Restoration of fertility by orthotopic transplantation of frozen adult mouse ovaries

BACKGROUND: Successful thawing and orthotopic transplantation of ovarian tissue has produced live offspring in mice, but until now has only been successful for very young ovary donors. METHODS: Whole and half ovaries from adult C3H/HeNCrlBR (C3H) and whole ovaries from B6129SF1/J were frozen-thawed and then grafted orthotopically into B6C3F1/CrlBR (B6C3F1) and B6129SF1/J recipients, respectively. In bilateral transplant groups (bilateral), recipients underwent a bilateral ovariectomy, followed by orthotopic grafting. In unilateral groups recipients either underwent bilateral ovariectomy followed by unilateral grafting (unilateral(ovx)) or had only one ovary removed and replaced with a graft (unilateral) along with complete transection of the remaining oviduct. RESULTS: Ovary size and number of follicles decreased dramatically in grafted compared with control groups, but the loss in the unilateral(ovx) group was significantly less than in the unilateral group. Similar numbers of litters and litter size were obtained in bilateral and unilateral grafts of fresh ovary. However, a much lower number of litters and litter size were derived from unilateral grafts than from unilateral(ovx) grafts of frozen ovary. CONCLUSIONS: Normal fertility can be restored by orthotopic grafting of fresh or frozen adult mouse ovaries and no significant difference between fresh and frozen ovaries was found. Grafting of half ovaries does not alter the overall fertility rate. Unilateral(ovx) grafting is an efficient procedure to produce live pups and removes the negative effect of recipient native ovaries on post-grafting fertility.     

Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells

BACKGROUND: The development of a spermatogonial transplantation technique has provided new possibilities for the treatment of male infertility. Previous studies have shown that spermatogonial stem cells could reinitiate spermatogenesis after cryopreservation and reintroduction into the seminiferous tubules of infertile recipient males, and this raised the possibility of banking frozen stem cells for male infertility treatment. It remains unknown, however, whether germ cells from freeze-thawed stem cells are fertile, leaving the possibility that the procedure compromises the integrity of the stem cells. METHODS AND RESULTS: Dissociated mouse testis cells were cryopreserved and transplanted into infertile recipient testes. The freeze-thawed testis cell populations contained higher concentrations of stem cells than fresh testis cell populations. Offspring were obtained from freeze-thawed stem cells transplanted into infertile males, and fertility restoration was more efficient in immature (5-10 days old) than in mature (6-12 weeks old) recipients. However, offspring were also obtained from infertile adult recipients using in-vitro microinsemination. CONCLUSIONS: This first successful application of frozen stem cell technology in the production of offspring by spermatogonial transplantation suggests the superiority of immature recipients for clinical applications. Thus, the combination of cryopreservation and transplantation of stem cells is a promising approach to overcome male infertility.     

Assessment of the developmental potential of frozen-thawed mouse oocytes

Mouse oocytes were cryopreserved by a protocol shown previouslytominimize damage tot the zona peelucida and cytoskeletal system.After thawing, the incidence of fertilization didnot differfrom that in control groups of oocytes, and after fertilizaiton,the ability of the fertilized frozen–thawed oocytes todevelop tothe balstocyst stage in vitro was only slightly less(77%) than that of the controls (87 and 89%). Transfer of frozen–thawedand fertilized oocytes after their culture to the balstocyststage in vitro resulted in a lower implantation rate (46%) thanfor the controls (68–73%), but of the implanting embryosthe same proportions in experimental and control groups survivedtoyield vaiable fetuses. In contrast, transfer after culturein vitro to the 2-to 4-cell stage rsulted in similar implantationrates for control and frozen–thawed fertilized oocytes(70–84%), but the spontaneous abortion rate was higherfor the embryos derived from frozen–thawed oocytes. Overallthe cumulative survival rate for frozen oocytes transferrd atthe 2-cell stage (36%) was better than after transfer at theblastocyst stage (30%), but both were less than for the transferat any stage of the control oocytes (47–55%). The cumulativesurvival of cryopreserved oocytes to viable fetuses was 30–40%less than that of the control oocytes. These results are comparedwith those form previous studies and the main remaining obstaclesto completely successful cryopreservation are identified     

Effects of cryopreservation on survival and development of interphase- and mitotic-stage 1-cell mouse embryos.

The effects of cryopreservation with 1,2-propanediol on two groups of 1-cell mouse embryos were studied in terms of survival after thawing, growth in vitro until the blastocyst stage and development in vivo assessed by the number of implantations and living fetuses. The two groups were divided according to different stages in the cell cycle: cells in (i) interphase with two distinct pronuclei or (ii) mitosis just prior to the first cleavage division. Zygotes in the interphase stage proved to be more resistant to freezing and thawing procedures, showing a significantly higher survival rate after thawing than zygotes in mitosis (78.5 versus 61.3%, P < 0.05). Blastocyst formation was similar in the two experimental groups: 72.7% for interphase and 60.8% for mitosis (P = 0.06), but for both groups fewer blastocysts formed when compared with the control group (86.7%) (P < or = 0.01). The implantation rates were not statistically different: 54.2% for the interphase cells and 47.4% for the control group and 44.0% for the mitotic cells and 49.4% for the control group. The formation of living fetuses was similar between the experimental and control groups: 36.5% for the interphase group (40.0% for its control group) and 22.6% for the mitotic group (38.8% for its control group). We conclude that freezing embryos during nuclear division is detrimental for their survival after thawing.     

Effect of cryoprotectants and their concentration on post-thaw survival and development of rapid frozen-thawed pronudear stage mouse embryos

Experiments have been conducted to develop a simple rapid-freezingprotocol for pronudear stage mouse embryos. The effect of typeof cryoprotectant (dimethyl-sulphoxide (DMSO) or 1,2-propanediol(PROH)) and concentration (ranging from 3.5 to 8.0 mol/1 with0.5 mol/1 sucrose) on the post-thaw morphological survival rate,on cleavage rate and on development to the blastocyst stagewere studied. Further, in-vivo viability of embryos frozen usingthe most effective cryoprotectant concentration (PROH at 7.0mol/1) was compared with viability of non-frozen embryos. Thetype of cryoprotectant and its concentration influenced thesurvival and development of embryos to the blastocyst stagein vitro. The best development with PROH was achieved at 7.0mol/1 (66%, 128/193), whereas with DMSO the best developmentwas achieved at 6.0 mol/1 (42%, 71/171). The rates of survivaland cleavage did not differ between the two best cryoprotectantconcentrations (P > 0.01) but the proportion of embryos whichdeveloped to blastocyst was higher (P > 0.001) with PROHat 7.0 mol/1 compared with DMSO at 6.0 mol/1. The rates of survivaland development were higher (P < 0.001) with DMSO at 3.5and 6.0 mol/1 compared with similar concentrations of PROH.The cleavage and development, however, was higher (P < 0.001)at 7.0 mol/1 PROH compared with the same concentration of DMSO.At 8.0 mol/1 the survival and development was not different(P > 0.01) between DMSO and PROH. The rate of implantationand the percentage of live fetuses at autopsy of the recipientsreceiving non-frozen embryos was higher (63 and 41% respectively)than in those receiving frozen–thawed embryos (53 and37% respectively), but not significantly different. It may beconcluded that the concentration range of cryoprotectants whichallows acceptable embryo viability after freezing and thawingis very narrow. The rapid protocol using dehydration in 0.25mol/1 and 0.5 mol/1 sucrose followed by exposure to 7.0 mol/1PROH and 0.5 mol/1 sucrose for 45 s was the most effective forcryopreservation of pronudear stage mouse embryos.     

Key factors in experimental mouse hematopoietic stem cell transplantation

The first mouse model of hematopoietic stem cell transplantation (HSCT) was developed more than 50 years ago. HSCT is currently being widely used in a broad range of research areas, which include studies of the engraftment process, the pathogenesis of graft-versus-host disease and possible ways of its treatment and prophylaxis, attempts to use the graft-versus-leukemia/tumor effect in treating hematological and oncological malignancies, cancer vaccine development, induction of transplanted organ tolerance, and gene therapy. However, although this model is widely distributed, many laboratories use different protocols for the procedure. There are a number of papers discussing different HSCT protocols in clinical work, but no articles summarizing mouse laboratory models are available. This review attempts to bring together different details about HSCT in the mouse model, such as the types of transplantation, possible pretreatment regimens and their combinations, methods and sources of graft harvesting and preparation for the transplantation procedure, the influence of graft cell dose and content on the engraftment process, the transplantation method itself, possible complications, symptoms and techniques of their prophylaxis or treatment, as well as follow-up and engraftment assessment. We have also tried to reflect current knowledge of the biology of the engraftment.     

Physiology: Transplantation of frozen--thawed mouse primordial follicles

Primordial follicles were isolated from juvenile mouse ovariesand cryopreserved by slow freezing with dimethylsulphoxide asthe cryoprotectant. After thawing, 80% of the oocytes and 65%of the somatic cells excluded Trypan Blue dye, indicating thatcell membranes were still intact. Frozen—thawed cellswere suspended in plasma clots and transplanted to the ovarianbursas of host animals that had been sterilized by oophorectomy.The grafts of frozen—thawed cells reorganized into morphologicallydistinguishable ovaries which produced signs of oestrogenicactivity. After natural mating, host females produced normaloffspring that were demonstrated by genetic markers to be derivedfrom the transplanted frozen—thawed primordial follicles.     

Mature oocytes derived from purified mouse fetal germ cells

BACKGROUND: Mouse fetal germ cells have been successfully purified from fetal gonads. However, there are no published reports describing a procedure for deriving mature oocytes from isolated fetal germ cells. The purpose of this present study is to explore whether purified fetal germ cells are able to differentiate into mature oocytes through an in vivo grafting procedure. METHODS AND RESULTS: First, intact 11.5 and 12.5 days post-coitum (dpc) female gonads with or without the attached mesonephros and the reaggregated female gonad cells were transplanted into the recipient mice. The results demonstrate both the gonad accompanied by mesonephroi and the innate gonad structure are not absolutely required for 11.5 dpc and 12.5 dpc oogonia to generate mature oocytes. Next, oogonia were purified from female gonads, aggregated with different ovarian somatic cells and transplanted into the recipient mice. Purified 12.5 dpc oogonia were able to generate mature oocytes by aggregating with 12.5 dpc ovarian somatic cells, but not with 16.5 dpc or 0 days postpartum ovarian somatic cells. We also tested 12.5 dpc male germ cells but they were unable to undergo oogenesis. CONCLUSIONS: Our study demonstrates that mature oocytes can be derived from purified fetal germ cells through an aggregation and transplantation procedure. It also suggests that the synchronized interactions between oogonia and gonadal somatic cells are important to ensure normal folliculogenesis.     

Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries

BACKGROUND: Ovarian tissue cryopreservation and transplantation can be used to restore fertility to sterile females. A question that warrants further investigation is whether the follicular content is affected by the freeze-thawing and grafting procedure, and if so, to what extent and by what mechanism. METHODS AND RESULTS: Intact newborn mouse ovaries were allografted under the kidney capsule or were cryopreserved by slow freezing with dimethylsulphoxide as the cryoprotectant prior to grafting. Estrogenic activity of ovariectomized recipient mice, as revealed by vaginal cytology, resumed after 11 days of transplantation. At 14 days after transplantation, ovarian grafts were recovered and processed histologically for follicle number counting. The follicular content of grafts of fresh ovaries was 58% of that from ovaries of age-matched 14 day old mice. In frozen-thawed ovarian grafts, the follicular content was only 9% lower than that of fresh grafted ovaries. Apoptosis of follicular cells was investigated by DNA nick end labelling. We observed a marked increase in the staining of fragmentation of DNA shortly after transplantation (2-12 h) of fresh newborn mouse ovaries. CONCLUSIONS: The results of the present study indicate that transplantation rather than cryopreservation accounts for the major and early loss of primordial follicles in grafted newborn mouse ovaries.     

人胚胎生殖干细胞移植对大鼠心肌梗死的影响

目的:探讨直接注射移植人胚胎生殖干细胞对大鼠心肌梗死的影响.方法:缝扎SD大鼠左冠状动脉前降支,建立心肌梗死模型.取5～10周人胚胎生殖腺嵴,组织块体外培养,生物学鉴定为人胚胎生殖干细胞(hEG).将其直接注射于大鼠急性心肌梗死边缘.于移植后1 d、1、2、4周处死大鼠,免疫组织化学方法观察心肌特异转录因子GATA-4和抗人细胞核抗体MAB1281在移植细胞的表达.结果:免疫组织化学显示移植组MAB1281检测阳性,GATA-4在移植细胞阳性表达.结论:hEG细胞直接注射移植大鼠心肌梗死处,细胞能存活并呈现向心肌细胞分化的表现,显示出正常心肌细胞的光镜结构.     

神经干细胞移植对小鼠脊髓损伤的影响

目的观察神经干细胞(NSCs)移植对脊髓损伤(SCI)小鼠功能恢复的影响。方法分离、培养、增殖和纯化E14-17d小鼠的NSCs,并通过免疫荧光法对其进行鉴定。将绿色荧光蛋白转基因小鼠的NSCs移植到小鼠脊髓损伤模型体内,术后进行行为学和病理学检测,观察小鼠功能恢复情况。运用改良Allen's法制备小鼠T10-T11脊髓损伤动物模型,动物分为假手术组(12只)、模型组(12只),治疗组(12只)和对照组6(12只)。治疗组每只小鼠自眶静脉注射NSCs悬液200μl(2×10个细胞),对照组只注射DMEM/F12培养基200μl。术后1d、3d、7d、14d、21d、28d和56d,进行BBB运动功能评分和病理学检测,观察植入区细胞生长变化情况。结果 BBB评分显示:治疗组明显高于对照组(<0.01),治疗组与假手术组相比没有明显差异(>0.05),说明NSCs移植后小鼠的行为学得到了明显改善,功能有所恢复。病理学检测发现,移植后NSCs不仅迁移到脊髓损伤区,而且与宿主细胞较好地整合。结论移植的E14-17d胚胎小鼠NSCs不仅可在脊髓损伤部位存活并和宿主细胞整合,而且可促进小鼠后肢运动功能恢复。     

Effects of supplementation with free radical scavengers on the survival and fertilization rates of mouse cryopreserved oocytes

BACKGROUND: This study was conducted to investigate the effects of supplementation with free radical scavengers on the survival and fertilization rates of freeze-thawed mouse oocytes. METHODS: Superovulated oocytes with cumulus cells were cryopreserved by slow freezing in propanediol combined with a rapid thawing protocol. The cryopreservation medium was supplemented with the antioxidant enzymes superoxide dismutase (SOD) and catalase, and with the nitric oxide (NO) scavenger, haemoglobin (Hb). RESULTS: The addition of 50 IU/ml SOD showed significantly higher survival and fertilization capabilities compared with control (P < 0.01). Oocyte survival was greatly increased by concomitant addition of SOD with 10 IU/ml catalase (P < 0.01). On the other hand, the NO donor (sodium nitroprusside) inhibited survival and fertilization rates (P < 0.05). Significantly decreased survival and fertilization rates were also observed following the addition of high concentrations (10(-3) to 10(-6) nmol/l) of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). In contrast, significantly better oocyte survival and fertilization rates were detected with low concentrations (10(-7) nmol/l) of L-NAME. Oocyte survival potential was significantly increased by addition of Hb (1 microg/ml, P < 0.05). Moreover, oocyte survival and fertilization rates were significantly promoted by the concomitant addition of SOD with Hb (P < 0.01). CONCLUSIONS: These results suggest that supplementation of free radical scavengers, particularly combinations of SOD with NO scavengers in freezing and thawing media, improved the post-thaw survival and fertilization rates of cryopreserved mouse oocytes.     

Comparison of ethylene glycol, 1,2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts

The aim of the study was to analyse the toxicity, the osmolar and cryoprotective activity of ethylene glycol (ETG) in terms of survival rate (SR), cleavage rate (CR) and expanded blastocysts percentage (EBP) of mouse embryos. Early mouse embryos and blastocysts were slowly cooled with ETG, 1,2-propanediol (PROH) or glycerol, and thawed. The Van t'Hoff curve for 1.5 mol/l ETG showed recovery of initial volume within 4 min. No differences were observed in CR and EBP of ETG-exposed compared with non-exposed mouse zygotes. The SR of zygotes frozen with PROH was significantly better than with ETG (92% and 60% respectively; P < 0.01), and a significantly better EBP was achieved for blastocysts frozen with glycerol compared with ETG (75% and 50% respectively; P < 0.05). For 4-cell stage embryos, no differences were observed in SR and EBP between ETG and PROH. Higher EBP was observed for 4-cell stage embryos (53%) frozen with ETG compared with pronucleate stage (19%) and blastocysts (48%). Low toxicity, good SR and EBP were observed for mouse embryos frozen with ETG, the best results being obtained at the 4-cell stage. At other embryonic stages, PROH and glycerol respectively seemed to provide better results.     

Successful uterine transplantation in the mouse: pregnancy and post-natal development of offspring

BACKGROUND: Uterine transplantation could serve as a tool in studies of the physiology of implantation/pregnancy, and is also a possible future treatment for patients with absolute uterine infertility. Here, the first live-born offspring in any uterine transplantation model is reported. METHODS: A syngeneic mouse model with a uterus transplanted, by end-to-side aorta/vena cava vascular anastomoses, alongside the native uterus was used. The cervix was attached to a cutaneous stoma. Pregnancy rate and offspring (birth weight, growth and fertility) was evaluated after blastocyst transfer to the native and the grafted uterus of transplanted mice and to controls. RESULTS: Pregnancy rates were comparable in the grafted uterus (8/12 animals became pregnant) and the native uterus (9/12 pregnant) of transplanted animals and controls (8/13 pregnant). In a separate set of animals, the native uterus was removed at transplantation to exclude influences from the native uterus on the pregnancy potential of the graft; two of four animals became pregnant after blastocyst transfer. The weights/lengths of fetuses (gestational day 18) and gestational lengths were similar in all groups. Offspring were delivered and the growth trajectories (up to 8 weeks) of offspring delivered from grafted or native uteri of transplanted mice were similar as compared with controls, and all were fertile. The second-generation offspring from transplanted animals were all fertile with normal birth weights. CONCLUSIONS: These observations document the capacity of a transplanted uterus to harbour pregnancies to term, and reveal that offspring from a transplanted uterus develop to normal fertile adults.     

间充质干细胞的免疫调节特性及其在干细胞移植中的应用

间充质干细胞具有独特的免疫负调节特性,能在不同层次作用于T淋巴细胞、B淋巴细胞、自然杀伤细胞（NK细胞）和树突状细胞,通过直接接触、分泌细胞因子转化生长因子（TGF-β）或调节这些细胞的代谢而发挥免疫调节作用,可望在干细胞移植后免疫重建及移植物抗宿主病的治疗中发挥重要作用。     

Reproductive capacity of sperm obtained after germ cell transplantation in a mouse model

BACKGROUND: The testicular stem cell transplantation technique has become an established research model in the mouse. This technique may also become useful for clinical applications. Therefore, it is necessary to investigate whether sperm obtained after testicular stem cell transplantation retain their full functional capacity and whether they are able to produce normally-developing embryos. This study aimed at evaluating the fertilizing and developmental abilities of sperm obtained after stem cell transplantation. METHODS: First, transplanted male mice were mated with females in order to evaluate in-vivo conception. Subsequently, functionality of sperm obtained after testicular germ cell transplantation was investigated by performing both IVF and ICSI. RESULTS: After in-vivo conception we found that in the control group 90% of the mice with a copulating plug became pregnant. In the experimental group only 35% of the mice with a copulating plug became pregnant (P = 0.006). After IVF, fertilization and blastocyst developmental rates were significantly lower in the transplanted group (P < 0.0001). Fertilization and blastocyst developmental rates after ICSI were comparable with control sperm. CONCLUSIONS: Our study showed that in the mouse, sperm obtained after stem cell transplantation are able to fertilize oocytes on the basis of assisted reproduction. It is recommended to further investigate this method in the human, as well as to investigate the post-implantation development of the embryo.     

促红细胞生成素对小鼠诱导多能干细胞源性神经干细胞向功能性神经元样细胞分化的影响

目的:探讨促红细胞生成素体外对小鼠诱导多能干细胞(iPSCs)来源的神经干细胞神经分化的影响。方法:神经干细胞培养基培养诱导iPSCs分化为神经干细胞,设4个浓度加入含促红细胞生成素的培养基,免疫细胞化学荧光鉴定神经元标志物微管相关蛋白2(MAP-2)、神经干细胞标志物巢蛋白的表达。FM 1-43染色技术观察FM 1-43染色颗粒消失的情况。结果:iPSCs在条件培养液的诱导下形成神经干细胞克隆,15 U/ml促红细胞生成素处理后神经干细胞克隆球向神经元方向分化,呈MAP-2阳性细胞。新分化的神经元有大量FM 1-43绿色阳性颗粒。结论:iPSCs体外诱导可获得神经干细胞,促红细胞生成素浓度为15 U/ml时可促进iPSCs源性神经干细胞向功能性的神经元方向分化。     

Germ cell transfer into rat, bovine, monkey and human testes.

Germ cell transplantation is a potentially valuable technique offering oncological patients gonadal protection by reinitiating spermatogenesis from stem cells which were reinfused into the seminiferous tubules. In order to achieve an intratubular germ cell transfer, intratubular microinjection, efferent duct injections and rete testis injections were applied on dissected testes of four different species: rat, bull, monkey and man. Ultrasound-guided intratesticular rete testis injection was the best and least invasive injection technique with maximal infusion efficiency for larger testes. Deep infiltration of seminiferous tubules was only achieved in immature or partially regressed testes. This technique was applied in vivo on two cynomolgus monkeys. In the first monkey a deep infusion of injected cells and dye into the lumen of the seminiferous tubules was achieved. In the second, transplanted germ cells were present in the seminiferous epithelium 4 weeks after the transfer. These cells were morphologically identified as B-spermatogonia and located at the base of the seminiferous epithelium. In summary, this paper describes a promising approach for germ cell infusion into large testes. The application of this technique is the first successful attempt of a germ cell transfer in a primate.     

自体外周血造血干细胞移植后淋巴细胞亚群的恢复

目的探讨自体外周血造血干细胞移植(auto-PBSCT)后免疫重建的规律。方法测定血液肿瘤患者auto-PBSCT前后淋巴细胞各亚群的绝对值。结果部分亚群在移植前低于正常。移植后各细胞亚群恢复时间不同,其中NK细胞在6个月恢复;B细胞在9个月恢复正常。T细胞总数在9个月恢复正常,其中主要为CD3 CD8 细胞。CD3 CD4 细胞在18个月恢复,而CD4 CD28 细胞在2年内仍未恢复。CD3 CD8 细胞在移植后早期有所下降,在3个月时恢复并高于正常,至9个月时逐渐下降至正常。CD3 CD8 细胞的主要部分是免疫表型呈活化的T细胞,即CD8 HLA-DR 细胞和CD8 CD38 细胞。两者分别在1个月和3个月时高于正常,在9个月时恢复正常。结论血液肿瘤患者移植前即存在淋巴细胞亚群异常的前提下,移植后NK细胞最先恢复,其次为B细胞,T细胞各亚群中CD3 CD8 细胞恢复较早,其中主要为活化T细胞。     

Assessment of functional integrity of frozen-thawed mouse embryos by albumin and leucine uptake

To assess the effects of freezing–thawing on metabolicfunctions of embryos prior to implantation, we measured theuptake of [¹²⁵I]bovine serum albumin (BSA) and [³H]leucine in2-cell mouse embryos, that were freshly collected (control),exposed to cryoprotectants (non-frozen), and frozen–thawed,and in morulae and blastocysts cultured from these 2-cell embryos.No significant difference in [¹²⁵I]BSA uptake by 2-cell embryoswas observed among the three groups. However, [¹²⁵I]BSA uptakeby blastocysts in the frozen–thawed group was significantlyreduced compared with the control and non-frozen groups. [³H]leucineuptake by 2-cell embryos in the frozen–thawed and non-frozengroups was significantly less than in the control group. Fluoresceindiacetate staining was performed in the control and frozen-thawed2-cell embryos. The intensity of fluorescence after fluoresceindiacetate exposure did not differ between the control and frozen–thawedembryos. The present study with mouse embryos suggests thatfreezing-thawing procedures impair the metabolic functions,in particular the membrane transport system, of embryos. Measurementsof BSA and leucine uptake in embryos may be useful for evaluatingthe quality of frozen–thawed embryos