Name: orthotopic ovarian transplantation procedures to investigate the life- and health-span influence of ovarian senescence in female mice
Uploaded: 2018-02-12T13:00:00.000+00:00
Duration: 6 min 49 s
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
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>

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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&quot; 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&quot; 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+&amp;trade; Cordless Detachable Blade Clipper and a CeramicEdge&amp;reg; #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

10.8K 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

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

科研

免疫与感染

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, ...

发育生物学

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

成人小鼠卵巢中的精确卵泡图解

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular mechanisms that maintain oocyte quality remain unclear, thus it is difficult to increase the birth rate of women over 35 years old at present. Oocytes contain more mitochondria than any type of cell in the body, and any mitochondrial dysfunction can lead to reduced oocyte quality. In the 1990s, oocyte cytoplasmic transfer resulted in great success in human reproduction but was accompanied by ethical controversies. Autologous mitochondrial transplantation is expected to be a useful technique to increase the quality of oocytes that have decreased due to age. In the present study, we used adipose-derived stem cells from aged mice as a mitochondria donor to increase the quality of oocytes of aged mice. Further development of autologous mitochondrial transfer technology will provide a new and effective treatment for infertility in aged women.

Here, we describe the isolation of mitochondria from mouse adipose-derived mesenchymal stem cells, and then transfer the mitochondria into aged mouse oocytes to improve the quality of the oocytes.

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

移植到小鼠卵巢脂肪垫

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

医学

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

In this protocol, the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method were optimized for the paired interrogation of the cell-specific ovarian transcriptome and epigenome using the NuTRAP mouse model crossed to a Cyp17a1-Cre mouse line.

小鼠卵巢表观基因组和转录组的细胞特异性配对询问

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the ...

生物学

The Chick Chorioallantoic Membrane (CAM) Model as a Tool to Study Ovarian Tissue Transplantation

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle loss occurring shortly after reimplantation due to abnormal follicle activation and death. Rodents are benchmark models for investigating follicle activation, but the cost, time, and ethical considerations are becoming increasingly prohibitive, thus driving the development of alternatives. The chick chorioallantoic membrane (CAM) model is particularly attractive, being inexpensive and maintaining&#160;natural immunodeficiency up to day 17 postfertilization, making it ideal to study&#160;short-term xenografting of human ovarian tissue. The CAM is also highly vascularized and has been widely used as a model to explore angiogenesis. This gives it a remarkable advantage over in vitro models and allows the investigation of mechanisms affecting the early post-grafting follicle loss process. The protocol outlined herein aims to describe the development of a CAM xenografting model for human ovarian tissue, with specific insights into the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

The Chick Chorioallantoic Membrane CAM Model as a Tool to Study Ovarian Tissue Transplantation

Here, we describe a protocol for developing a chick chorioallantoic membrane (CAM) xenografting model for human ovarian tissue and demonstrate the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

鸡绒毛膜尿囊膜 （CAM） 模型作为研究卵巢组织移植的工具

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle ...

开发 FSH 水平升高的卵巢切除小鼠模型

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

作者聚焦:研究围绝经期女性 FSH 与病理生理变化之间的关系 - 来自小鼠模型的见解 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

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

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此视频中的章节

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

卵巢组织培养对小鼠卵巢现象的可视化

成人小鼠卵巢中的精确卵泡图解

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

移植到小鼠卵巢脂肪垫

小鼠卵巢表观基因组和转录组的细胞特异性配对询问

鸡绒毛膜尿囊膜（CAM）模型作为研究卵巢组织移植的工具

在小鼠模型中探索卵泡刺激素在体内的独立作用

在小鼠模型中探索卵泡刺激素在体内的独立作用

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

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

卵巢组织培养对小鼠卵巢现象的可视化

成人小鼠卵巢中的精确卵泡图解

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

移植到小鼠卵巢脂肪垫

小鼠卵巢表观基因组和转录组的细胞特异性配对询问

鸡绒毛膜尿囊膜 （CAM） 模型作为研究卵巢组织移植的工具

在小鼠模型中探索卵泡刺激素 在体内 的独立作用

在小鼠模型中探索卵泡刺激素 在体内 的独立作用

鸡绒毛膜尿囊膜（CAM）模型作为研究卵巢组织移植的工具

在小鼠模型中探索卵泡刺激素在体内的独立作用

在小鼠模型中探索卵泡刺激素在体内的独立作用