A modified protocol for <i>in vitro</i>maturation of mouse oocytes from secondary preantral follicles

A 2-step culture system was designed and tested for the in vitro maturation efficiency of oocytes from pre-puberty preantral follicles of FVB/N inbred mice. The following modifications were made: 1) The concentration of ITS was reduced by half in the basal MIF medium to minimize uncoordinated growth between oocyte and GC cells;2) Heterogeneous preantral follicles were cultured in groups of 3 - 5 follicles in hanging drops of medium with reduced concentration of ITS for six days to induction follicular aggregation. This hanging drop method mimics a 3-D IVM culture system at the early stage of cultivation in which the sphere structure of each follicle is well maintained. It also enables follicles in each aggregate to communicate with each other, synchronize their growth, and thus prevent immature follicular rupture. 3) Medium was further supplemented with retinoic acid to enhance developmental capacity of meiotically arrested oocytes. After a 14-day culture in vitro, ~37% of the collected inbred preantral follicles completed nuclear maturation. Approximately 94% of the mature oocytes tested were able to be fertilized;and 77% of them developed into healthy embryos. These results demonstrate that our IVM system is reliable to produce a satisfactory number of high quality oocytes. In addition, multiple cytoplasmic parameters, including gene expression of key regulators, chromosome/spindle organization, mitochondrial proliferation and distribution, and total ATP content were explored to characterize the supportive and limiting components of our IVM system so that the culture system can be further optimized.

Effects of Estradiol on Cashmere Goat Primary Hair Follicles Cultured in vitro

[Objective] The aim of this study was to preliminarily explore the effects of estradiol on morphology and growth of cashmere goat primary hair follicles. [Method] Cashmere goat primary hair follicles were cultured in serum-free Williams E media supplemented with different doses of 17 β-E2 (0, 0.1, 1.0, 10.0, 100.0 nmol/L), and their growth rates and morphological changes were observed. [Result] The growth rate of 0.1 nmol/L 17 β-E2 group was quite comparable with that of the control group(0 nmol/L), but the 17 β-E2 with concentrations of 1.0, 10.0 and 100.0 nmol/L displayed different degrees of inhibition on the growth of hair follicles. Different morphological changes of hair follicles could also be discovered in different concentration treatments. [Conclusion] The study laid a certain foundation for exploring the regulation mechanism of estrogen on growth of cashmere goat hair follicles.

Effects of Different Culture Condition in vitro on Growth of Cashmere Goat Primary Hair Follicles

[Objective] The aim of this study is to lay a foundation for illustrating the biological characteristics and growth regulation mechanism of hair follicles.[Method]Cashmere goat primary hair follicles were separated under aseptic condition and cultured in serum-free DMEM and serum-free Williams E media respectively;subsequently,the growth rate and morphological changes were observed under the inverted microscope.[Result]Hair follicles cultured in serum-free DMEM media showed a growth rate of 0.034 mm/d during the first 3 days,whose structure and morphological characteristics could maintian a stable status for a long time in the growth process.Hair follicles grew much faster in the serum-free Williams E media with a growth rate of 0.077 mm/d during the first 3 days.[Conclusion]There were significant differences(P<0.05)between the growth of cashmere goat hair follicles cultured in the 2 kinds of media.Serum-free Williams E medium was superior to serum-free DMEM medium.

Effects of N-acetylcysteine on growth,viability and reactive oxygen species levels in small antral follicles cultured in vitro

Objective:To investigate the effects of different concentrations of N-acetylcysteine on follicular growth and morphology,as well as on viability,levels of reactive oxygen species(ROS)and meiotic progression of oocytes from in vitro cultured bovine early antral follicles.Methods:Isolated early antral follicles(about 500μm)were cultured in TCM-199+alone or supplemented with 1.0,5.0 or 25.0 mM N-acetylcysteine at 38.5℃with 5%CO_(2) for 8 days.Follicle diameters were evaluated at day 0,4 and 8 of culture.At the end of culture,the levels of ROS,chromatin configuration and viability(calcein-AM and ethidium homodimer-1 staining)were investigated in the cumulus-oocyte complexes.Comparisons of follicle diameters between treatments were performed.Data on percentages of morphologically normal follicles,growth rates and chromatin configuration in different treatments were compared.Results:An increase in follicular diameters after culture in all treatments was observed,except for follicles cultured with 25.0 mM N-acetylcysteine.Fluorescence microscopy showed that oocytes cultured in all treatments were stained positively with calcein AM,and that 5.0 mM N-acetylcysteine reduced fluorescence for ethidium homodimer-1.Intracellular levels of ROS in oocytes from follicles cultured with 1.0 mM N-acetylcysteine showed a significant reduction compared to other treatments.The presence of N-acetylcysteine in culture medium did not influence the rates of oocyte at the germinal vesicle stage.Conclusions:N-acetylcysteine at concentrations of 1.0 and 5.0 mM reduces ROS levels and staining for ethidium homodimer-1 in in vitro cultured follicles,respectively,while 25.0 mM N-acetylcysteine decreases follicular growth and the percentages of continuously growing follicles.

Melatonin promotes the development of the secondary hair follicles by regulating circMPP5

Background The quality and yield of cashmere fibre are closely related to the differentiation and development of secondary hair follicles in the skin of cashmere goats.The higher the density of secondary hair follicles,the higher the quality and yield of cashmere from the fleece.Development of secondary hair follicles commences in the embryonic stage of life and is completed 6 months after birth.Preliminary experimental results from our laboratory showed that melatonin(MT)treatment of goat kids after their birth could increase the density of secondary hair follicles and,thus,improve the subsequent yield and quality of cashmere.These changes in the secondary hair follicles resulted from increases in levels of antioxidant and expression of anti-apoptotic protein,and from a reduction in apoptosis.The present study was conducted to explore the molecular mechanism of MT-induced secondary hair follicle differentiation and development by using whole-genome analysis.Results MT had no adverse effect on the growth performance of cashmere kids but significantly improved the character of the secondary hair follicles and the quality of cashmere,and this dominant effect continued to the second year.Melatonin promotes the proliferation of secondary hair follicle cells at an early age.The formation of secondary hair follicles in the MT group was earlier than that in the control group in the second year.The genome-wide data results involved KEGG analysis of 1044 DEmRNAs,91 DElncRNAs,1054 DEcircRNAs,and 61 DEmiRNAs which revealed that the mitogen-activated protein kinase(MAPK)signaling pathway is involved in the development of secondary hair follicles,with key genes(FGF2,FGF21,FGFR3,MAPK3(ERK1))being up-regulated and expressed.We also found that the circMPP5 could sponged miR-211 and regulate the expression of MAPK3.Conclusions We conclude that MT achieves its effects by regulating the MAPK pathway through the circMPP5 sponged the miR-211,regulating the expression of MAPK3,to induce the differentiation and proliferation of secondary hair follicle cells.In addition there is up-regulation of expression of the anti-apoptotic protein causing reduced apoptosis of hair follicle cells.Collectively,these events increase the numbers of secondary hair follicles,thus improving the production of cashmere from these goats.

Isolated lymphoid follicles in colon: Switch points between inflammation and colorectal cancer?

Gut-associated lymphoid tissue is supposed to play a central role in both the organization of colonic repair mechanisms and colorectal carcinogenesis. In inflammatory conditions, the number, diameter and density of isolated lymphoid follicles (ILFs) increases. They are not only involved in immune surveillance, but their presence is also indispensable in normal mucosal regeneration of the colon. In carcinogenesis, ILFs may play a dual role. On the one hand they may support tumor growth and the metastatic process by vascular endothelial growth factor receptor signaling and producing a specific cytokine and cellular milieu, but on the other hand their presence is sometimes associated with a better prognosis. The relation of ILFs to bone marrow derived stem cells, follicular dendritic cells, subepithelial myofibroblasts or crypt formation, which are all involved in mucosal repair and carcinogenesis, has not been directly studied. Data about the putative organizer role of ILFs is scattered in scientific literature.

Three Dimensional <i>In Vitro</i>Culture of Murine Secondary Follicles in a Defined Synthetic Matrix

Ovarian follicle growth in three dimensional (3D) matrices in vitro has limitations: a) matrices don’t expand as follicles grow, b) requirements for enzyme-mediated retrieval, and c) animal-derived components prevent clinical application. Therefore, we evaluated N-Isopropylacrylamide (SFX-1), a novel synthetic 3D culture matrix, for follicle culture. Groups of three murine secondary follicles were encapsulated in 50 μL of DMEM/F12-1%ITS-10%FCS (DMEM/F12) or SFX-1 (3:2 v/v DMEM/F12) or Matrigel (1:1 DMEM/F12) and cultured for 48 h. Matrigel contains growth factors but SFX-1 has no animal-derived factors. Each culture condition was examined in 6 wells containing 18 follicles, in four replicate experiments (n = 4). Photomicrographs were used to determine follicle diameters and morphological integrity. Follicles were Live-Dead (LD) stained or disaggregated to generate cells for viability assessment using Trypan Blue (TB). Estradiol, progesterone and anti-mullerian hormone (AMH) in conditioned media were measured using Enzyme-linked Immunoassay. All culture conditions supported similar increases in follicle diameter. DMEM/F12 did not maintain morphological integrity which prevented follicle retrieval after 48 h;25% were retrieved from DMEM/F12, but 44% and 41% follicles were retrieved from SFX-1 and Matrigel respectively. Follicles retrieved from Matrigel could not be disaggregated, which prevented TB viability assessment. LD estimations of viable cells/follicle were lower than TB, but culture conditions had no effect on viability;SFX-1 64% ± 8% and DMEM/F12 69% ± 9%. SFX-1 and Matrigel supported similar levels of progesterone synthesis, only Matrigel supported estrogen synthesis, but none of the culture conditions supported AMH production. SFX-1 was not cytotoxic and was comparable to Matrigel. Further development of SFX-1 for use with human follicles is supported.

体外培养牛腔前卵泡卵母细胞的超微结构

直径约100μm牛腔前卵泡在无血清下体外培养15d。超微结构研究显示,培养前腔前卵泡卵母细胞微绒毛短小,分布均匀。胞质内细胞器致密而均匀,线粒体多为长或圆形,嵴较少,高尔基体、脂滴稀少,未见有皮质颗粒出现。培养6d时,微绒毛略变粗长,但数量减少。胞质内细胞器,由近核分布向胞质中央区转移,线粒体形态丰富,基质电子密度极高。培养15d腔前卵泡卵母细胞微绒毛增多,变长,斜向或垂直插入已经形成的透明带中。线粒体增多,大小不等,形态各异,胞质中区有溶酶体样物质及零星的皮质颗粒出现。电镜研究表明,体外培养与体内正常发育腔前卵泡卵母细胞超微结构变化规律基本相似。

Identification of Bulge Stem Cells in Mouse and Human Hair Follicles

The skin contains various populaions of stem cells, but its characterization has been hampered by lack of markers and unclear location. The hair follicle has a niche for stem cells called a “bulge” which acts as a reservoir of multipotent stem cells. In the study reported here, an immunohistochemical and immunofluorescence analysis was performed on mouse and human tissues in order to determine the possible presence of stem cells of hair follicle through cytokeratin 15 (CK15), CD34, and CD200 markers identified as crucial to the stem cells and to identify the bulge region. Mouse (n = 7) and human (n = 7) skin samples were used. The expression of proteins was determined by the indirect immunoperoxidase technique and a secondary antibody bound to a fluorochrome. The specificity of staining was evaluated by negative controls. The results revealed that the stem cells associated with CD34 and CD200 antibodies were differentially expressed in the interfollicular epidermis, sebaceous glands, and bulge region, indicating that, in mice, CD34 and, in humans, CD200 are more specific than CK15 in detecting bulge cells. It also suggests that CD34 is specific for mouse bulge cells, while CD200 might have specificity for progenitor cells and partially differentiated cells in humans.

人毛囊外根鞘细胞的分离及培养

目的建立毛囊外根鞘细胞的分离和培养方法。方法利用中性蛋白酶分离得到单个毛囊,采用组织块法和消化法进行细胞培养,倒置显微镜、扫描及透射电镜观察细胞形态,培养各代细胞做生长曲线,RT-PCR检测干细胞相对特异性标识分子K15和细胞分化标识分子外皮蛋白的mRNA表达情况。结果组织块法和消化法均能培养出毛囊外根鞘细胞,并可传代培养,组织块法细胞生长慢,不易汇合,消化法细胞增殖快。各代细胞中K15和外皮蛋白mRNA均有表达。结论消化法适合毛囊外根鞘细胞的体外培养,该方法的建立为进一步分离纯化培养毛囊干细胞奠定了基础。

Dynamic expression of Wnt signaling molecules and stem-cell marker CK15 in the growth cycle of rat whisker hair follicles

Objective To investigate the distribution and dynamic changes of both Wnt signaling molecules and CK15 throughoutthe three phases of the follicular cycle,and to explore the relationship between Wnt/β-catenin signaling and CK15 in rat whisker hair follicle(HF)growth cycles.Methods Hematoxylin-Eosin(HE)and immunofluorescence stains were used to characterize the expression patterns,including sites and levels of some representative proteins of both canonical and non-canonical Wnt signaling molecules,as well as HF epithelial stem cell marker CK15.Results The expression patterns of bothβ-catenin and Wnt5a were correlated with that of CK15.CK15 was only expressed in anagen.In catagen,β-catenin showed a massive depletion while Wnt5a noticeably increased.In telogen,high level expression ofβ-catenin and low level of Wnt5a were detected.Wnt10b and TCF3 were detected during the entire HF growth cycle.Conclusion These results suggest that Wnt5a is associated with the transition of anagen-catagen phase,accompanied by broad deletion ofβ-catenin and loss of CK15.WntlOb is important for the maintenance of HF activity and is related to the telogenanagen transition.

Stochastic Modelling of Relative Light Sensitivity of Hair Follicles

Relative light sensitivity (RLS) of HFs was mathematically described as the ratio of two stochastic variables presenting the durations of light sensitive and light insensitive sub-phases of the cycle according to a new theory of HF light sensitivity formulated in our previous article (Kruglikov, Am J Cosm Surg, 2012, 29:266 - 272). RLS gives possibility to rank the HFs from different body regions according to their light sensitivities. Application of proposed method for estimation of the light sensitivity of scalp hairs predicts remarkable difference in light sensitivities of HFs in alopecic and non-alopecic patients.

Follicle-stimulating hormone is expressed in ovarian follicles of chickens and promotes ovarian granulosa cell proliferation

Follicle-stimulating hormone(FSH),an important hypothalamic-pituitary-gonadal axis(HPG)hormone,is secreted by the pituitary gland.This study confirms that FSH is expressed in chicken follicles at different stages,and positive FSHβ mRNA signals were stronger(P<0.05)in granulosa cells than in oocytes.The 369 bp coding sequence of FSHβ in ovaries is 100%identical to that in the pituitary gland.The experiment in vitro revealed that the ovary possessed FSH secretory capacity.Further,FSHβ mRNA was significantly upregulated(P<0.05)in follicles and significantly higher(P<0.05)than that in the pituitary gland by approximately 2–23 times with the development.The number of granulosa cells decreased significantly(P<0.05)in the cells with siRNA treatment,confirming that the ovarian FSH could promote granulosa cell proliferation.This view was supported by cell cycle analysis and CCND2 and CCNE2 expression.Further research indicated that no difference(P>0.05)was observed between the number of granulosa cells treated with FSHβ siRNA and in exogenous FSH.However,the number of granulosa cells without FSHβ siRNA transfection was significantly higher(P<0.05)for exogenous FSH.This finding suggests that the proliferative effect of exogenous FSH on ovarian granulosa cells depend on endogenous FSH.This study demonstrated that the FSH gene was expressed in chicken follicles and promoted ovarian granulosa cell proliferation,which enriched the theory on HPG axis.

卵巢卵泡膜细胞瘤的CT表现与病理对照分析

目的:分晰卵巢卵泡膜细胞瘤CT特征并与病理结果进行对照,以提高对卵巢卵泡膜细胞瘤CT表现的认识。方法:回顾性分析11例卵巢卵泡膜细胞瘤临床资料和CT表现。结果:11例共12个病灶,单发10例,多发1例。肿瘤边界清楚8个,边界部分清楚部分模糊4个,见蒂结节2例,术中发现肿瘤蒂扭转。肿瘤大小31mm×38mm～126mm×140mm,8个肿瘤平扫与子宫肌层密度相仿,9个肿瘤内可见散在小片状囊变区,对应大体标本上肿瘤内的囊变区。增强扫描肿瘤实质区呈缓慢渐进性轻度强化,囊变区未见强化。1例卵泡膜细胞瘤动脉期及实质期可见显著强化的纤细肿瘤血管影,而肿瘤实质仍为轻微强化。少量腹水征9例。9例合并有雌激素刺激相关的疾病。镜下肿瘤细胞呈旋涡状排列,周围被大量胶原纤维分隔,细胞浆内含有大量脂肪小滴,细胞核类圆形,未见明显核分裂。结论:卵巢卵泡膜细胞瘤的CT表现能在一定程度上反映此瘤的病理特征,对本病的诊断和鉴别诊断具有重要价值,但确诊仍需依赖病理学检查。

某些中药煎剂对阿霉素抑制的猪毛囊真皮鞘细胞增殖的影响

目的:探讨何首乌、女贞子等中药煎剂对阿霉素抑制的猪毛囊真皮鞘细胞增殖的影响。方法:用MTT法通过比色分析测定吸光度值(OD),检测不同剂量(0、5、10、20)mg/L阿霉素作用24h和10mg/L阿霉素作用不同时间(4、8、12、24、48h)对猪毛囊真皮鞘细胞增殖的影响;用流式细胞仪检测不同剂量的阿霉素作用24h后猪毛囊真皮鞘细胞的凋亡率,从而找出用于以下研究中阿霉素的最适浓度。用MTT法检测不同剂量中药煎剂和10mg/L阿霉素同时给药共同作用24h以及先给予不同剂量中药煎剂作用24h,然后给予10mg/L阿霉素作用24h两种条件下对猪毛囊真皮鞘细胞增殖的影响。结果:阿霉素抑制体外培养的猪毛囊真皮鞘细胞增殖,其抑制效应随阿霉素浓度的增大和作用时间的延长而增强;细胞凋亡率以阿霉素浓度为10mg/L时最高。中药煎剂和阿霉素10mg/L同时给药条件下,前者不能有效升高OD值(P>0.05);如果先给予中药煎剂处理,后给10mg/L阿霉素,则中等剂量的中药煎剂即能有效升高OD值(P<0.001)。结论:阿霉素抑制体外培养的猪毛囊真皮鞘细胞增殖,先给予适当剂量的中药煎剂处理细胞,能在一定程度上减轻阿霉素对细胞的损伤。

Coexistence of esophageal blue nevus, hair follicles and basaloid sqamous carcinoma:A case report

We present the case of a 57-year-old man who underwent esophagectomy for esophageal carcinoma found at barium meal and gastroscopic examination. He was diagnosed as esophageal basaloid squamous carcinoma （BSC） and gastric stromal tumor, which were associated with focal proliferation of melanocytes/ pigmentophages and hair follicles in esophageal mucosa. Melanocytic hyperplasia （melanocytosis） has previously been recognized as an occasional reactive lesion, which can accompany esophageal inflammation and invasive squamous carcinoma. The present case is unusual because of its hyperplasia of not only melanocytes but also hair follicles. To our knowledge, this is the first report of esophageal blue nevus and hair follicle coexisting with BSC.

Evaluation of glucose metabolism in women with multiple ovarian follicles

Objective ：To investigate glucose metabolism in women with multiple ovarian follicles （MOF） and explore the relationship between glucose metabolism, insulin resistance and body weight. Methods：We evaluated 46 women with MFO and 30 normal women as controls. All the subjects were given 75g of glucose orally in order to perform the oral glucose tolerance test （OGTT） and insulin releasing test （IRT）, and they were also evaluated for insulin resistance using the insulin resistance index with homeostatic model assessment （HOMA）. Results：The occurrence of impaired glucose tolerance in women with MOF was 10.87%, which was significantly higher than that in the control group （3.33% ,P 〈 0.05）. The rate of insulin resistance was 30.43% in the study group as compared to 10.00% in the control group. The results showed that there was significant difference between the two groups（P 〈 0.05）. The levels of FSH,LH,PRL,E2,T and P between the two groups had no significant difference （P 〉 0.05）. BMI in women with impaired glucose tolerance was correlated positively to insulin resistance （r = 0.567, P 〈 0.05）. Conclusion：Abnormal glucose metabolism was observed in women with unitary multiple ovarian follicles, and this could be attributed to obesity and insulin resistance. Women with MOF and associated obesity should be subjected to OGTT so that their glucose levels can be monitored as a preventive measure.

Justicia insularis Improves the in vitro Survival and Development of Ovine Preantral Follicles Enclosed in Ovarian Tissue

Objectives： Evaluating the addition effect of./. insularis extract and FSH on the survival, activation and ROS production after in vitro culture of ovine preantral follicles enclosed in ovarian tissue. Methods： In the first experiment, ovarian fragments were fixed （non-cultured control） or in vitro cultured in α-MEM＋ （cultured control）, α-MEM＋ supplemented with FSH 50 ng/mL, or in α-MEM＋supplemented with J. insularis （JUS0.3; 1.25 or 5 mg/mL） for 1 or 7 days, at 39℃, 5% CO2. In the second experiment, fragments were fixed or cultured in α-MEM＋ supplemented with anethole 300 μg/mL ＋ FSH 50 ng/mL or in α-MEM＋ supplemented with anethole 300μg/mL ＋ 0.3 mg/mL JUS. Key findings： JUS0.3 was the only treatment that maintained the percentage of morphologically normal follicles similar to non-cultured control even after 7 days of culture. After 7 days of culture, a higher （p 〈 0.05） percentage of developing follicles was observed in JUS5 treatment compared with the other treatments except JUS 1.25. In the second experiment, FSH maintained the percentage of normal follicles and promoted activation of primordial follicles. A reduction （p 〈 0.05） of stromal cell density was observed in MEM＋＋ANE supplemented with JUS or FSH. Conclusions： J. insularis in a concentration-dependent manner maintained the levels of ROS and improved in vitro follicular survival and activation of ovine primordial follicles.

自噬在毛发再生中的作用

背景:在生物医学界,自噬是一个快速发展的研究热点,许多研究人员正在积极探究多种疾病中自噬的分子调控机制,但是自噬在毛发生长中所起的作用仍存在许多未知。目的:综述目前自噬在毛发生长与再生中的研究进展及应用价值,了解自噬在毛发生长中的作用,以探究自噬在病理性脱发中的发病机制,为研究治疗脱发的药物提供新思路。方法:应用计算机在中国知网(CNKI)、PubMed数据库中,以“毛囊生长,毛发生长,毛发再生,自噬相关蛋白,自噬活性,自噬相关基因,自噬”或“hair growth,hair follicle,hair regeneration,autophagy”为检索词进行检索。查阅国内外近年来关于自噬、毛发生长和自噬在毛发生长中作用的研究进展进行总结,排除与文章主题内容不相符的文献,最终纳入78篇文献进行结果分析。结果与结论:(1)自噬是真核生物的正常代谢过程,具有复杂的分子机制和功能特性,既有利于细胞生存,又可促进细胞死亡。(2)脱发相关疾病与自噬活性的改变有关,且自噬活性可调控毛发生长周期;敲除或过表达自噬相关基因可改变毛发生长状态,已发现有多条自噬相关信号通路与毛囊生长有关;自噬的激活剂或抑制剂可用于治疗或预防脱发疾病。

Effect of adrenalectomy on rat epididymidis

AIM: To investigate the effect of adrenalectomy (ADX) on the epididymidis of Sprague-Dawley rats. METHODS: The histological, biochemical (cholesterol protein, zinc, copper, alkaline and acid phosphatase aryl sulphatase, lactic dehydrogenase and leucine amino peptidase) and hormonal (FSH, LH and testosterone) changes of caput and cauda epididymis in ADX rats were observed. RESULTS: Organ wet weight, histological studies and morphometric measurements indicated a cellular degeneration in caput and cauda epididymis of ADX rats. Serum testosterone level was significantly lower in ADX than in sham-operated rats, while the serum FSH and LH were below the detection limit of 1 mIU/mL. The enzymatic activity was higher in ADX than in sham-operated rats. Epididymal zinc level increased whereas copper level decreased in ADX rats compared to the sham-operated. CONCLUSION: Adrenalectomy leads to degeneration of caput and cauda epididymidis epithelial cells as a result of decreased supply of testosterone.