Name: orthotopic ovarian transplantation procedures to investigate the life- and health-span influence of ovarian senescence in female mice
Uploaded: 2018-02-12T13:00:00
Description: 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

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
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<ol>
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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>

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 ...

Research

JoVE Journal

Developmental Biology

Culture and Co-Culture of Mouse Ovaries and 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

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

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

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

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

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

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

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

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 ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

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

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

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 ...