这份出版物中的研究报告得到了来自国立老龄研究所、老龄啮齿动物群体 (南 Nadon) 和犹他州立大学、兽医学院、动物、乳品学系的14月龄女子 CBA 小鼠的慷慨捐赠。和兽医科学。

这里描述的所有方法都是根据国家研究委员会的指导方针在实验室动物的护理和使用指南中制定的, 并已得到犹他州立大学动物保育和使用委员会的批准。
注: 卵巢移植程序可能涉及小鼠 (CBA/J 菌株) 从21天的年龄到18月的年龄。CBA/J 菌株的小鼠在45和60天的年龄和繁殖衰老之间成为繁殖能力在10和12月之间。
1. 完整卵巢移植治疗老龄小鼠
注意: 第一个协议描述了在将卵巢从幼鼠 (60 天) 转移到同一自交系的 postreproductive 雌性 (12 月) 的过程中使用的程序。同一程序用于卵巢移植两个雌性小鼠之间。
<ol><li>通过腹腔 (ip, 27 ga 针) 注射来称量和麻醉捐赠动物, 例如使用氯胺酮、甲苯噻嗪和乙酰丙嗪鸡尾酒 (65 毫克/千克氯胺酮, 13 毫克/千克甲苯噻嗪2.0 毫克/千克, 乙酰丙嗪; 鸡尾酒剂量-6.5 毫升/千克)。将动物送回加热的家庭笼子, 直到麻醉生效。</li>
	<li>一旦麻醉, 把动物从笼子里取出, 放在加热的纸巾上。使用剪刀 (#40 刀片首选), 删除的头发几毫米侧向中线两侧, 开始刚刚下方的肋骨和移动远端, 准备一个修剪 ' 补丁 ' 约2-3 厘米正方形的任何一侧的容易老鼠离开一条头发大约1-2 厘米覆盖中线。</li>
	<li>用70% 乙醇轻轻擦拭修剪过的区域, 用棉尖拭子将 betadine 溶液涂抹在修剪过的部位。</li>
	<li>应用抗生素凝胶进行眼部保护, 并管理 meloxicam (2.0 毫克/千克) 皮下 (南卡罗来纳州, 27 ga 针) 在动物颈部的侧面, 用于术后疼痛缓解。</li>
	<li>将动物置于易受加热的无菌手术领域, 用70% 乙醇浸泡的2厘米 x 2 厘米棉纱布擦拭修剪过的区域。</li>
	<li>做一个 1.0-1.5 厘米 paralumbar 切口和坦率地解剖皮肤从底层筋膜。再通过筋膜切开切口。坦率地解剖在这个筋膜之下尽可能表面上尽可能, 直到到达腹腔</li>
	<li>在切口上放置一个小的手术悬垂 (从2厘米 x 2 厘米的纱布切开), 用无菌生理盐水湿润褶皱。</li>
	<li>使用小钳, 定位周围的卵巢在腹腔的脂肪组织, 并轻轻地提取脂肪垫和卵巢, 并把它放在湿纱布悬垂。卵巢脂肪垫将出现更多的 ' 白色 ' 比周围的脂肪组织。</li>
	<li>放置一个1.5 英寸斗牛犬钳在脂肪垫 (注意不要阻碍卵巢囊), 以保持在该过程中的地方。</li>
	<li>将解剖显微镜移到原处, 明确辨认卵巢囊。用两个制表钳抓住囊, 切开卵巢脐对面的卵巢囊, 露出卵巢。如果囊中的预期进入区是高度血管化的, 用注射器将一滴肾上腺素 (1:100,000) 放在囊壁表面, 以减少撕裂时的出血。</li>
	<li>轻轻地从卵巢囊中取出卵巢, 用制表钳夹在卵巢脐上, 以防止出血。</li>
	<li>将供体卵巢放在观察玻璃填充 4° C 无菌生理盐水, 松散覆盖和预留, 直到准备移植。理想情况下, 将手表玻璃置于额外的解剖显微镜下, 以便外科医生在移植时能很容易地掌握卵巢。</li>
	<li>此时, 无论是弄死还是搁置捐赠鼠标 (必须不断监测), 以接收移植的卵巢, 组织, 细胞或占位玻璃珠在以后的时间点。</li>
	<li>用干净的手术场地和仪器, ovariectomize 的受体动物。年长的老鼠往往有增加的脂肪组织数量, 可能需要增加麻醉剂量 (高达 20%)。</li>
	<li>切除后的卵巢从接受者, 放置一个小块止血泡沫垫内的空囊, 以控制出血和援助凝血和放置一小块盐水浸泡纱布在暴露的地方。</li>
	<li>一只手找到一个捐献者卵巢 (或组织, 细胞, 占位玻璃珠) 与钟表匠钳, 进入显微镜视野。另一钳在相反的手, 取出纱布, 同时从囊中取出止血泡沫垫, 同时放置新的卵巢囊内。</li>
	<li>如果新的卵巢不适合在接受者囊内, 切开捐赠子房在一半 (或1/3 或 1/4) 和安置入囊在片断。如果囊不完全覆盖整个卵巢, 放置一个缝合通过囊, 通过卵巢和背部通过囊, 以举行卵巢在地方 (参见讨论部分)。 注: 玻璃的地方持有人珠可能难以处理钟表匠的镊子。</li>
	<li>当在囊中放置一个玻璃珠, 拿起大钳的珠子和下降的脂肪垫旁边的卵巢囊。把珠子滚入囊中。</li>
	<li>拿起一个 Castroviejo 针持有人已经加载了锥形针和9-0 或10-0 单丝缝合, 找出撕裂边缘的囊和关闭囊使用一个或两个霍尔斯特德缝合。 注: 缝合可以与钟表匠或其他小钳一起执行。</li>
	<li>用无菌生理盐水轻轻冲洗部位, 取出脂肪垫钳, 并将卵巢/脂肪垫送回腹部。</li>
	<li>取下悬垂, 用6-0 铬肠或 vicryl 关闭腹壁。这一步可以使用或没有显微镜。</li>
	<li>将显微镜移到一旁 (如果不在上一步移动), 并关闭皮肤与9毫米伤口剪辑。</li>
	<li>将动物放在一个加热的恢复笼中, 从光线中遮蔽, 直到麻醉恢复后才能持续监测动物。额外的 meloxicam 可在手术后24小时使用, 如果表明。手术后九天左右取出伤口夹。 注: 未采用免疫抑制技术, 移植后或死亡时未发现移植物抗宿主病的证据。</li>
</ol>2. 将生殖细胞耗尽的卵巢移植到完整的小鼠体内。
注意: 这个协议包括将卵巢从一个年轻的, 生殖细胞耗尽的老鼠转移到一个 postreproductive 的雌性。在本议定书中, 卵巢接受者和卵巢切除术和移植程序是一样的, 除了移植的卵巢已被化学耗尽的原始和主要卵泡/生殖细胞。其余的二级和窦卵泡将进行招募/闭锁离开卵巢缺乏生殖细胞。4-vinylcyclohexene diepoxide (VCD) 的生殖细胞损耗很好地描述为16。VCD 是一种毒素。由于 vcd 处理的卵巢被移植到一个未被治疗的小鼠, 这一程序得益于一个 vcd 处理的卵巢在鼠标不暴露的毒性影响 vcd。本议定书描述了幼鼠、卵巢捐献者的胚胎细胞耗尽的过程。
<ol><li>稀释 VCD (64 毫克/毫升) 在芝麻油注射和存储在 4° C, 直到使用。</li>
	<li>每天注射一次卵巢捐献者的 VCD (160 毫克/千克), 15 天开始在28天的年龄。在相同的时间安排下, 用芝麻油注射控制小鼠。</li>
	<li>在50天的年龄, 检查 VCD 治疗和油只处理小鼠停止生殖周期性10天以上。在60天的年龄, VCD 治疗小鼠应该不显示性腺输入的细胞学证据。</li>
	<li>恢复卵巢从60天老鼠和移植如上。</li>
	<li>通过检测缺乏生殖循环和提高循环 androstenedione 的水平, 确定成功移植生殖细胞衰竭卵巢的方法。雄激素: 与 VCD17的生殖细胞耗尽后雌激素比值显著增加。</li>
	<li>术后10-14 天开始阴道细胞学检查, 收集数据至少连续10天, 以确保准确测定周期性。</li>
</ol>

<ol>
	<li>National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Fertility and Infertility Branch. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Fertility+Status+as+a+Marker+of+Overall+Health'.+"Support+studies+that+investigate+fertility+status+as+a+marker+of+overall+health+for+both+men+and+women".">'Fertility Status as a Marker of Overall Health'. "Support studies that investigate fertility status as a marker of overall health for both men and women".</a> New Research Priorities. , (2016).</li><li>Eisenberg, M. L., Li, S. F., Behr, B., Cullen, M. R., Galusha, D., Lamb, D. J., Lipshultz, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semen+quality,+infertility+and+mortality+in+the+USA.">Semen quality, infertility and mortality in the USA.</a> Hum. Reprod. 29 (7), 1567-1574 (2014).</li><li>Thom, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Heart+Association+Statistics+Committee+and+Stroke+Statistics+Subcommittee.+Heart+disease+and+stroke+statistics+-+2006+update+-+A+report+from+the+American+Heart+Association+Statistics+Committee+and+Stroke+Statistics+Subcommittee.">American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics - 2006 update - A report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee.</a> Circulation. 113 (6), E85-E151 (2006).</li><li>Shuster, L. T., Rhodes, D. J., Gostout, B. S., Grossardt, B. R., Rocca, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+menopause+or+early+menopause:+Long-term+health+consequences.">Premature menopause or early menopause: Long-term health consequences.</a> Maturitas. 65 (2), 161-166 (2010).</li><li>Jacobsen, B. K., Knutsen, S. F., Fraser, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+at+natural+menopause+and+total+mortality+and+mortality+from+ischemic+heart+disease:+The+Adventist+health+study.">Age at natural menopause and total mortality and mortality from ischemic heart disease: The Adventist health study.</a> J Clin Epidemiol. 52 (4), 303-307 (1999).</li><li>Kulaksizoglu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+Factors+for+Diabetes+Mellitus+in+Women+with+Primary+Ovarian+Insufficiency.">Risk Factors for Diabetes Mellitus in Women with Primary Ovarian Insufficiency.</a> Biol Trace Elem Res. 154 (3), 313-320 (2013).</li><li>Johnell, O., Kanis, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+estimate+of+the+worldwide+prevalence+and+disability+associated+with+osteoporotic+fractures.">An estimate of the worldwide prevalence and disability associated with osteoporotic fractures.</a> Osteoporos Int. 17 (12), 1726-1733 (2006).</li><li>Rosario, E. R., Chang, L., Head, E. H., Stanczyk, F. Z., Pike, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+levels+of+sex+steroid+hormones+in+men+and+women+during+normal+aging+and+in+Alzheimer's+disease.">Brain levels of sex steroid hormones in men and women during normal aging and in Alzheimer's disease.</a> Neurobiol Aging. 32 (4), 604-613 (2011).</li><li>Cargill, S. L., Medrano, J. F., Anderson, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infertility+in+a+line+of+mice+with+the+high+growth+mutation+is+due+to+luteal+insufficiency+resulting+from+disruption+at+the+hypothalamic-pituitary+axis.">Infertility in a line of mice with the high growth mutation is due to luteal insufficiency resulting from disruption at the hypothalamic-pituitary axis.</a> Biol Reproduction. 61 (1), 283-287 (1999).</li><li>Mason, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+young+ovaries+restored+cardioprotective+influence+in+postreproductive-aged+mice.">Transplantation of young ovaries restored cardioprotective influence in postreproductive-aged mice.</a> Aging Cell. 10 (3), 448-456 (2011).</li><li>Mason, J. B., Terry, B. C., Merchant, S. S., Mason, H. M., Nazokkarmaher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+Ovarian+Function+Significantly+Influenced+Trabecular+and+Cortical+Bone+Volume,+Architecture+and+Density+in+Mice+at+Death.">Manipulation of Ovarian Function Significantly Influenced Trabecular and Cortical Bone Volume, Architecture and Density in Mice at Death.</a> Plos One. 10 (12), 15 (2015).</li><li>Peterson, R. L., Parkinson, K. C., Mason, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+Ovarian+Function+Significantly+Influenced+Sarcopenia+in+Postreproductive-Age+Mice.">Manipulation of Ovarian Function Significantly Influenced Sarcopenia in Postreproductive-Age Mice.</a> J. Transplant. , (2016).</li><li>Peterson, R. L., Parkinson, K. C., Mason, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+immune+and+renal+function+in+aged+females+by+re-establishment+of+active+ovarian+function.">Restoration of immune and renal function in aged females by re-establishment of active ovarian function.</a> Reprod. Fertil. Dev. , (2017).</li><li>Parkinson, K. C., Peterson, R. L., Mason, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+behavior+and+sensory+function+were+significantly+influenced+by+restoration+of+active+ovarian+function+in+postreproductive+mice.">Cognitive behavior and sensory function were significantly influenced by restoration of active ovarian function in postreproductive mice.</a> Exp. Geront. 92, 28-33 (2017).</li><li>Cargill, S. L., Carey, J. R., Muller, H. G., Anderson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+of+ovary+determines+remaining+life+expectancy+in+old+ovariectomized+mice.">Age of ovary determines remaining life expectancy in old ovariectomized mice.</a> Aging Cell. 2 (3), 185-190 (2003).</li><li>Brooks, H. L., Pollow, D. P., Hoyer, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+VCD+Mouse+Model+of+Menopause+and+Perimenopause+for+the+Study+of+Sex+Differences+in+Cardiovascular+Disease+and+the+Metabolic+Syndrome.">The VCD Mouse Model of Menopause and Perimenopause for the Study of Sex Differences in Cardiovascular Disease and the Metabolic Syndrome.</a> Physiology. 31 (4), 250-257 (2016).</li><li>Rivera, Z., Christian, P. J., Marion, S. L., Brooks, H. L., Hoyer, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroidogenic+Capacity+of+Residual+Ovarian+Tissue+in+4-Vinylcyclohexene+Diepoxide-Treated+Mice.">Steroidogenic Capacity of Residual Ovarian Tissue in 4-Vinylcyclohexene Diepoxide-Treated Mice.</a> Biol. Reprod. 80 (2), 328-336 (2009).</li><li>Yuan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+in+inbred+strains+of+mice:+study+design+and+interim+report+on+median+lifespans+and+circulating+IGF1+levels.">Aging in inbred strains of mice: study design and interim report on median lifespans and circulating IGF1 levels.</a> Aging Cell. 8 (3), 277-287 (2009).</li><li>Mason, J. B., Cargill, S. L., Anderson, G. B., Carey, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+Young+Ovaries+to+Old+Mice+Increased+Life+Span+in+Transplant+Recipients.">Transplantation of Young Ovaries to Old Mice Increased Life Span in Transplant Recipients.</a> J. Gerontol. A Biol. Sci. Med. Sci. 64 (12), 1207-1211 (2009).</li><li>Harrison, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acarbose,+17-alpha-estradiol,+and+nordihydroguaiaretic+acid+extend+mouse+lifespan+preferentially+in+males.">Acarbose, 17-alpha-estradiol, and nordihydroguaiaretic acid extend mouse lifespan preferentially in males.</a> Aging Cell. 13 (2), 273-282 (2013).</li><li>Manay, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=17&#945;-Estradiol+Attenuates+Neuron+Loss+in+Ovariectomized+Dtg+A&#946;PP/PS1+Mice.">17&#945;-Estradiol Attenuates Neuron Loss in Ovariectomized Dtg A&#946;PP/PS1 Mice.</a> J. Alzheimers Dis. 23 (4), 629-639 (2011).</li><li>Parker, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+conservation+at+the+time+of+hysterectomy+and+long-term+health+outcomes+in+the+nurses'+health+study.">Ovarian conservation at the time of hysterectomy and long-term health outcomes in the nurses' health study.</a> Obstet Gynecol. 113, 1027-1037 (2009).</li></ol>

作者没有什么可透露的。

在21天至18个月的小鼠中进行了卵巢切除和完整和生殖细胞衰竭卵巢的移植。在进行卵巢切除手术的时候, 最好在适当的情况下, 在切开该囊组织时要注意。在年龄在六月以上的小鼠中, 大部分的囊膜与脂肪垫覆盖, 这限制了可用于进入的区域。在这些情况下, 通常有利于撕裂囊打开接近脂肪垫边界。这种方法经常留下一个更容易接近的 "口袋" 的法氏囊组织 "塔克" 新的卵巢内囊。这种方法也可以减少...

生殖对健康和寿命的影响常常被误认为仅限于生育能力的时期。虽然与繁殖的生命周期的相关阶段的生活方式和表型很容易区分从前和 postreproductive 阶段, 生殖状态影响健康在整个生命周期的所有阶段。生殖能力的时期通常被称为生殖生命的跨度, 区别于时间的生命跨度。这一术语在现实中是对两个生命跨度的指定进行人工分离, 两者是紧密联系在一起的, 影响一个人的事件, 往往也会影响到另一个。
过去十年的证据表明, 个人的生殖状况与发展慢性健康状况的风险增加有关1。一项研究记录了一组男性的短寿命和生殖衰竭之间的关联2。这个协会在女性中更引人注目。绝经前妇女的心血管疾病是罕见的3, 但卵巢衰竭增加心血管疾病急剧在更年期4和年轻妇女过早卵巢衰竭5。胰岛素抵抗6和绝经后骨质疏松症7和接近2/3 的美国老年痴呆症患者为女性8。卵巢信号的定义明确的变化标志着从绝经前, 抗病的状态, 到绝经后, 疾病负担状态的转变。这种稳定的过渡, 提供了一个机会, 以确定机制, 加速疾病风险的老龄化。
最近, 保存的卵巢组织移植已被用于恢复妇女的生育能力, 这可能危及生殖细胞, 如化疗。卵巢组织移植还可用于在非卵巢基不孕的动物中产生可行的后代, 包括转基因小鼠9。卵巢移植是一种有效的方法, 以分离的影响, 生殖功能或生殖衰老的时间老化本身。
卵巢激素在女性健康的许多方面都得到了良好的支持, 这就导致了在更年期引起的疾病风险增加的主要原因, 即积极循环生殖细胞产生的激素损失。虽然卵巢激素在女性健康中的价值是不容置疑的, 但在绝经后妇女中取代这些荷尔蒙对恢复年轻卵巢的年轻妇女的健康福利是不可靠的。这些健康好处得到可靠地恢复通过 heterochronic 移植年轻卵巢到 postreproductive 雌性小鼠10,11,12,13,14。
捐献者和受赠小鼠的生殖状态应在移植或卵巢切除术之前和之后确定。假定 postreproductive 小鼠在12月的手术前显示性腺输入的迹象, 这些实验不包括在内。性腺输入是由阴道细胞学决定的。阴道细胞学资料收集至少连续10天前手术, 以确保 postreproductive 状态的受体小鼠和积极循环卵巢的捐助小鼠。一个发情周期的定义是从当天有核上皮细胞第一次出现的时期 (即, 发情前期) 的前一天出现有核上皮细胞在阴道涂片, 只要有一个时期的白细胞介于两者之间的存在 (即、diestrus)。发情是由存在的大, 鳞状上皮细胞, 有或没有细胞核。在手术前和/或术后10天内不显示周期性活动的小鼠, 确定没有性腺输入。在手术前和/或术后10天内显示至少一个完整的发情周期的小鼠, 确定有性腺输入的期间。雌性小鼠应安置在雄性小鼠的面前, 或暴露于从雄性笼子里弄脏的床上用品上, 以确保持续暴露于循环诱发的刺激。
这些移植程序是最有效的完成与两个外科医生同时工作的捐助者和接受的老鼠。然而, 程序很容易完成的一个外科医生, 与捐赠卵巢最经常恢复之前开始的卵巢切除手术的收件人鼠标。该协议描述了单个外科医生的程序。暴露的生殖道和关闭的网站通常是没有显微镜, 而删除和更换卵巢是在显微镜下完成。与学习任何外科手术一样, 切口部位可能更大, 手术可能需要更长的时间, 而首先学习的过程, 并通常会减少与经验。

<table>
	<tbody>
		<tr>
			<td>Dissecting microscope</td>
			<td>Any dissecting scope with an adjustable working distance.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm Water Recirculator and appropriate pad for your size surgical/recovery field.</td>
			<td>Stryker/Gaymar</td>
			<td>TP700</td>
			<td>Circulating water pads produce even and consistent temperature operating and recovery fields.</td>
		</tr>
		<tr>
			<td>Surgical instruments-dissection instruments for access to the reproductive tract</td>
			<td></td>
			<td></td>
			<td>Choice of specific surgical instruments is highly surgeon-specific</td>
		</tr>
		<tr>
			<td>Surgical instruments-fine instruments for the transplantation procedure.</td>
			<td></td>
			<td></td>
			<td>Choice of specific surgical instruments is highly surgeon-specific</td>
		</tr>
		<tr>
			<td>Suture material for abdominal wall</td>
			<td>eSutures</td>
			<td>G3756K: 6-0 MILD CHROMIC GUT 18&#34; HE-7 CUTTING, DOUBLE ARMED</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture material for ovarian bursa</td>
			<td>eSutures</td>
			<td>2809G: 9-0 or 2810G: 10-0 ETHILON BLACK 5&#34; BV130-5 TAPER</td>
			<td>10-0 suture can be difficult to locate if dropped on the surgery field.</td>
		</tr>
		<tr>
			<td>Absorbable gelatin sponge</td>
			<td>PFIZER.</td>
			<td>Gelfoam- PFIZER 09-0315-08.</td>
			<td>cut into small (~2mm) pieces under sterile conditions.</td>
		</tr>
		<tr>
			<td>Staples for skin closure</td>
			<td>Becton Dickinson</td>
			<td>MikRon Autoclip 9mm (BD 427631)</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment.</td>
			<td>MWI Veterinary Supply Co.</td>
			<td>Puralube sterile opthalmic ointment</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes for anesthesia, analgesia and epinephrine</td>
			<td>MWI Veterinary Supply Co.</td>
			<td>BD 305536</td>
			<td>27g x 3/8in,Intradermal Bevel 1/2ml</td>
		</tr>
		<tr>
			<td>Syringes for saline.</td>
			<td>MWI Veterinary Supply Co.</td>
			<td>BD 309657.</td>
			<td>3ml syringe, luer-lok tip, 27g needle</td>
		</tr>
		<tr>
			<td>Clippers/blade</td>
			<td>Andis</td>
			<td>Super AGR+&#8482; Cordless Detachable Blade Clipper and a CeramicEdge&#174; #40 blade.</td>
			<td>Any clippers with a fine blade.</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>University chemical supply</td>
			<td></td>
			<td>dilute to 70% for use.</td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>MWI Veterinary Supply Co.</td>
			<td>003233</td>
			<td></td>
		</tr>
		<tr>
			<td>2x2 gauze pads</td>
			<td>MWI Veterinary Supply Co.</td>
			<td>003073</td>
			<td>autoclaved with surgery packs</td>
		</tr>
		<tr>
			<td>Borosilicate glass beads</td>
			<td>Sigma-Aldrich Co.</td>
			<td>Z273619</td>
			<td>Autoclave in sintilation vials</td>
		</tr>
		<tr>
			<td>4-vinylcyclohexene diepoxide</td>
			<td>Sigma-Aldrich Co.</td>
			<td>94956</td>
			<td></td>
		</tr>
		<tr>
			<td>Sesame oil</td>
			<td>Sigma-Aldrich Co.</td>
			<td>S3547</td>
			<td></td>
		</tr>
	</tbody>
</table>

orthotopic ovarian transplantation procedures to investigate the life- and health-span influence of ovarian senescence in female mice

卵巢移植在犹他州立大学于1963年首次进行。在最近的工作中, heterochronic 移植哺乳动物卵巢被用来调查年轻女性卵巢的健康保护作用。目前的手术方法采用原位移植法, 将同种异体卵巢移植回卵巢囊中的原位置。这与更常用的卵巢/卵巢组织皮下或肾囊下的异位移植不同。所有三个地点都为移植组织提供高效的血管重建。然而, 原位移植为卵巢提供了最自然的信号环境, 是唯一的程序, 提供的机会, 动物繁殖自然后手术。在去卵巢手术过程中, 必须注意去除所有内源性卵巢组织。如果任何内源组织保持或如果只有一个卵巢被删除, 移植的组织将保持休眠状态, 直到现有的组织变得衰老。虽然移植卵巢的血管重建很快发生, 移植接受者可能需要相当长的时间适应新的下丘脑/垂体/性腺/肾上腺 (HPG/a) 轴信号机制与移植组织.这通常需要大约100天的鼠标。因此, 移植实验的设计应适应这一适应期。卵巢移植的典型结果将包括接受者健康状况的变化, 反映移植卵巢的年龄, 而不是接受者的按年代年龄。

在21天的卵巢切除将避免在青春期开始时对生殖系统进行重大的调控, 并消除雌性性腺除了对性腺激素的直接影响之外还可能产生的其他影响。CBA/J 小鼠的生殖衰退通常以8-10 月的不规则周期开始。在11月的年龄, 大多数雌性在这条线的老鼠已经达到了一个繁殖失败的点15与完全失去反应性卵母细胞12月的年龄。这些年龄不同的小鼠品种。双侧卵巢切除/卵巢?...

Watch this Scientific Journal Video about Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice at JoVE.com

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

这里描述的是原位卵巢移植协议, 改变卵巢对受体小鼠生理的影响。这种手术方法是用来暴露雌性小鼠对各种类型的卵巢影响, 可能包括 heterochronic 移植和卵巢移植已经经历了各种前体治疗。

原位卵巢移植术对雌性小鼠卵巢衰老的影响及寿命及健康范围研究

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is ...

orthotopic-ovarian-transplantation-procedures-to-investigate-life

Research

JoVE Journal

Developmental Biology

10.7K 次观看.  Utah State University. 这里描述的是原位卵巢移植协议, 改变卵巢对受体小鼠生理的影响。这种手术方法是用来暴露雌性小鼠对各种类型的卵巢影响, 可能包括 heterochronic 移植和卵巢移植已经经历了各种前体治疗。

视频: Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

文化和共同培养小鼠的卵巢和卵巢卵泡

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

科研

发育生物学

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

利用共聚焦分析<em&gt;非洲爪蟾</em&gt;调查的Wnt和Shh形态发生素梯度的调节剂

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. This morphological alteration is also recognized as epithelial to mesenchymal transition (EMT). Dysregulation of EMT is associated with fibrosis and cancer metastasis. There is emerging evidence that EMT is controlled by a number of molecular mechanisms. As such, many key genes that control apical constriction are also known to be important factors in the EMT observed in cancer metastasis. Like EMT during Drosophila gastrulation, epithelial cells can be induced to change their shape and be reprogrammed to redirect cell fate towards various other cell types. Here we provide a robust imaging method of Drosophila gastrulation to assay the initiation of morphogenetic cellular movements and cell fate identification during this stage of embryonic development. Using this method, we identify cell rearrangement at the time of gastrulation and demonstrate the importance of apical constriction during gastrulation using GFP labeled DE-cadherin.

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

细胞形态改变与细胞运动的影像<em&gt;果蝇</em&gt;原肠胚形成使用DE-cadherin的记者转基因果蝇

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called liquefaction. In this study, we show how to collect the post-ejaculated semen from the female reproductive tract in a mouse model. First, adult female mice in the estrus stage were housed in a male&#39;s cage overnight. The next morning, copulation was confirmed by the presence of copulatory plug at the vaginal opening. Female mice with copulatory plugs were euthanized, and each reproductive tract was collected as a whole (vagina, uterus, oviducts, ovaries), ensuring a closed system to contain the semen. The reproductive tract was placed in a 1.5 mL microcentrifuge tube, and the vagina was cut off to release the semen into the tube. To ensure maximum semen volume for analysis, toothless forceps were used to squeeze the uterine horns from ovarian end to vaginal end expelling remaining semen. The whole reproductive tract was then discarded. The semen-containing tube was briefly spun down. A 25 &#956;L capillary pipette was placed into the tube at a 180&#176; angle (parallel to the tube wall). The amount of time used to fill the capillary tube to the 25 &#956;L line was recorded. Semen from a proven male breeder usually takes approximately 60-180 s to fill a 25 &#956;L capillary tube. This semen collection technique can also be used in other downstream applications such as sperm imaging and motility analysis.

Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

雌性生殖道交配后精子的收集及对小鼠精子液化的测定

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

原发性细胞衰老的诱导与鉴定

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

卵巢组织培养对小鼠卵巢现象的可视化

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

基因型衰老过程中正常人成纤维细胞活性氧和衰老相关分泌表型的定量测定

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

原位卵巢移植术对雌性小鼠卵巢衰老的影响及寿命及健康范围研究

摘要

探索更多视频

此视频中的章节