Effect of immunization against OPN5 on the reproductive performance in Shan Partridge ducks under different photoperiods.

Photoperiod is an important environmental factor that influences seasonal reproduction behavior in birds. Birds translate photoperiodic information into neuroendocrine signals through deep brain photoreceptors (DBPs). OPN5 has been considered candidate DBPs involved in regulating seasonal reproduction in birds. We found that OPN5 could mediate light to regulate the follicle development in ducks. In this study, we further verified the effect of OPN5 on follicular development in Shan Partridge ducks by immunizing against the extracellular domain (ECD) of OPN5. We investigated the specific regulatory mechanism of photoperiod mediated by OPN5 on the reproductive activity of ducks. The trial randomly divided 120 Shan Partridge ducks into 3 groups with different treatments: the immunization of OPN5 group was done at d0, d15, d30, and d40 with 1 mL of vaccine containing OPN5 protein (thus containing 1, 1, 0.5, and 0.5 mg of OPN5-KLH protein), and the control group (CS and CL groups) was injected at the same time with the same dose of OPN5-uncontained blank vaccine. The group of CS (900 lux), OPN5 (600 lux), and CL (600 lux) lasted for 40 d in 12 L:12 D photoperiods, respectively. Then, the groups of CS, OPN5, and CL subsequently received 12 L:12 D, 12 L:12 D, and 17 L:7 D light treatments for 33 d, respectively. The ducks were caged in 3 constant rooms with the same feeding conditions for each group, free water, and limited feeding (150 g per duck each day). Duck serum and tissue samples were collected at d 40, d 62, and d 73 (n = 12). It was found that before prolonged light, the group of immunization (group OPN5) and the group of strong light intensity (group CS) were higher than the group of CL in egg production. Subsequent to prolonged light, the group CL in egg production rose about the same as the group immunization, while the strong light group (group CS) was lower. Group OPN5 increased the ovarian index of ducks, and both the immunization of group OPN5 and group CL (extended light) increased the thickness of the granular layer and promoted the secretion of E2, P4, LH, and PRL hormones. Compared with group CS, group CL and OPN5 increased the mRNA level and protein expression of OPN5 in the hypothalamus on d 62 and d 73 (P < 0.05). The gene or protein expression patterns of GnRH, TRH, TSHß, DIO2, THRß, VIP, and PRL were positively correlated with OPN5, whereas the gene expression patterns of GnIH and DIO3 were negatively correlated with OPN5. The results showed that immunization against OPN5 could activate the corresponding transmembrane receptors to promote the expression of OPN5, up-regulate the expression of TSHß and DIO2, and then regulate the HPG axis-related genes to facilitate the follicular development of Shan Partridge ducks. In addition, in this experiment, prolonging the photoperiod or enhancing the light intensity could also enhance follicle development, but the effect was not as significant as immunizing against OPN5. Our results will offer beneficial data and more supportive shreds of evidence in favor of elucidating the role of OPN5 in relation to photoperiods and reproduction.

Drp1 regulated PINK1-dependent mitophagy protected duck follicular granulosa cells from acute heat stress injury.

The mitochondrial quality control system is crucial in maintaining cellular homeostasis during environmental stress. Granulosa cells are the main cells secreting steroid hormones, and mitochondria are the key organelles for steroid hormone synthesis. The impact of the mitochondrial quality control system on granulosa cells' steroid hormone synthesis and survival under heat stress is still unclear. Here, we showed that acute heat stress induces mitochondrial damage and significantly increases the number of mitophagy-like vesicles in the cytoplasm of duck ovary granulosa cells at the ultra-structural level. Meanwhile, we also found heat stress significantly increased mitochondrial fission and mitophagy-related protein expression levels both in vivo and in vitro. Furthermore, by confocal fluorescence analysis, we discovered that LC3 was distributed spot-like manner near the nucleus in the heat treatment group, and the LC3 spots and lysosomes were colocalized with Mito-Tracker in the heat treatment group. We further detected the mitophagy-related protein in the cytoplasm and mitochondria, respectively. Results showed that the PINK1 protein was significantly increased both in cytoplasm and mitochondria, while the LC3-Ⅱ/LC3-Ⅰ ratio increase only occurred in mitochondrial. In addition, the autophagy protein induced by acute heat treatment was effectively inhibited by the mitophagy inhibitor CysA. Finally, we demonstrated that the alteration of cellular mitophagy by siRNA interference with Drp1 and PINK1 inhibited the steroid synthesis of granulosa cells and increased cell apoptosis. Study provides strong evidence that the Drp1 regulated PINK1-dependent mitophagy pathway protects follicular granulosa cells from acute heat stress-induced injury.

Heat enhances the inhibitory effect of lipopolysaccharide on duck granulosa cell proliferation and steroid biosynthesis in vitro.

Lipopolysaccharide (LPS) reduces the reproductive performance of laying ducks, especially during the hot summer months. To study the underlying mechanisms, we investigated the effects of different LPS concentrations and heat on duck granulosa cell (GC) proliferation and steroid biosynthesis in vitro. We investigated GC proliferation, secretion, and activation of the MAPK pathway. The cell cycle results showed that LPS treatment alone did not significantly affect cell proliferation, whereas the mRNA expression levels of IGF2, IGFBP2, and CyclinD1 were downregulated and p27kip1 was significantly upregulated after 2000 ng/mL LPS treatment when compared to untreated cells. In steroid hormone synthesis, although LPS increased the expression of most steroid biosynthesis genes, it inhibited the expression of CYP11A1 at high LPS concentrations. High temperatures enhanced the inhibitory effect of LPS on the expression of proliferation-promoting genes. Heat significantly reduced CYP11A1 and CYP19A1 expression. In addition, the phosphorylation of P38 was significantly upregulated by high temperatures combined with LPS, whereas the phosphorylation of ERK1/2 and JNK was downregulated. The relative protein expression of Bax/BCL-2 was upregulated at high temperatures in combination with LPS. Heat treatment enhanced the inhibitory effects of LPS on the proliferation and hormone biosynthesis of duck GCs in vitro.

Effects of melatonin on testicular function in adult male mice under different photoperiods.

Photoperiod is an important environmental factor affecting animal physiological function. Melatonin is an endogenous hormone that plays an important role in circadian and seasonal (or cyclical) rhythms and seasonal reproduction in mammals. To investigate the effects of melatonin on the reproductive performance of adult male mice under different photoperiods, sixty mice were randomly allotted to six groups: control (Light Dark, 12 L:12 D), control plus melatonin (MLD, 12 L:12 D), 24-hour continuous light (LL, 24 L:0 D), 24-hour continuous light plus melatonin (MLL 24 L:0 D), constant darkness (DD, 0 L:24 D), and constant darkness plus melatonin (MDD, 0 L:24 D). Normal saline (100 µL) was injected into the LD, LL, and DD groups at noon each day; the MLD, MLL, and MDD groups were injected with melatonin (1 mg/mL; 2 mg/kg·body weigh). After 24 hours of prolonged light exposure, testis morphology decreased, convoluted seminiferous tubules became sparse, the diameter of convoluted seminiferous tubules decreased, and the level of sex hormones decreased. After the administration of exogenous melatonin, testicular morphology and sex hormone levels decreased in the MLD group under normal light conditions. In the MLL group, the testicular tissue morphology returned to normal, the diameter of convoluted tubules increased, the hormone levels of LH (Luteinizing hormone) and MTL (melatonin) significantly increased (P<0.05), and th0e gene expressions of LHß and Mtnr1A (Melatonin receptors 1A) increased. There was almost no difference in the MDD group under continuous darkness. In conclusion, melatonin can damage the reproductive performance of male mice under normal light conditions, while exogenous melatonin can alleviate and protect the testicular injury of male mice under continuous light conditions.

Long-day photoperiods affect expression of OPN5 and the TSH-DIO2/DIO3 pathway in Magang goose ganders.

This study sought to understand the regulation mechanism of OPN5 through the TSH-DIO2/DIO3 pathway mediated photoperiod on the breeding activity of short-day breeding birds. In this study, the reproductive activity of Magang goose was regulated by artificial light, and the reproductive activity of the ganders were determined according to the daily laying rate of female geese. The testicular development and the serum reproductive hormone concentrations of ganders were measured during the reproductive period (d 0), the reproductive degeneration period (d 13 and 27) and the resting period (d 45). The mRNA and protein expression patterns of OPN5, the HPG axis reproductive genes, and TSH-DIO2/DIO3 pathway related genes were examined. Results showed that the laying rate of geese and the gonadal indices (GSI) decreased gradually after the photoperiod increased. Histological observation found that the spermatogenic function of the testis was normal on d 0 and 13, while degeneration occurred by d 27 and 45. Serum testosterone, FSH, and LH concentration showed a slight increase on d 13, followed by a sharp decrease on d 27 and 45 (P < 0.01), while PRL concentrations were low on d 0 and 13, and increased rapidly on d 27 and 45 (P < 0.01).The expression pattern of GnRH, FSH, LH, and THRß mRNA were similar, with high levels on d 0 and 13 and a decreasing trend on d 27 and 45 (P < 0.05 or P < 0.01); and GnRHR mRNA levels were higher on d 13 (P < 0.05), but then had decreased by d 27 and 45 (P < 0.01). The expression pattern of GnIH and GnIHR was similar, which was opposite to that of GnRHR. VIP, PRL, and PRLR increased gradually and peaked on d 45 (P < 0.01). The expression trend of TRH, TSHß, and DIO2 was similar to that of GnRHR, and the expression abundance increased on d 13, and then decreased on d 27 and 45. GnRH protein expression was significantly higher than during the other 3 periods (P < 0.01) while the GnIH protein levels were extremely low on d 0, had gradually increased by d 13, and significantly increased by d 27 and 45 (P < 0.01). The protein expression trends of THR and DIO2 were similar to that of GNIH. DIO3 protein expression was low on d 0 and 13, and increased by d 27 and 45. These results suggest that when the photoperiod increased, the hypothalamus OPN5 gene and protein were upregulated and the pituitary TSHß, TSHR, and hypothalamus THRß, TRH, and DIO2 were downregulated, and thus the reproductive activity of geese was inhibited.

Effects of stocking density on ovarian development and maturation during the rearing period in Shan-ma ducks.

Stocking density critically affects the growth and subsequent performance of animals in modern poultry production. This study investigated the effects of stocking density on ovarian development, ovarian maturation, and the mRNA expression of key genes in the reproductive axis during the rearing period of Shan-ma ducks. The experiments involved 180 healthy 7-wk-old Shan-ma ducks and randomly divided into low stocking density (LSD; n = 30, density = 5 birds/m2), medium stocking density (MSD; n = 60, density = 10 birds/m2) and high stocking density groups (HSD; n = 90, density = 15 birds/m2), for rearing. After examining ovarian development and measuring hormone levels in the plasma and expression levels of key regulatory genes in the reproductive axis at 19 wk of rearing, analysis of the gonad index analysis, reflecting stocking density, uncovered statistically significant differences. The gonad index of the LSD group was significantly higher than those of the MSD and HSD groups (P < 0.01), while no significant difference was observed between the MSD and HSD groups. pre-ovulatory follicles (POFs) and small yellow follicles (SYFs) development was only apparent in the LSD group, with the large white follicles (LWFs) number of this group being significantly higher than that of the MSD group (P < 0.05). The blood levels of E2 (estradiol), P4 (progesterone), and T (testosterone) were significantly higher in the LSD group than in the MSD and HSD groups (P < 0.05 or 0.01). Also, the levels of both P4 and T were significantly higher in the MSD group than in the HSD group (P < 0.01). The gene expression levels of GnRHR, FSH, AMHR, and FSHR were significantly increased in the LSD group compared to the MSD and HSD groups (P < 0.05 or 0.01), while the expression levels of GnIHR and GDF9 were significantly decreased in the LSD and MSD groups compared to the HSD group (P < 0.05 or 0.01). Steroid biosynthesis pathway genes such as StAR, CYP11A1, 3ß-HSD, CYP19A1, and BMP15 were significantly downregulated at greater stocking densities (P < 0.05 or 0.01). Likewise, the protein expression of StAR, 3ß-HSD, and CYP19A1 was also significantly decreased (P < 0.05 or 0.01). These results demonstrate that both medium and high stocking densities suppressed the expression of the key reproduction-promoting factors, while the expression level of the key reproductive inhibitory factors was enhanced. Therefore, rates of ovarian development and maturation could be reduced by a high stocking density leading to a delay in reproduction performance during the rearing period of Shan-ma ducks.

Resveratrol sustains intestinal barrier integrity, improves antioxidant capacity, and alleviates inflammation in the jejunum of ducks exposed to acute heat stress.

Resveratrol, a natural antioxidant, anti-inflammatory plant extract, was found to have a protective effect in poultry subjected to heat stress. In this study, we strove to characterize resveratrol on intestinal of duck exposed to acute heat stress and investigate the underlying mechanism. A total of 120 Shan-ma ducks (60 days old) were randomly divided into 2 groups. The control group was fed a basal diet, and the resveratrol group was fed a basal diet supplemented with 400 mg/kg resveratrol. Animals in 2 groups were kept at a temperature of 24°C ± 2°C for 15 d. Then, animals of both groups were placed in an artificial climate room at 39°C. Twelve ducks of each group were sacrificed for sampling at 0, 30, and 60 min, respectively. Results indicated that resveratrol increased the ratio of villus height to crypt depth, increased the number of goblet cells, and reduced the histopathological damage of jejunum caused by acute heat stress. Furthermore, the gene expression of heat shock proteins (HSP60, HSP70, and HSP90) and tight junction proteins (CLDN1 and OCLN) was significantly increased in the resveratrol group compared to that in the control groups. Simultaneously, resveratrol significantly activated the SIRT1-NRF1/NRF2 signaling pathways, improved ATP level of jejunum, and increased SOD and CAT antioxidant enzymes activities. In addition, we found that the NF-κB/NLRP3 inflammasome signaling pathways were repressed under acute heat stress. Meanwhile, supplement resveratrol further inhibited the NLRP3 inflammasome pathway, decreased protein level of NLRP3 and caspase1 p20, reduced the secretion of IL-1ß. Taken together, our results indicate that resveratrol against the oxidative damage and inflammation injury in duck jejunum induced by heat stress via active SIRT1 signaling pathways.

The effect of heat stress on proliferation, synthesis of steroids, and gene expression of duck granulosa cells.

Granulosa cells (GCs) play an important role in the development of follicles. In this study, we investigate the impact of heat stress at 41°C and 43°C on duck GCs' proliferation and steroids secretion. And, the transcriptomic responses to heat treatment were examined using RNA-sequencing analysis. Digital gene expression profiling was used to screen and identify differentially expressed genes (fold change ≥ 2 and Q value < 0.05). Further, the differential expression genes (DEGs) were classified into GO categories and KEGG pathways. The results show that duck GCs blocked in the G1 phase were increased on exposure to heat stress. Meanwhile, the expression of proliferative genes, which were essential for the transition from G1 to S phase, was inhibited. At the same time, heat stress inhibited the estradiol synthesis of GCs by decreasing CYP11A1 and CYP19A1 gene expression. A total of 241 DEGs including 181 upregulated and 60 downregulated ones were identified. Transcriptome result shows that heat shock protein and CXC chemokines gene were significantly activated during heat stress. While collagenases (MMP1 and MMP13) and strome lysins (MMP3) were downregulated. And, the hedgehog signaling pathway may be a prosurvival adaptive response under heat stress. These results offer a basis for better understanding the molecular mechanism underlying lay-eggs-less in ducks under heat stress.

Reference gene selection for expression studies in the reproductive axis tissues of Magang geese at different reproductive stages under light treatment.

In quantitative PCR research, appropriate reference genes are key to determining accurate mRNA expression levels. In order to screen the reference genes suitable for detecting gene expression in tissues of the reproductive axis, a total of 420 (males and females = 1:5) 3-year-old Magang geese were selected and subjected to light treatment. The hypothalamus, pituitary and testicular tissues were subsequently collected at different stages. Ten genes including HPRT1, GAPDH, ACTB, LDHA, SDHA, B2M, TUBB4, TFRC, RPS2 and RPL4 were selected as candidate reference genes. The expression of these genes in goose reproductive axis tissues was detected by real-time fluorescent quantitative PCR. The ΔCT, geNorm, NormFinder and BestKeeper algorithms were applied to sort gene expression according to stability. The results showed that ACTB and TUBB4 were the most suitable reference genes for the hypothalamic tissue of Magang goose in the three breeding stages; HPRT1 and RPL4 for pituitary tissue; and HPRT1 and LDHA for testicular tissue. For all three reproductive axis tissues, ACTB was the most suitable reference gene, whereas the least stable reference gene was GAPDH. Altogether, these results can provide references for tissue expression studies in geese under light treatment.

Identification of recombination in novel goose parvovirus isolated from domesticated Jing-Xi partridge ducks in South China.

Outbreaks of short beak and dwarfism syndrome (SBDS), caused by a novel goose parvovirus (NGPV), have occurred in China since 2015. This rapidly spreading, infectious disease affects ducks in particular, with a high morbidity and low mortality rate, causing huge economic losses. This study analyzed the evolution of NGPV isolated from Jing-Xi partridge duck with SBDS in South China. Complete genome sequences of the NGPV strains GDQY1802 and GDSG1901 were homologous with other GPV/NGPV and Muscovy duck parvovirus (MDPV) strains. Phylogenetic analysis showed that the NGPV isolated from mainland China was related to the Taiwan 82-0321v strain of GPV. In contrast to 82-0321v and the SDLC01 strain, which was first isolated from China, the two isolates showed no deletions in the inverted terminal repeat (ITR) region. Further, in these isolates, 24 amino acid sites of the replication protein were different compared to that of GPV live vaccine strain 82-0321v, and 12 sites were unique across all NGPV isolates. These isolates also showed differences in 17 amino acid sites of the capsid protein from that of 82-0321v, two of which were the same as those in MDPV. Recombination analysis identified the major parents of GDSG1901 and GDQY1802 as the NGPV-GD and NGPV-Hun18 strains, and the minor parents as the classical GPV 06-0329 and GPV LH strains, respectively. GDQY1802 and GDSG1901 are recombinant GPV-related parvovirus isolated from domesticated partridge duck. Recombination is evident in the evolution of NGPV, and as such, the use of live attenuated vaccines for NGPV requires further study.

Polysaccharide of Atractylodes macrocephala Koidz Enhances Cytokine Secretion by Stimulating the TLR4-MyD88-NF-κB Signaling Pathway in the Mouse Spleen.

Polysaccharide of Atractylodes macrocephala Koidz (PAMK) has been reported to have beneficial effects on regulation of immune responses in mammals and poultry. Nonetheless, the immunoregulatory mechanism of action of PAMK remains unclear. The Toll-like receptor 4 (TLR4) signaling cascade has been proved as a classic polysaccharide-regulated pathway. The aim of this study was to explore the effects of PAMK on the TLR4 signaling pathway in the regulation of spleen function in mice. Ninety-six 5-week-old BALB/c female mice were randomly allocated into four groups with three replicates per group and eight mice per replicate in a single-factor completely randomized experimental design. The control group was fed a basic diet (PAMK free); the other three groups were fed 100, 200, or 400 mg/kg PAMK for 28 days. The spleen index, concentrations of cytokines, and mRNA and protein expression levels of genes related to TLR4 signaling were determined in spleen tissue. Compared with the control group, the spleen index significantly increased in all treatment groups. Concentrations of interleukin 2 (IL-2), IL-4, interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α) in the medium-PAMK group also increased significantly. PAMK in the medium-PAMK group significantly increased both mRNA and protein expression of TLR4, myeloid differentiation factor 88 (MyD88), TNFR-associated factor 6 (TRAF6), TRAF3, and nuclear factor kappa B (NF-κB) in the spleen. In conclusion, PAMK may increase immune-response capacity of the spleen in mice via TLR4-MyD88-NF-κB signaling.

Rapid and sensitive detection of goose parvovirus and duck-origin novel goose parvovirus by recombinase polymerase amplification combined with a vertical flow visualization strip.

Goose parvovirus (GPV) is one of the most serious viral pathogens in goslings. Recently, a new pathogen to the Chinese mainland-duck-origin novel goose parvovirus (N-GPV)-was found to be 90.8-94.6% identical to the nucleotide sequence of GPV, and typically causes growth disorders and high infection rates in meat ducks. The spread of both of these viruses hinders the healthy development of the waterfowl breeding industry. In this study, recombinase polymerase amplification (RPA) was combined with a vertical flow (VF) visualization strip to develop a universal assay for the rapid detection of GPV and N-GPV. A set of specific primers and probes were designed to target the VP3 gene. Detection was possible at a constant temperature of 37 °C within 5-10 min. The assay successfully detected GPV and N-GPV with high-specificity and did not exhibit cross-reactivity with other waterfowl viruses and bacteria. The analytical sensitivity of the GPV-RPA-VF assay was 2 × 102 copies of GPV plasmid. Validation of the GPV-RPA-VF assay-using 60 samples from the field--confirmed 100% similarity between the results of GPV-RPA-VF and conventional qPCR. The results indicate that the GPV-RPA-VF assay was accurate, sensitive, and specific. This assay can be performed with minimal equipment and training to rapidly detect GPV and N-GPV during the early phase of an outbreak, especially when timely veterinary diagnoses are needed in the field and in rural areas.

Identification of Goose PKR Gene: Structure, Expression Profiling, and Antiviral Activity Against Newcastle Disease Virus.

Double-stranded RNA-dependent protein kinase (PKR) is an important antiviral IFN-stimulated gene (ISGs) that recognizes double-stranded RNA (dsRNA) and mediates inhibition of translation initiation and protein synthesis in various types of viral infection. In this study, the complete coding sequence (CDS) of goose PKR (goPKR) is identified and characterized. The open reading frame (ORF) of goPKR is 1668 bp, which encodes a polypeptide of 555 amino acids. The sequence identity results demonstrate that the goose PKR is most closely related to duck PKR gene, with nucleotide identities of 91.6%, whereas nucleotide identity of the goose PKR to chicken, human, and mouse PKR is 76.4%, 51.9%, and 52.0%, respectively. Interestingly, the deduced amino acid sequence of goose PKR contains 3 main structure domains, including 2 double-strand RNA-binding motif (dsRBM) domains and one serine/threonine protein kinase domain. This is similar to the chicken and mammals, whereas it is different from duck PKR protein, which contains only one dsRBM1 domain and one serine/threonine protein kinase domain. Quantitative real-time PCR analysis indicates that goose PKR mRNA is widely expressed in all sampled tissues. It is highly expressed in the blood, spleen, lung, and bursa of Fabricius and jejunum and is slightly expressed in heart, muscle, trachea, and brain. The results of confocal microscopy suggest that PKR-EGFP is mainly localized in the cytoplasm, and overexpression of goPKR protein significantly reduces Newcastle disease virus (NDV) replication (viral copies and viral titer) in goose embryo fibroblasts. These findings show that goose PKR is an important antiviral ISG, involved in the antiviral innate immune defense to NDV in geese.

Effects of Armillariella tabescens mycelia on the growth performance and intestinal immune response and microflora of early-weaned pigs.

This study was performed to evaluate effects of Armillariella tabescens (A. tabescens) on the growth performance and intestinal immune response and microflora in early-weaned pigs when used as feed additive. A. tabescens mycelia were added to basal diets at concentrations of 0%, 0.1%, 0.3% or 0.9% (w/w). A total of 144 commercial cross-bred piglets were randomly allocated to one of these four diets and fed for 30 days. The growth performance of early-weaned piglets displayed improvement with diets containing 0.1% and 0.3% dried mycelia powder from A. tabescens. Supplementing with 0.1% or 0.3% A. tabescens mycelia induced a 2.6- and three-fold increase in secretory immunoglobulin A (sIgA) content in the jejunal mucosa, respectively, but had only a marginal effect on sIgA in the ileal mucosa. Expression of interleukin-2, interferon-γ, and tumor necrosis factor-α in the jejunal mucosa were elevated with A. tabescens mycelia administration. Increased amounts of Lactobacillus spp. and Bifidobacterium spp. in the jejunum, and decreased amounts of Escherichia coli in the jejunum and ileum were observed with the administration of A. tabescens-containing diets. This study demonstrated that A. tabescens had beneficial effects on the growth performance and intestinal microflora of early-weaned pigs.

Preliminary ex vivo feasibility study on targeted cell surgery by high intensity focused ultrasound (HIFU).

High intensity focused ultrasound (HIFU) has become a new noninvasive surgical modality in medicine. A portion of tissue seated inside a patient's body may experience coagulative necrosis after a few seconds of insonification by high intensity focused ultrasound (US) generated by an extracorporeal focusing US transducer. The region of tissue affected by coagulative necrosis (CN) usually has an ellipsoidal shape when the thermal effect due to US absorption plays the dominant role. Its long and short axes are parallel and perpendicular to the US propagation direction respectively. It was shown by numerical computations using a nonlinear Gaussian beams model to describe the sound field in a focal zone and ex vivo experiments that the dimension of the short and long axes of the tissue which experiences CN can be as small as 50µm and 250µm respectively after one second exposure of US pulse (the spatial and pulse average acoustic power is on the order of tens of Watts and the local acoustic spatial and temporal pulse averaged intensity is on the order of 3×10(4)W/cm(2)) generated by a 1.6MHz HIFU transducer of 12cm diameter and 11cm geometric focal length (f-number=0.92). The concept of thermal dose of cumulative equivalent minutes was used to describe the possible tissue coagulative necrosis generated by HIFU. The numbers of cells which suffered CN were estimated to be on the order of 40. This result suggests that HIFU is able to interact with tens of cells at/near its focal zone while keeping the neighboring cells minimally affected, and thus the targeted cell surgery may be achievable.

The polymorphisms of UCP1 genes associated with fat metabolism, obesity and diabetes.

Uncoupling protein 1 (UCP1), a 32-kDa protein located in the inner mitochondrial membrane, is abundant in brown adipose tissue, as a proton transporter in mitochondria inner membrane which uncouples oxidative metabolism from ATP synthesis and dissipates energy through the heat. UCP1 has been reported to play important roles for energy homeostasis in rodents and neonate of larger mammals including human. Recently, numerous candidate genes were searched to determine the genetic factors implicated in the pathogenesis of obesity, related metabolic disorders and diabetes. UCP-1, which plays a major role in thermogenesis, was suggested to be one of the candidates. This review summarizes data supporting the existence of brown adipocytes and the role of UCP1 in energy dissipation in adult humans, and the genetic variety association with the fat metabolism, obesity and diabetes.

[Treatment of nonneoplastic epithelial disorders of skin and mucosa of vulva with focused ultrasound].

OBJECTIVE: To explore the clinical efficacy of focused ultrasound for patients with white lesions of the vulva, as well as its safety and feasibility. METHODS: Clinical data of 941 patients with white lesions of the vulva treated with focused ultrasound from June 2003 to December 2005 were retrospectively reviewed. The mean age of the patients was 40.8 years (18 - 70 years) and the median course of the disease was 6.2 years (3 months - 45 years). Meanwhile, pathological diagnosis was performed in all the patients before treatment, in which 498 cases were squamous hyperplasia, 342 cases were lichen sclerosus and 101 cases were lichen sclerosus with squamous hyperplasia. Patients were followed up and therapeutic effects of focused ultrasound was evaluated at 6 and 12 months after the treatment, respectively. The symptoms of pruritus in the vulva and the changes in the color and elasticity of the vulvar lesions were observed. RESULTS: Of all the patients, 900 were followed up after the treatment, and the ratio of effectiveness was 94.9%. Only 46 patients (5.1%) had no response to the therapy. Of the effective patients, 434 cases were completely cured (48.2%), and 420 cases were improved (46.7%). Pruritus of vulva recurred in 101 patients (11.2%) one year after treatment; however, these patients still had a response to the second or third treatment. CONCLUSIONS: Focused ultrasound therapy is a highly effective instrument in treatment of white lesions of the vulva. It can not only relieve the symptoms of itching, but is also helpful in recovering the color and elasticity of the vulva.

[Investigation of follicular development and oocyte maturation after cryopreservation and xenograft of newborn mouse ovaries].

In order to explore the feasibility of cryopreserving primordial follicles in attaining their developmental competence following freezing and thawing, ovaries from newborn mice were cryopreserved and the thawed ovaries were xenografted into kidney capsules of adult female mice. Ovaries were isolated from newborn B6C2F(1) female mice, infiltrated by Leibovitz 15 (L-15) medium containing 10% (V/V) fetal bovine serum (FBS) and 1.5 mol/L dimethylsulfoxide (DMSO), and then packed into 0.25 ml plastic straws. The ovaries contained in straws were frozen under nitrogen vapour at -40 degrees C in Cryocell 1200 programmable freezer, and stored in liquid nitrogen for periods ranging from 1 week to 6 months. Upon thawing, the straws were dipped into room temperature water for 10~20 s, after which the ovaries were collected and washed in L-15 buffer containing 10% (V/V) FBS without DMSO to remove cryoprotectant. The thawed ovaries were transplanted into kidney capsules of 8~12-week old adult B6C2F(1) female recipient mice by two protocols, with either 1 or 2 ovaries in each capsule. Upon withdrawal after at least 14 d of transplantation, only 45.00% (72/160) of the ovaries were recovered from 40 recipients transplanted with 2 ovaries in each capsule, compared to 82.50% (33/40) in 20 recipients with only 1 ovary in each capsule. The grafted ovaries exhibited similar follicular developmental progression to that of natural ovaries. There were antral follicles present in the transplanted ovaries on day 14, whose number increased more substantially on day 19 after transplantation. Following stimulation of the recipient mice with 10 IU PMSG on day 19 after xenografting, follicles further developed to preovulatory stage with appearance of cumulus oocytes and enlarged antrum. Oocytes from these fully grown antral follicles were collected and matured in vitro in modified essential medium-alpha (MEMalpha). After 16~17 h of culture, 40.90% of the oocytes exhibited germinal vesicle breakdown (GVBD) and among which 89.02% proceeded to the metaphase II (MII) stage as indicated by exclusion of the first polar body. The remaining oocytes were further cultured and 50.83% of which initiated GVBD by 20~21 h of culture, but only 21.40% of which proceeded to MII. The above results demonstrated that the primordial follicles in newborn mouse ovaries were capable of sustaining freezing and thawing, and reinitiating development following xenograft into kidney capsule in adult recipient female mice. Production of mature oocytes from such re-developed follicles following gonadotrophin priming and the subsequent oocyte in vitro maturation implied immense prospect of application of this method to preserve female germ cells, conserve endangered species, establish animal gene stock, and utilize oocytes in assisted reproductive techniques.

[Evaluation of white lesions of vulva treated with focused ultrasound].

OBJECTIVE: To explore the feasibility and efficacy of focused ultrasound for the treatment of vulvar dystrophy, including squamous hyperplasia (SH) and lichen sclerosus (LS). METHODS: A total of 76 eligible patients with vulvar dystrophy (45 SH and 31 LS) were treated with focused ultrasound between 1999 and 2002. Before and after ultrasound therapy, both ultrasonography and biopsies of the lesions were performed to monitor and evaluate the changes of the lesion being treated. The positive expressions of CD34 (cluster of differentiation of endothelial cells), a marker of the epithelial cells of blood vessels and myelin basic protein (MBP), a marker of the oligodendrocytes and Schwann cells were tested using the streptavidin-peroxidase (SP) immunohistochemistry method before and after the ultrasound procedure to evaluate the effects of ultrasound treatment. RESULTS: In two years, follow-up, 49 of 76 cases (32 SH and 17 LS) were cured, 23 (11 SH and 12 LS) improved, and 4 (2 SH and 2 LS) persisted. The response rate was 94.7% (72/76). The positive expression of CD34 and MBP significantly increased at the treated region (P < 0.05). Grey-scale ultrasound imaging showed a localized hypoechoic region after the treatment, which recovered to normal appearance within 7 - 10 days. CONCLUSION: Vulvar dystrophy can be treated with focused ultrasound effectively and safely. This approach appears to be a new promising treatment method, although further studies are still needed.