symptoms, and other complications, better stabilize blood pressure, and enhance sedation effect.

[A case of chronic ulcer with ectopic ossification in lower right extremity].

On January 1st, 2019, one male patient, aged 55 years, with chronic ulcer in right lower extremity was admitted to Northern Jiangsu People's Hospital. After admission, ulcer debridement and vacuum sealing drainage (VSD) surgery was performed in lower right extremity. Two pieces of bone-like tissue was removed from the lateral space between the Achilles tendon and fibula in the right leg, which was confirmed as ossification tissue by histopathological sections after surgery. The wounds were treated with VSD technique and dressing changes. On the 49th day after surgery, split-thickness skin graft was taken from the lateral left thigh and grafted to the wound area, which was then treated with VSD. The skin graft of patient survived well. The wound healed completely and the patient was discharged from hospital on day 77 post hospitalization. This case suggests that the patients with chronic ulcer should complete all related examinations in time, and improving the blood circulation of the wound and clearing the ectopic ossification in the wound are critical for wound healing.

Genomic origin and EGFR-TKI treatments of pulmonary adenosquamous carcinoma.

BACKGROUND: Adenosquamous carcinoma (ASC) of the lung is a heterogeneous disease that is composed of both adenocarcinoma components (ACC) and squamous cell carcinoma components (SCCC). Their genomic profile, genetic origin, and clinical management remain controversial. PATIENTS AND METHODS: Resected ASC and metastatic tumor in regional lymph nodes (LNs) were collected. The ACC and SCCC were separated by microdissection of primary tumor. The 1021 cancer-related genes were evaluated by next-generation sequencing independently in ACC and SCCC and LNs. Shared and private alterations in the two components were investigated. In addition, genomic profiles of independent cohorts of adenocarcinomas and squamous cell carcinomas were examined for comparison. We have also carried out a retrospective study of ASCs with known EGFR mutation status from 11 hospitals in China for their clinical outcomes. RESULTS: The most frequent alterations in 28 surgically resected ASCs include EGFR (79%), TP53 (68%), MAP3K1 (14%) mutations, EGFR amplifications (32%), and MDM2 amplifications (18%). Twenty-seven patients (96%) had shared variations between ACC and SCCC, and pure SCCC metastases were not found in metastatic LNs among these patients. Only one patient with geographically separated ACC and SCCC had no shared mutations. Inter-component heterogeneity was a common genetic event of ACC and SCCC. The genomic profile of ASC was similar to that of 170 adenocarcinomas, but different from that of 62 squamous cell carcinomas. The incidence of EGFR mutations in the retrospective analysis of 517 ASCs was 51.8%. Among the 129 EGFR-positive patients who received EGFR-TKIs, the objective response rate was 56.6% and the median progression-free survival was 10.1 months (95% confidence interval: 9.0-11.2). CONCLUSIONS: The ACC and SCCC share a monoclonal origin, a majority with genetically inter-component heterogeneity. ASC may represent a subtype of adenocarcinoma with EGFR mutation being the most common genomic anomaly and sharing similar efficacy to EGFR TKI.

[Advances in the research of heterotopic ossification caused by burns].

Heterotopic ossification is a rare complication of burns, and its incidence and risk factors are still unclear. Through summarizing the literature on heterotopic ossification caused by burns at home and abroad, the author searched for the risk factors of heterotopic ossification after burn and the new progress of its prevention and treatment. It was realized that the size, depth and healing time of burn wounds were related to heterotopic ossification; the nonsteroidal anti-inflammatory drugs, radiation therapy, and their combination therapy can be used for the prevention of heterotopic ossification; surgery is an effective means of treating heterotopic ossification.

Use of Raman spectroscopy to identify active spermatogenesis and Sertoli-cell-only tubules in mice.

Microdissection testicular sperm extraction (micro-TESE) has become the first line therapy to harvest spermatozoa for men with nonobstructive azoospermia. However, the pitfall is that the selection of seminiferous tubules depends on subjective assessment of the colour and size of tubules, which cannot guarantee successful retrieval of spermatozoa. The aim of this study was to determine whether Raman spectroscopy (RS) could distinguish tubules with spermatogenesis from Sertoli-cell-only (SCO) tubules, and potentially serve as a useful tool to improve sperm retrieval rates. Fourteen male adult mice were divided into two groups: SCO group received a single intraperitoneal injection of busulfan (40 mg per kg body weight), and the control group received a placebo dose of 0.9% saline solution. Mice were sacrificed after 4 weeks, and the testicular tissue was assessed by RS and then confirmed with histopathology. The results indicated that tubules with spermatogenesis had intensified Raman peaks at 748, 1124, 1309, 1446 and 1658 cm-1 compared to SCO tubules, except a decreased peak at 1582 cm-1 . RS was able to distinguish the two groups with a sensitivity of 91.2% and specificity of 82.9%. In conclusion, RS may serve as a useful diagnostic tool prior to sperm retrieval.

Tumour budding is a reproducible index for risk stratification of patients with stage II colon cancer.

AIM: High-risk patients with Stage II colon cancer may benefit from adjuvant chemotherapy, but it is difficult to identify such a patient group. A robust and reproducible index would be helpful to select the subset of Stage II colon cancer patients at high risk. This study investigated the potential prognostic significance of tumour budding in Stage II colon cancer. METHOD: In all, 135 Stage II colon cancer patients with known outcome were identified. The degree of tumour budding was assessed by two individual observers and was classified, according to the number of tumour buds in the area with the greatest budding intensity on haematoxylin and eosin slides, as high-grade budding (10 or more tumour buds) and low-grade budding (0-9 buds). Inter-observer agreement for two observers was assessed by using the kappa test. Progression-free and cancer-specific survivals were analysed using the Kaplan-Meier method and Cox regression. RESULTS: The 5-year progression-free survival rates for patients with high-grade tumour budding (n = 36) and those with low-grade budding (n = 99) were 57.6% and 89.0% (P < 0.001). The 5-year cancer-specific survival rates were 66.7% vs 92.0% (P < 0.001). Cox regression analyses demonstrated tumour budding as an independent predictor of disease progression (hazard ratio 4.982, P < 0.001) and cancer-related death (hazard ratio 4.142, P = 0.003). The two observers agreed on the classification of tumour budding in 118 cases (87.4%) and the inter-observer agreement was good (κ = 0.692). CONCLUSION: Tumour budding is a strong and reproducible prognostic factor for adverse outcome in Stage II colon cancer, which may serve as a prognostic marker to identify patients with a high risk of recurrence who may benefit from adjuvant therapy.

Electric-field control of nonvolatile magnetization in Co40Fe40B20/Pb(Mg(1/3)Nb(2/3))(0.7)Ti(0.3)O3 structure at room temperature.

We report a large and nonvolatile bipolar-electric-field-controlled magnetization at room temperature in a Co(40)Fe(40)B(20)/Pb(Mg(1/3)Nb(2/3))(0.7)Ti(0.3)O(3) structure, which exhibits an electric-field-controlled looplike magnetization. Investigations on the ferroelectric domains and crystal structures with in situ electric fields reveal that the effect is related to the combined action of 109° ferroelastic domain switching and the absence of magnetocrystalline anisotropy in Co(40)Fe(40)B(20). This work provides a route to realize large and nonvolatile magnetoelectric coupling at room temperature and is significant for applications.

A decision analysis of treatments for obstructive azoospermia.

BACKGROUND: Treatments for post-vasectomy obstructive azoospermia include vasectomy reversal, microsurgical epididymal sperm aspiration (MESA) or percutaneous testicular sperm extraction (TESE) with IVF/ICSI. We examined the cost-effectiveness of these treatments. METHODS: A decision analytic model was created to simulate treatment. Outcome probabilities were derived from peer-reviewed literature and the Society for Assisted Reproductive Technologies database. Procedural costs were derived from a sampling of high-volume IVF centers and the Medicare Resource Based Relative Value Scale. Indirect costs of complications, lost productivity and multiple gestation pregnancies were considered. Sensitivity analyses were performed. RESULTS: Vasectomy reversal was more cost-effective than either MESA or TESE under all probability conditions. In 1999, vasectomy reversal demonstrated superior cost-effectiveness to TESE and MESA ($19,633 versus $45,637 and $48,055, respectively, equivalent to $25,321 versus $58,858 and $61,977 in 2005 dollars). In 2005, vasectomy reversal ($20,903) remained the most cost-effective treatment over TESE ($54,797) and MESA ($56,861). The cost-effectiveness of all treatments improved over projections by inflation. The relative cost-effectiveness of the therapies was unchanged over time. CONCLUSIONS: Vasectomy reversal appears more cost-effective than percutaneous TESE and MESA for treatment of obstructive azoospermia when the impact of indirect costs is considered. The absolute cost-effectiveness of all therapies improved over time. These results may be tailored with institution-specific data to allow more individualized results.

Subcutaneous extralesional triamcinolone acetonide injection versus conservative management in the treatment of chalazion.

OBJECTIVE: To compare the efficacy of subcutaneous extralesional triamcinolone acetonide injection versus conservative treatment for chalazion. DESIGN: Randomised controlled trial. SETTING: Eye clinics of two regional hospitals in Hong Kong. PATIENTS: Patients over 18 years old presenting with primary chalazion were randomised into two groups. In group 1, 12 patients were treated with lid hygiene, warm compresses, and chloramphenicol 1% ointment 4 times a day. In group 2, 16 patients were treated with 0.3 mL triamcinolone acetonide (10 mg/mL) injection to the subcutaneous tissue extralesionally via the percutaneous route. Exclusion criteria were: acutely infected chalazion with preseptal cellulitis, recurrent chalazion, small chalazion (< or =2 mm), and prior treatment to chalazion. MAIN OUTCOME MEASURES: Size of chalazion, recurrence of chalazion, intra-ocular pressure, and complications from treatment, including skin pigmentary change or atrophy and pyogenic granuloma. RESULTS: There was a clinically and statistically significant difference between the success rates in group 1 (58.3%) and group 2 (93.8%). In group 1, the mean prior duration of chalazion before treatment was significantly shorter in success cases than in failed cases. One patient with multiple chalazia in group 2 developed hypopigmentary skin changes at one treatment site. CONCLUSION: Subcutaneous extralesional triamcinolone acetonide injection was more effective than conservative treatment for chalazion.

Heterogeneous mutations in the SLC3A1 and SLC7A9 genes in Chinese patients with cystinuria.

Cystinuria is a recessively inherited aminoaciduria that leads to recurrent urolithiasis. It is caused by the defective transport of cystine and dibasic amino acids in the proximal renal tubules and intestinal epithelium. Two genes responsible for this, SLC3A1 and SLC7A9, are known. Patients with two SLC3A1 mutations are classified as type A cystinuria, whereas patients with two SLC7A9 mutations are classified as type B cystinuria. Few clinical and molecular data have been reported for Asian cystinuria patients. In this study, we determined the molecular basis of cystinuria in eight unrelated Chinese subjects. Coding exons and flanking introns of the SLC3A1 and SLC7A9 genes were directly sequenced after amplification by polymerase chain reaction. Five different SLC3A1 mutations were found. Two missense mutations, D210G and S547L, were novel. The other three SLC3A1 mutations (IVS6+2T>C, R181Q and R365W) have been described previously. In addition, four novel SLC7A9 mutations, C137R, c.730delG, IVS10+2_3delTG and IVS12+3insT, together with two previously reported mutations (A70V and G195R) were found. All patients except one carried compound heterozygous mutations. IVS12+3insT was detected in patients from two families. This is the first molecular genetic study on Chinese cystinuria patients. Three patients with type A cystinuria, two with type B cystinuria, and three carriers of type B cystinuria were identified. Our results suggest that the molecular basis of cystinuria is heterogeneous in our local population.

Robotic microsurgical vasovasostomy and vasoepididymostomy in rats.

Robotic and robotic assisted procedures are rapidly increasing in popularity amongst practicing surgeons and urologists. Robotic techniques are now commonly used to perform radical prostatectomies for prostate cancer and pyeloplasties for uretero-pelvic junction obstruction. Other robotic procedures include robotic assisted coronary artery bypass and partial nephrectomies. We initially studied the da Vinci surgical robot in order to determine the feasibility of adapting the robot to microsurgical vasovasostomy and vasoepididymostomy. In this paper, we describe our experience in developing a set of practice exercises using the da Vinci robot to prepare for these microsurgical procedures. We also review the design and results of our studies of robotic microsurgical vasovasostomy and vasoepididymostomy in rats.Finally, we discuss the potential future roles of robotic microsurgery in urology and surgery.

Correlation of ultrasonographic and orchidometer measurements of testis volume in adults.

OBJECTIVE: To determine the correlation between testicular volume measured with an orchidometer or high-resolution scrotal ultrasonography (US) with colour-flow Doppler analysis. PATIENTS AND METHODS: In all, 159 men (mean age 36.6 years) presenting for infertility evaluation underwent both a physical examination by a one experienced examiner and high-resolution US with colour-flow Doppler analysis. An orchidometer was also used to measure testicular volume after stretching the scrotal skin tightly over the testis and after warming with a heating pad. The US was interpreted by a radiologist who had no knowledge of the orchidometer estimates. The volume was calculated as 0.71 x length x width x height. RESULTS: For the right testes the mean orchidometer and US estimates were 18.4 and 18.3 mL, yielding a correlation coefficient of 0.72 (r (2) = 0.52, P < 0.01). On the left the respective values were 17.1 and 16.9 mL, with a correlation coefficient of 0.69 (r (2) = 0.48, P < 0.01). CONCLUSION: Orchidometer estimates of testicular volume correlate closely and very significantly with US estimates in adults. In the hands of an experienced examiner orchidometer measurements provide an accurate, rapid and inexpensive assessment of testicular volume.

Is bag urine culture useful in monitoring urinary tract infection in infants?

OBJECTIVES: We studied the false-positive rate of bag urine cultures in diagnosing bacteriuria in infants and examined the factors responsible for contamination. METHODS: One hundred asymptomatic patients with previous urinary tract infections (UTI; age range 5-23 months; sex ratio M : F 73:27) were screened by bag urine cultures and confirmed by suprapubic aspiration or catheterization. Those producing contaminated and clean samples were interviewed with a standard questionnaire. RESULTS: Sixty patients had insignificant growth and 40 had doubtful or mixed growth. After further interview and instructions, the latter group produced a second bag urine sample with negative results in 23 and positive results in 17. Among the latter, UTI was confirmed in five and excluded in 12 cases. The false-positive rate of first urine collection was 36.8%. Comparing patients with contaminated urine to those with negative urine samples, we found the former were associated with waiting for more than one void to complete urine collection, and with uncircumcised boys. Other factors did not reach statistical significance. Proper counselling and repeating a second culture reduced the overall false-positive rate to 12.6%. CONCLUSION: In our population, the contamination rate of one bag urine culture was 36.8%, which is unacceptable and alternative methods need to be sought. Contamination was associated with improper collection procedures and with uncircumcised boys. Proper instructions and doing a second urine culture reduced the overall rate to 12.6%, but at the expense of delaying diagnosis and treatment.

Expression of human BRE in multiple isoforms.

BRE, a putative stress-modulating gene, found able to down-regulate TNF-alpha-induced NF-kappaB activation upon overexpression, is now shown in human cells expressed as multiple mRNA isoforms. A total of six isoforms are produced by alternative splicing predominantly at either end of the gene. Predicted from the cDNA sequences of these isoforms, three of them (alpha(a), alpha(b), and alpha(c)) code for BRE of different C-terminus, and the other three (beta(a), beta(b), and beta(c)) may possibly be the nonfunctional counterparts. All human cells examined coexpress all the predominant splice variants, albeit at different ratios. Comparing with normal cells, immortalized human cell lines uniformly express higher levels of BRE. Interestingly, peripheral blood monocytes responded to LPS by down-regulating the expression of all the BRE isoforms, which was however less obvious in the cell line counterpart, THP-1. Isoform alpha(a), which codes for the canonical BRE with a C-terminal peroxisomal targeting sequence, is the most abundant transcript. We propose that the function of BRE and its isoforms is to regulate peroxisomal activities.

Self-expandable chitosan stent: design and preparation.

Stents are largely used in surgical procedures to relieve pathological obstructions. The purpose of the present study was to design and prepare a biocompatible stent with a self-expandable mechanism. Thin films were prepared from deacetylated chitosan (4% w/v) dissolved in acetic acid solution (2% v/v). The chitosan films were tested by a calibrated tensiometer to measure the Young's module (E). The films were used to manufacture stents by pulling and winding them around a cylindrical rod in a helical fashion. Thirteen stents (diameter = 0.5 +/- 0.05 mm, length approximately 4 mm) were inserted into the vas deferens of wistar rats. Upon stent insertion, the vasal anastomosis was achieved with a laser-soldering technique. The animals were sacrificied 8 weeks later. The stress test showed that the chitosan film was elastic (maximum strain = 105% +/- 6%, E = 0.7655 +/- 0.0288 Mpa). The stents self-expanded by releasing their elastic energy. All the stents but one remained open inside the vasa despite high incidence of sperm granuloma. A biocompatible and self-expandable stent with a helical design is proposed.

Evaluation of Th1/Th2 ratio and cytokine production profile during acute exacerbation and convalescence in asthmatic children.

BACKGROUND: Th2-type cytokines, such as IL-4 and IL-5, are generally believed to play an important role in the pathogenesis of allergic asthma. In contrast, Th1-type cytokine, especially interferon (IFN)-gamma, is thought to have a downregulatory effect on Th2 immune response cells. Thus, the imbalance of Th1 and Th2 cells may be a key factor in relation to disease severity. OBJECTIVE: The aim of this study is to examine the changes in the Th1/Th2 ratios and cytokine production profiles of asthmatic children at acute attacks and convalescent stages. METHODS: Twelve asthmatic patients were included in this study. The percentages of IFN-gamma- or IL-4-producing cells were determined with a flow-cytometric method of intracellular protein detection. Fresh whole blood obtained from normal controls and patients at two stages was stimulated with brefeldin A, phorbol myristate acetate, and ionomycin for 4 hours. Cells were fixed and stained intracellularly with fluorescein isothiocyanate- or phycoerythrin-conjugated antibody specific to each cytokine in combination with anti-CD4. ELISA assays were applied to measure cytokine concentrations of supernatant from peripheral blood mononuclear cells (PBMC) activated with phorbol myristate acetate and ionomycin for 24 hours. RESULTS: Among CD4+ cells, the percentage of IL-4+ cells decreased significantly from 8.18 +/- 4.77% at acute attacks to 4.18 +/- 1.26% (P < .020) at convalescence. The percentage of IFN-gamma+ cells also decreased from 13.49 +/- 4.21% to 9.03 +/- 5.42% (P < .052). The Th1/Th2 ratios of patients at the two stages were similar, and both were lower than the normal controls. Significantly higher IL-5 and lower IFN-gamma production were detected from activated PBMC of asthmatic patients than normal controls. CONCLUSIONS: The decrease of IFN-gamma+ and IL-4+ cells detected at the single-cell level may explain the potential mechanism of convalescence from acute asthma attacks. High Th1/Th2 ratio and low IL-5 production from the PBMC of normal controls support the idea of a biased Th2 immune response in asthmatic patients.

Protein phosphatase inhibitor cantharidin inhibits steroidogenesis and steroidogenic acute regulatory protein expression in cultured rat preovulatory follicles.

The effect of cantharidin, a natural toxicant of blister beetles and a strong inhibitor of protein phosphatases types 1 and 2A, on luteinizing hormone (LH)-induced synthesis of steroidogenic acute regulatory (StAR) protein was studied in a serum-free culture of preovulatory follicles. StAR protein is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage (P450scc) enzyme. Treatment of preovulatory follicles dissected from ovaries of immature rats primed with pregnant mares' serum gonadotropin (10 IU) with LH for 24 h resulted in a dose-dependent increase in the level of StAR protein that reached a maximum at 100 ng LH/ml. This increase was associated with an increase in progesterone production. Treatment of follicles with increasing concentrations (10 - 1000 ng/ml) of cantharidin suppresssed LH (100 ng/ml)-induced StAR protein levels and progesterone production in a dose-dependent manner. The amount of P450scc protein and the conversion of 22R-hydroxycholesterol to progesterone were not affected by cantharidin. This indicates that cantharidin did not interfere with the activity of P450scc. Cantharidin also decreased StAR protein levels and progesterone production induced by the adenylate cyclase activator forskolin (10(-5) M) or a cAMP analog 8-Br-cAMP (0.5 mM). These results demonstrate that cantharidin inhibits the LH-induced StAR protein levels, and, thus, suggest that phosphoprotein phosphatase activity is required for the cAMP-protein kinase A-stimulated steroidogenic activity of the preovulatory follicle.

Dexamethasone enhances follicle stimulating hormone-induced P450scc mRNA expression and progesterone production in pig granulosa cells.

The effect of dexamethasone on follicle-stimulating hormone (FSH)-stimulated expression of cholesterol side-chain cleavage (P450scc) enzyme and production of progesterone by ovarian granulosa cells was studied in vitro. Granulosa cells from 3- to 5-mm pig antral follicles were cultured for 48 h in the presence or absence of FSH and/or dexamethasone. Treatment with FSH resulted in a dose-dependent increase in the level of P450scc mRNA that reached a submaximum at 100 ng FSH/ml. This increase was associated with an increase in progesterone production. Treatment of the cells with increasing concentrations (10(-9)-10(-6) M) of dexamethasone for 48 h increased constitutive and potentiated FSH-stimulated P450scc mRNA levels and progesterone production in a dose-dependent manner. Increasing duration (12-48 h) of treatment with dexamethasone (100 nM) led to a time-dependent increase in basal and FSH-stimulated progesteorne production, achieving statistical significance by 48 and 24 h, respectively. Dexamethasone also increased P450scc mRNA level and progesterone production induced by the adenylate cyclase activator forskolin (10 microM) or a cAMP analog 8-Br-cAMP (1 mM). The effects of dexamethasone on FSH-induced progesterone production were blocked by cotreatment of the cells with glucocorticoid receptor antagonist RU-486. These results demonstrate that dexamethasone potentiates FSH actions on steroidoogenesis in the pig ovary. Possible mechanisms for this potentiation include the ability of dexamethasone to stimulate P450scc gene expression.