The effect of paternal heat stress on protein profiles of pre-implantation embryos in the mouse

The study was undertaken to compare the protein profiles of [35S]-methionine-labelled control-sired embryos with heat-sired embryos at 7, 14 or 21 days after mature fertile B6CBF F1 male mice were kept at 36 +/- 0.3 degrees C and 62 +/- 2.7% relative humidity for 24 h. One-dimensional gel electrophoresis and autoradiographs were used to examine the protein profiles between the two-cell embryos and the blastocysts. The results obtained demonstrate that paternal heat stress 7 or 14 days earlier did not apparently affect protein patterns of two-cell embryos, four-cell to eight-cell embryos, morulae or blastocysts. However, 21 days earlier, there were changes in protein patterns of two-cell embryos and abnormal embryos, but not the morulae. To further support and extend these results, two-dimensional gel electrophoresis and phosphorimaging were employed and the results obtained show that paternal heat stress 21 days before mating affected protein profiles of two-cell embryos and morulae in the mouse. Together, these findings have indicated that paternal heat stress affects most but not all protein patterns of pre-implantation embryos, which strongly supports our previous results demonstrating that paternal heat stress significantly reduced the developmental proportion of pre-implantation embryos in the mouse.     

Sperm Wash in Three Culture Media: Maximization of Motile Sperm Recovery During Swim-Up Incubation

Three different culture media commonly used during in vitro gamete manipulations were studied for their efficacy in sperm wash procedure. Highest numbers of motile sperm were recovered at 6 hours following incubation in WT-6 and Ham's F-10 media. However, WT-6 yielded higher motile sperm numbers than Ham's F-10. Swim-up sperm number reached a peak at 3 hours following incubation in BWW. A period of 2 to 6 hours of incubation of sperm pellets overlayed with sperm wash media resulted in highly enriched motile sperm fractions free of dead spermatozoa and seminal debri.     

In vitro culture of mouse embryos in human amniotic fluid

Human amniotic fluid was compared with Ham's F-10 culture medium as a possible alternative for use in in vitro fertilisation. The cleavage success of mouse embryos in human amniotic fluid (experimental group) was 92% compared with 86% in Ham's F-10 medium. It is concluded that human amniotic fluid is a viable alternative culture medium for mouse embryos.     

Comparative functional study of clonal insulin-secreting cells cultured in five commercially available tissue culture media

The electrofusion-derived rat insulin-secreting cell line BRIN-BD11 was cultured in five different commercially available media to determine the optimum medium for the in vitro maintenance of such clonal cell lines. Cells were cultured in RPMI-1640, DMEM, McCOY'S, F-12K, or MEM culture medium supplemented with 10% (v/v) fetal bovine serum and antibiotics (100 U/ml penicillin and 0.1 g/L streptomycin). Insulin secretion studies performed after 10 days revealed RPMI-1640 to be the best performing medium in terms of insulin secretory responsiveness to a range of stimuli including glucose, L-alanine, L-arginine, carbachol, and glibenclamide. Insulin release was significantly decreased (p < 0.01 to p < 0.05) in all other media compared to RPMI-1640. Only the cells cultured in RPMI-1640 and DMEM showed a significant glucose-induced insulin secretory response (p < 0.01 and p < 0.05). McCOY'S gave the next best result followed by F-12K and MEM. After the 10-day culture period, the highest insulin content was found in cells cultured in RPMI-1640 and DMEM with significantly lower levels of insulin in cells cultured in McCOY'S, F-12K, and MEM (p < 0.01 to p < 0.001). RPMI-1640 was used for further studies to investigate the effects of 5.6-16.7 mmol/L glucose in culture on the secretory responsiveness of BRIN-BD11 cells. Significant responses to a number of nonglucidic secretagogues were seen following culture at 5.6 and 16.7 mmol/L glucose, although responsiveness was less than after culture with 11.1 mmol/L glucose. At 16.7 mmol/L glucose culture, glucose-stimulated insulin release was abolished.     

Maternal diabetes and retarded preimplantation development of mice

The streptozocin-induced diabetic (STZ-D) mouse was found to be a suitable model for studying the effects of maternal diabetes on the preimplantation embryo. This study looked at the effects of maternal diabetes on embryonic growth. Female Quakenbush mice were made diabetic (plasma glucose levels greater than 20 mM) by injection of 190 mg/kg i.p. STZ and were superovulated by standard methods. The blastocysts collected on day 4 from diabetic mothers had 8.5% fewer cells and a 35% lower protein synthetic rate than control embryos. Their cellular protein synthetic rate was 19% less than that in controls. Morulae from diabetic mothers also displayed a reduced protein synthetic rate, but this reduction was not seen in the two-cell embryo. Furthermore, blastocysts cultured in vitro from two-cell embryos from diabetic and control mothers displayed similar protein synthetic rates. This infers that the two-cell embryos from diabetic mothers are normal, and the retardation seen in later development in vivo occurs after the two-cell stage while the embryo is still free in the oviductal and uterine environment. Treatment of the diabetic mice with ultralente insulin every 12 h raised the protein synthetic rate of those blastocysts toward control levels, whereas treatment with lente insulin every 8 h recovered the embryo to the same rate as the control embryos. Because insulin has been shown to be mitogenic and stimulates protein synthesis of morulae and blastocysts in vitro, the absence of insulin in the diabetic mothers may be the cause of the retardation observed in their preimplantation embryos.     

应用b-FGF刺激的软骨细胞构建自体组织工程化软骨的研究

目的　探索碱性成纤维细胞生长因子 (b FGF)对体外软骨细胞增殖和体内组织工程化软骨构建的影响。　方法　细胞取自猪耳软骨 ,用Ham′sF 12培养液体外培养 ,实验分两组 :培养液含b FGF组 (b FGF组 ) ,培养液无b FGF组 (对照组 )。培养过程中观察细胞形态 ,于 (12 75± 1 2 6 )d后分别收集 2组第 2代细胞 ,进行细胞增殖倍数比较 ,并按 5 0× 10 6 cell ml浓度与聚环氧乙烯 (Plu ronic)混合 ,制成细胞 生物材料复合物 ,按每一样本 0 5ml注射到猪自体皮下 ,8周后取材 ,从重量、体积、GAG含量和组织学等方面进行分析。结果　b FGF组细胞贴壁呈类成纤维细胞样 ,对照组细胞贴壁呈多角形 ;各组第 2代细胞数和原代细胞数相比 ,b FGF组扩增 70倍 ,对照组为 5 4倍 ,b FGF组是对照组的 12 7倍 ;8周后取材 ,b FGF组形成的组织工程化软骨的平均重量和体积为 0 371g 0 370cm3,约是对照组的 2倍 ;GAG含量和组织学上 ,两者均非常接近正常猪耳软骨。结论　培养液中应用b FGF ,原代软骨细胞能在 2周内大量扩增 ,并能在体内构建良好的软骨 ,这有助于解决软骨组织工程种子细胞缺乏问题。     

人输卵管上皮细胞共培养系统对小鼠胚胎体外发育的影响

为了检测人输卵管上皮细胞对早期胚胎体外分裂与生长的影响，将２１０个小鼠２细胞期胚胎与传代的人输卵管上皮细胞进行共培养，另以１４５个胚胎培养在单纯的培养液中作为对照。共培养的胚胎有８９．０％发育到桑椹胚，８７．１％形成囊胚，而对照组仅为６３．４％和４９．０％（Ｐ＜０．０１）。共培养胚胎发育速度也较对照组快，且发育过程中，共培养胚胎出现碎片及不规则分裂率明显低于对照组。提示人输卵管上皮细胞共培养系统可以促进胚胎体外发育，能在体外获得数量较多的高质量胚胎。     

Comparison of the effects on penetration capacity of human spermatozoa between using Ham's F-10 medium and a medium based on the composition of human tubal fluid

The penetration ability of human spermatozoa on zona-free hamster ovum when using Ham's F-10 (HF-10) medium was compared with that when using a medium based on the composition of human tubal fluid. Forty semen specimens were each divided into two aliquots; one aliquot was washed with and incubated in HF-10 medium and the other in HTF medium. The penetration rate of sperm with motility between 30 to 60 percent was significantly increased when HTF medium was used (67.9 +/- 6.9%) compared with the rate when HF-10 medium was used (49.2 +/- 7.3%) (P less than 0.05). This study shows that the ZFHO penetration ability of human spermatozoa with poor motility can be improved by using HTF medium as compared with the use of HF-10 medium.     

Enhancement of In Vitro Hair Shaft Elongation in Follicles Stored in Buffers That Prevent Follicle Cell Apoptosis

BACKGROUND: Viability and survival of stored micrografts during hair follicle transplantation are important limitations of micrograft transplantation procedures. In this study, we investigated the effect of different storage solutions and inhibitors of apoptotic cell death (ACD) on hair follicle cell viability by measuring in vitro hair shaft elongation (HSE) for 5 days. METHODS: Micrografts from informed patients undergoing routine micrograft transplantation were stored for 5 hours at room temperature in phosphate-buffered salt solution (PBS) or HEPES-buffered Dulbecco's modified Eagle's medium (DMEM), containing different concentrations of the ACD-inhibitors aminoguanidine (AMG), hormones (insulin, hydrocortisone), 14,15-epoxy-eicosatrienoic acid (14,15-EET), or combinations of these. RESULTS: In vitro, HSE was significantly increased in micrografts stored in DMEM compared with PBS (2.3%+/-0.6% vs. 28.4%+/-3.9%, P<0.0001). DMEM supplemented with AMG (10 microg/mL) or 14,15-EET (1 ng/mL) further increased in vitro HSE (33.9%+/-7.1%, p=0.01, and 32.8%+/-6.1%, P=0.02, respectively). Evaluation of ACD in stored micrografts, performed by determination of cytoplasmic histone-associated DNA fragments, confirmed the results found by HSE. ACD was detectable after a 36-hour culture in serum-containing medium and was higher in micrografts stored in PBS compared with micrografts stored in DMEM (A405nm/A492nm: 1.63+/-0.21 vs. 1.42+/-0.07, respectively; P<0.01). The addition of AMG further decreased serum-induced ACD in the micrografts (DMEM 1.42+/-0.07 vs. DMEM/AMG 0.90+/-0.11, P<0.0001). CONCLUSION: Our study demonstrated an important role of ACD in micrograft transplantation surgery. Preconditioning of micrografts with storage buffers containing inhibitors of ACD could prevent serum-induced ACD after transplantation and might increase the viability of micrografts and the clinical outcome in micrograft transplantation.     

The effect of serum on motility of human spermatozoa in culture

The motility of spermatozoa was higher at 6-22 h in Ham's F10 culture medium supplemented with 20-30% human serum than with lower proportions of serum or with 1% human serum albumin. Heat treatment (56 degrees C, 1 h), charcoal extraction and dialysis (18,000 molecular weight cut-off) of the serum did not reduce sperm motility suggesting that high molecular weight components are responsible for maintenance of motility. Renewing the medium (Ham's F10 with 30% serum) at 7 h resulted in better sperm motility and velocity at 22 h. At 22 h the pO2, pCO2, pH and sodium concentrations were not different in replenished and control cultures, but the concentration of glucose was higher and that of potassium lower if the medium was changed. These results suggest that addition of 20-30% human serum and renewal of medium at intervals is beneficial for sperm culture and may be of use in in vitro fertilization.     

Viability of cultured nerve grafts: An assessment of proliferation of Schwann cells and fibroblasts

Previous studies demonstrated that the viability of nerve grafts had a positive effect on nerve regeneration, while the cold storage of nerve grafts obtained few viable cells at the later stage. The purpose of this study was to examine the cellular activities of Schwann cells and fibroblasts in cultured nerve grafts prior to transplantation. 2.5-cm long sciatic nerve grafts were harvested from 75 male Lewis rats. Two different media were utilized to culture the nerve grafts up to 3 weeks: Dulbecco's modified eagle medium (DMEM) only or DMEM supplemented with 2 microM forskolin and 10 microg/ml pituitary exact (mitogen medium for Schwann cells). In vivo predegenerated and normal nerve grafts were used as positive and negative controls, respectively. We employed a 5-bromo-2'-deoxyuridine (BrdU) incorporation method to evaluate the proliferating cells in the cultured nerve grafts. S-100 and vimentin immunostaining were used to estimate the presence of Schwann cells and fibroblasts in all nerve grafts at different intervals. The results showed that the proliferating cells increased progressively under culture conditions. The proliferating cells distributed evenly in small fascicles (average diameter 251 +/- 71.5 microm), whereas they appeared mainly in the margin of large fascicles (average diameter 624 +/- 87.3 microm). The mitogen medium stimulated Schwann cell multiplication more significantly in comparison with DMEM after 3 days of culture (P < 0.01), however, there were fewer fibroblasts present in the mitogen medium than in DMEM after 2 days of culture (P < 0.01). It is suggested that the viability of nerve grafts can be preserved under culture conditions. Furthermore, the cellular activity of the Schwann cells and fibroblasts in nerve grafts can be manipulated in in vitro Wallerian degeneration.     

Cryopreservation of human adipose tissue-derived stem/progenitor cells using the silk protein sericin

Adipose tissue-derived stem/progenitor cells (ASCs) have attracted attention as a cell source that replaces marrow stromal cells (MSCs); ASCs may thus have applications in both regenerative medicine and cell transplantation. These medical treatments, however, require a high-quality supply of human ASCs. Therefore, the cryopreservation methods have been improved by changing a component of a cryopreservation medium. Sericin, a protein hydrolysate (with an average molecular weight of 30 kDa) is very rich in serine. The viability and the adipogenic/osteogenic potential of human ASCs were tested after freezing in a cryopreservation medium containing sericin. After thawing, the viability of the human ASCs frozen in the cryopreservation medium was found to be more than 95%. The proliferation rate of human ASCs frozen in CELLBANKER 2, and DMEM/Ham's F-12 medium (serum free) + 10% DMSO, 0.1 mol/L maltose, and 1% sericin was higher than that of the cells frozen in the maintenance medium + 10% DMSO. The adipogenic/osteogenic differentiation capabilities of frozen human ASCs were examined by Oil Red O staining/Von Kossa's method. The human ASCs were frozen using CELLBANKER 2, and DMEM/Ham's F-12 medium (serum free) + 10% DMSO, 0.1 mol/L maltose, and 1% sericin were positive. In conclusion, the cryopreservation medium containing sericin is therefore considered to have a beneficial effect on freezing human ASCs. This serum-free cryopreservation medium should be widely used in regenerative medicine, cell transplantation, and biological research.     

In vitro cartilage regeneration from proliferated adult elastic chondrocytes

The purpose of this study was to investigate cellular feasibility in the proliferation and differentiation status of adult chondrocytes for cartilage regeneration in comparison to fetal chondrocytes. Primary cells were isolated from adult (n = 6) and fetal (n = 6) sheep ear cartilages and expanded in 10% fetal bovine serum (FBS) containing Ham's F12 medium, in which adult and fetal cell proliferation rates were compared using a WST-1 assay kit. Approximately 4 million cells were seeded onto each 1 x 1 x 0.2-cm (200 microL) nonwoven fabric scaffold made from polyglycolic acid. Cell/polymer constructs were cultured in serum-free DMEM/F12 medium supplemented with 5 ng/mL TGF-beta2 and 5 ng/mL des(1-3)IGF-I (adult chondrocytes, group A) or in 10% FBS containing Ham's F12 medium (adult chondrocytes, group B, and fetal chondrocytes, group C) as controls in a rotating bioreactor for 6 weeks. The proliferation assay showed that fetal cells had a significantly better growth potential than did adult cells. Histology and extracellular matrix analyses revealed that groups A and C qualitatively displayed better matrix deposition than did group B. In conclusion, although adult sheep elastic chondrocytes had less growth potential than did fetal cells, the serum-free medium supplemented with growth factors significantly enhanced the production of cartilage matrix secreted from proliferated adult sheep elastic chondrocytes.     

Successful treatment of infertility due to retrograde ejaculation by instillation of serum-containing medium into the bladder. A case report

The treatment of infertility caused by retrograde ejaculation has often been ineffective. The unphysiological composition of the urine is the main reason complicating the recovery of motile sperm. We describe a case of retrograde ejaculation caused by a congenital defect of the internal sphinchter muscle of the urinary bladder. In this case, motile sperm were only recovered if the bladder had been filled previously with artificial culture medium (Ham's F-10) supplemented with the patient's own serum. Two pregnancies followed intrauterine insemination with sperm recovered in this way.     

The effect on cleavage of two-cell mouse embryos after a delay in embryo retrieval in a human in vitro fertilization programme

Two-cell mouse embryos are used for quality control in a human in vitro fertilization programme. A controlled experiment was designed to evaluate the effect on cleavage of two-cell mouse embryos after a delay in embryo retrieval. In the test group, two fallopian tubes were incubated in Whittingham's T6 medium for 2 1/2 hours per experiment before the embryos were removed for culture. In the control group embryos were removed from the fallopian tubes immediately after the mice were sacrificed. Five experiments were performed. Eight of 141 two-cell embryos (5,7%) reached the blastocyst stage in the test group, and in the control group 143 of 151 two-cell embryos (94,7%) reached the blastocyst stage after 72 hours. Embryos must be removed immediately after the mice are sacrificed to obtain constant results. If not, poor cleavage can lead to unnecessary confusion in the laboratory.     

Prednisone, azathioprine, and cyclosporine A toxicity on human fetal pancreas

The limited clinical success of human fetal pancreas (HFP) transplantation may be related to graft toxicity caused by immunosuppressant agents. This study describes the effects of prednisone (PRED), azathioprine (AZA), and cyclosporine A (CSA) on HFP tissue in vitro and in vivo. To assess in vitro function, fresh HFP explants (1-2 mm3; 16-21 weeks gestational age) were prepared and cultured 72 hr in supplemented Ham's medium containing varying concentrations of each drug. Insulin release in response to high glucose (17 mM) and theophylline (10 mM) challenge was determined and compared to basal release in low glucose (3 mM) buffer. No significant difference in insulin release was observed between culture control tissue and drug-cultured tissue throughout the concentration range (10(-8) -10(-4) M; P greater than 0.05). To assess in vivo function, cyropreserved HFP explants were transplanted under the kidney capsule of streptozotocin-induced diabetic nude mice. Mice were immunosuppressed with PRED (1 mg/kg), AZA (1 mg/kg), CSA (30 mg/kg), or combined triple drug therapy (COMBO), and glucose levels followed weekly. Hyperglycemia reversal and insulin withdrawal were observed in all drug groups [PRED (4/6), AZA (4/6), CSA (2/4), COMBO, (2/4)] and were not statistically different from control (5/8; P greater than 0.8). Time to insulin withdrawal was significantly different from control (12.2 +/- 2.2 weeks; P less than 0.05) only for AZA (10 +/- 0 weeks; PRED, 12.3 +/- 2.6 weeks; CSA, 11 +/- 0 weeks; COMBO, 15 +/- 0 weeks). Additionally, oral glucose tolerance tests in all groups were equivalent to nondiabetic controls. We were unable to demonstrate PRED, AZA, or CSA toxicity on HFP.(ABSTRACT TRUNCATED AT 250 WORDS)     

成年大鼠骨骼肌干细胞的纯化与培养

目的:建立一种骨骼肌干细胞的纯化及培养方法。方法:取成年雌性SD大鼠前肢肱三头肌,用胶原酶和dispase进行消化,200目的筛网滤过。采用差速贴壁法纯化骨骼肌干细胞,用含20%胎牛血清的HamsF10培养基进行培养。以α-骨骼肌肌动蛋白免疫细胞化学染色进行细胞鉴定。结果:成功地分离培养了成年大白鼠的骨骼肌干细胞。结论:采用差速贴壁的方法可以成功分离、纯化骨骼肌干细胞,为未来以自体骨骼肌干细胞注射治疗压力性尿失禁打下了基础。     

Hyperactivated motility is coupled with interdependent modifications at axonemal and cytosolic levels in human spermatozoa.

Whether the motility characteristics of hyperactivated spermatozoa were determined by stable changes at the axonemal level and whether the presence of cytosolic factors was required for the expression of these changes was investigated. Different degrees of sperm hyperactivation were produced in Percoll-washed spermatozoa after incubation for 1 hour to 3 hours at 37 degrees C in Ham's F-10 supplemented with human blood plasma or fetal cord serum. Decomplemented fetal cord serum induced the highest percentage of hyperactivation (19 +/- 3%), followed by human plasma (13 +/- 2%). Fetal cord serum that was not decomplemented did not induce a level of hyperactivation (1.7 +/- 0.2%) significantly different from control levels (0.9 +/- 0.2%). Dialyzed fetal cord serum induced intermediate levels of hyperactivation (6 +/- 1%). The motility characteristics of demembranated sperm models of hyperactivated spermatozoa induced by decomplemented fetal cord serum and nonhyperactivated spermatozoa were compared by videomicroscopy and computer-assisted digital image analysis. After demembranation with Triton X-100 and reactivation of motility by Mg. adenosine triphosphate (Mg.ATP), hyperactivated and nonhyperactivated spermatozoa showed similar motility characteristics. However, hyperactivated spermatozoa that were demembranated and reactivated in cytosolic extracts from hyperactivated spermatozoa had significantly higher (P less than 0.05) linear velocity (33 +/- 4 mu/sec) and lower linearity (0.23 +/- 0.04) than control spermatozoa that were demembranated and reactivated in control cytosolic extracts (velocity = 24 +/- 1 mu/sec; linearity = 0.32 +/- 0.02). The data suggest that the expression of hyperactivated motility requires interdependent changes at the axonemal and cytosolic levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

成骨细胞与诱导剂对骨髓基质干细胞的增殖与成骨分化的影响

目的探索一种新的骨髓基质干细胞(marrow stromal stem cells,MSCs)增殖与成骨分化的体外诱导培养体系. 方法乳大鼠颅骨来源的第3代成骨细胞与诱导剂(1 nmol/L地塞米松、10 mmol/L β-甘油磷酸钠、50μg/ml抗坏血酸)对大鼠股骨、胫骨来源的MSCs生长的影响.于8块24孔板上培养MSCs,每孔接种5×104个第3代MSCs.按培养成分不同分为4组,每组2块.DMEM培养为对照组;诱导剂培养为诱导剂组;成骨细胞培养为成骨细胞组;联合使用成骨细胞与诱导剂培养为联合诱导组.计数诱导1～8 d各组MSCs的数量并绘制细胞生长曲线,检测诱导10 d的MSCs的碱性磷酸酶活性,采用RT PCR检测诱导2周时MSCs骨钙素mRNA的表达水平.结果原代及传代MSCs形态正常.细胞生长曲线示MSCs数量均随时间延长增加.成骨细胞组增殖最快,诱导剂组增殖最慢,5～8 d成骨细胞组及诱导剂组细胞数量与对照组比较,差异有统计学意义(P＜0.05).联合诱导组碱性磷酸酶活性为2.01±0.56 U与对照组0.68±0.14 U、诱导剂组1.27±0.43 U及成骨细胞组0.77±0.19 U比较,差异均有统计学意义(P＜0.05).对照组不表达骨钙素mRNA,诱导剂组为0.783±0.094、联合诱导组为0.814±0.071与成骨细胞组0.302±0.026比较,差异均有统计学意义(P＜0.05).结论联合使用成骨细胞和诱导剂诱导MSCs,不影响MSCs的正常增殖而促进MSCs的成骨分化,诱导效果较好,可望成为一种新的骨组织工程种子细胞的体外培养体系.