Name: ovarian tissue culture to visualize phenomena in mouse ovary
Uploaded: 2018-06-19T14:30:00
Description: Watch this Scientific Journal Video about Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary at JoVE.com

박사 Naojiro 미나미 (교토대학)는 Oog1pro3.9 마우스를 제공 하는 감사 합니다. 이 연구는 JSP (KAKENHI # JP15H06275)와 닛토 재단에 의해 지원 되었다.

마우스는 23 ± 1 ° C 12 h 빛/12 h 어두운 사이클 환경 제어 실에서 보관 되어 있었다. 동물 보호 프로토콜 및 실험 아이치 의과대학에서 동물 실험에 대 한 지침에 따라 실시 했다 고 현 동물 관리 및 사용 위원회에 의해 승인 했다.
1입니다. 배양 및 요리 준비
<ol><li>기본적인 문화 매체를 준비 하려면 추가 태아 둔감 한 혈 청 (FBS, 5 %v / v), FSH (100 mIU/mL), LH (10 mU/mL), 그리고 페니실?...

<ol>
	<li>Myers, M., Britt, K. L., Wreford, N. G., Ebling, F. J., Kerr, 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=Methods+for+quantifying+follicular+numbers+within+the+mouse+ovary.">Methods for quantifying follicular numbers within the mouse ovary.</a> Reproduction. 127 (5), 569-580 (2004).</li><li>Adams, G. P., Jaiswal, R., Singh, J., Malhi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+understanding+ovarian+follicular+dynamics+in+cattle.">Progress in understanding ovarian follicular dynamics in cattle.</a> Theriogenology. 69 (1), 72-80 (2008).</li><li>Palma, G. A., 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=Biology+and+biotechnology+of+follicle+development.">Biology and biotechnology of follicle development.</a> Sci World J. , (2012).</li><li>Knight, P. G., Glister, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+superfamily+members+and+ovarian+follicle+development.">TGF-beta superfamily members and ovarian follicle development.</a> Reproduction. 132 (2), 191-206 (2006).</li><li>Picton, H. M., Harris, S. E., Muruvi, W., Chambers, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+growth+and+maturation+of+follicles.">The in vitro growth and maturation of follicles.</a> Reproduction. 136 (6), 703-715 (2008).</li><li>Spears, N., de Bruin, J. P., Gosden, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+establishment+of+follicular+dominance+in+co-cultured+mouse+ovarian+follicles.">The establishment of follicular dominance in co-cultured mouse ovarian follicles.</a> J Reprod Fertil. 106 (1), 1-6 (1996).</li><li>Baker, S. J., Srsen, V., Lapping, R., Spears, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+effect+of+follicle-follicle+interactions+and+declining+follicle-stimulating+hormone+on+murine+follicle+health+in+vitro.">Combined effect of follicle-follicle interactions and declining follicle-stimulating hormone on murine follicle health in vitro.</a> Biol Reprod. 65 (4), 1304-1310 (2001).</li><li>Komatsu, 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=Analysis+of+the+Effect+of+Leukemia+Inhibitory+Factor+on+Follicular+Growth+in+Cultured+Murine+Ovarian+Tissue.">Analysis of the Effect of Leukemia Inhibitory Factor on Follicular Growth in Cultured Murine Ovarian Tissue.</a> Biol Reprod. 93 (1), 18 (2015).</li><li>Komatsu, K., Masubuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+concentration-dependent+effect+of+progesterone+on+follicle+growth+in+the+mouse+ovary.">The concentration-dependent effect of progesterone on follicle growth in the mouse ovary.</a> J Reprod Dev. 63 (3), 271-277 (2017).</li><li>Murase, 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=Follicle+dynamics:+visualization+and+analysis+of+follicle+growth+and+maturation+using+murine+ovarian+tissue+culture.">Follicle dynamics: visualization and analysis of follicle growth and maturation using murine ovarian tissue culture.</a> J Assist Reprod Genet. , 1-5 (2017).</li><li>Ishida, 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=The+promoter+of+the+oocyte-specific+gene,+Oog1,+functions+in+both+male+and+female+meiotic+germ+cells+in+transgenic+mice.">The promoter of the oocyte-specific gene, Oog1, functions in both male and female meiotic germ cells in transgenic mice.</a> PLoS One. 8 (7), 68686 (2013).</li><li>Minami, N., 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=Oogenesin+is+a+novel+mouse+protein+expressed+in+oocytes+and+early+cleavage-stage+embryos.">Oogenesin is a novel mouse protein expressed in oocytes and early cleavage-stage embryos.</a> Biol Reprod. 69 (5), 1736-1742 (2003).</li><li>Nilsson, E. E., Skinner, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+factor-9+stimulates+progression+of+early+primary+but+not+primordial+rat+ovarian+follicle+development.">Growth and differentiation factor-9 stimulates progression of early primary but not primordial rat ovarian follicle development.</a> Biol Reprod. 67 (3), 1018-1024 (2002).</li><li>Nilsson, E. E., Kezele, P., Skinner, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukemia+inhibitory+factor+(LIF)+promotes+the+primordial+to+primary+follicle+transition+in+rat+ovaries.">Leukemia inhibitory factor (LIF) promotes the primordial to primary follicle transition in rat ovaries.</a> Mol Cell Endocrinol. 188 (1-2), 65-73 (2002).</li><li>Nilsson, E. E., Skinner, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+protein-4+acts+as+an+ovarian+follicle+survival+factor+and+promotes+primordial+follicle+development.">Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development.</a> Biol Reprod. 69 (4), 1265-1272 (2003).</li><li>Nilsson, E. E., Skinner, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kit+ligand+and+basic+fibroblast+growth+factor+interactions+in+the+induction+of+ovarian+primordial+to+primary+follicle+transition.">Kit ligand and basic fibroblast growth factor interactions in the induction of ovarian primordial to primary follicle transition.</a> Mol Cell Endocrinol. 214 (1-2), 19-25 (2004).</li><li>Abe, 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=Establishment+of+conditional+reporter+mouse+lines+at+ROSA26+locus+for+live+cell+imaging.">Establishment of conditional reporter mouse lines at ROSA26 locus for live cell imaging.</a> Genesis. 49 (7), 579-590 (2011).</li><li>Lan, Z. J., Xu, X., Cooney, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+oocyte-specific+expression+of+Cre+recombinase+activity+in+GDF-9-iCre,+Zp3cre,+and+Msx2Cre+transgenic+mice.">Differential oocyte-specific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2Cre transgenic mice.</a> Biol Reprod. 71 (5), 1469-1474 (2004).</li><li>Payer, 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=Generation+of+stella-GFP+transgenic+mice:+a+novel+tool+to+study+germ+cell+development.">Generation of stella-GFP transgenic mice: a novel tool to study germ cell development.</a> Genesis. 44 (2), 75-83 (2006).</li><li>Ohinata, Y., Sano, M., Shigeta, M., Yamanaka, K., Saitou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive,+non-invasive+visualization+of+primordial+germ+cell+development+in+mice+by+the+Prdm1-mVenus+and+Dppa3-ECFP+double+transgenic+reporter.">A comprehensive, non-invasive visualization of primordial germ cell development in mice by the Prdm1-mVenus and Dppa3-ECFP double transgenic reporter.</a> Reproduction. 136 (4), 503-514 (2008).</li></ol>

이 연구에서 우리는 마우스 난소의 여 포 개발을 공부 하는 두 가지 새로운 방법 개발. 첫 번째 방법은 포함 한다 여 포 개발의 분석 뒤 슬라이스 난소 성인 쥐 조직의 문화 그리고 생식 샘 자극 호르몬 독립 단계 초기 여 포 개발을 시각화 하 시간 경과 화상 진 찰의 사용을 포함 하는 두 번째. 이전에 우리 여 포 개발8,9, 백혈병 금지 요인 및 호르몬의 효?...

여성 마우스 난소 포함 몇 천 포1, 그리고 주기적인 배 란 각 발 정기 주기에서 약 10 oocytes를 성숙. 낭 여러 발달 단계로 분류 됩니다: 원시, 기본, 보조, antral, 및 각 oocyte 둘러싸고 granulosa 세포 층의 형태에 따라 Graafian 낭. 대부분 원시 낭은 휴면, 그들 중 일부는 활성화 하 고 각 발 정기 주기2주 낭으로 성장. 보조 소 낭 모양 단계 후 여 포 개발은 주로 gonadotropins, follicular 자극 호르몬 (FSH)과 황 호르몬 (LH)에 의해 규제 됩니다. 그러나, 원시 및 기본 모 근 개발 생식 샘 자극 호르몬, 독립적 이며 이러한 단계를 제어 하는 규제 메커니즘 남아 제대로 inderstood3,,45. 성장 인자와 호르몬, 원시 및 기본 여 포 여 포6,7간의 상호 작용에 의해 통제 된다. 따라서, 우리가 마우스 난소 조직, 뿌리 역동성의 분석을 수행 하 고 난소 조직 문화8,,910을 사용 하 여 관련된 규제 메커니즘을 조사 합니다.
여기, 두 개의 난소 조직 문화 모델 방법을 소개합니다. 첫 번째 follicular 분야의 측정에 의해 여 포 개발 분석 하 사용 되 고 두 번째는 유전자 변형 마우스 보조 뿌리 단계로 원시에서 초기 여 포 개발 하는 동안 규제 메커니즘을 연구 하는 데 사용 됩니다. 여 포 개발 분석, 우리가 주로 사용 4 주 오래 된 여성 쥐의 난소 낭의 쉽게 시각화를 허용 하기 때문에. 정기 배 란 및 모델 vivo에서 여 포 개발 유도, 우리 LH 서 지 및 관찰된 배 란, 여 포는이 폐쇄 증, 및 조직 배양 조건에서 estradiol의 분 비를 재현. 교양된 난소의 이미지 체포 되었다, 그리고 여 포 개발 프로세스 follicular 지역 변경 내용을 추적 하 여 분석 되었다. 그러나, 밝은 분야 현미경 검사 법 분석, 원시 및 초기 기본 모 공 사이의 구별 불분명 했다. 따라서, 우리 작은 낭을 감지 하 고 원시, 기본, 사이 구별 하는 방법을 개발 하 고 보조 뿌리에 배양 난소 조직을 사용 하 여 Oogenesin1 (Oog1) pro3. 9 그리고 0와 4 일 탄생11후 R26-H2B-mCherry 유전자 변형 쥐 난소. Oog1 식 oocytes에서 감지 감수 분열, 입국 후 고 주 낭 교양된 난소 조직의11 시간 경과 이미지를 사용 하 여 원시에서 전환의 관찰 수 있도록 뿌리 발달로 점차 증가 ,12. 형태학 상 방법을 휴면 원시 낭13,14,,1516활성화 요인 연구에 사용 되었습니다, 하지만 난소에 생리 여 포 개발은 관찰 하기 어려운 다양 한 요인의 효과 유지 새롭거나. 현재 문화 방법 실시간으로 분석 대상 요소의이 부족을 해결 하기 위해 설계 되었습니다.
현재 연구에서 우리가 follicular 개발 시간 경과 화상 진 찰 방법을 사용 하 여 추적 하 고 여 포 개발 과정 특징. 우리의 소설 메서드는 전례 없는 난소의 생리학을 조사 하기 위한 도구를 제공 합니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:1176px;" width="1175">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Follicle stimulating hormone from human pituitary</td>
			<td style="width:224px;">SIGMA</td>
			<td style="width:119px;">F4021</td>
			<td style="width:451px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lutenizing hormone from equine pituitary</td>
			<td>SIGMA</td>
			<td>L9773</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-streptomycin solution</td>
			<td>Wako Pure Chemical Industries</td>
			<td>168-23191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM a, GlutaMax, no nucleotides</td>
			<td>Thermo Fisher</td>
			<td>32561037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-0-10-C</td>
			<td>35mm dish, No. 0 coverslip, 10mm glass diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millicell cell culture insert</td>
			<td>Merck Millipore</td>
			<td>PICM0RG50</td>
			<td>Diameter: 315 mm, pore size: 0.4 mm, material: hydrophilic PTTE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">3.5cm cell culture dishes</td>
			<td style="width:224px;">greiner bio-one</td>
			<td style="width:119px;">627160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">50ml / centrifuge tube with triple seal cap</td>
			<td style="width:224px;">IWAKI</td>
			<td style="width:119px;">2345-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Low-profile disposable blades 819</td>
			<td style="width:224px;">Leica</td>
			<td style="width:119px;">14035838925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM 710</td>
			<td>Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>Confocal microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellVoyager, CV1000</td>
			<td>Yokogawa Electric Corporation</td>
			<td style="width:119px;"></td>
			<td>Time-lapse imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BZ-X700</td>
			<td>KEYENCE</td>
			<td style="width:119px;"></td>
			<td>Time-lapse imaging</td>
		</tr>
	</tbody>
</table>

ovarian tissue culture to visualize phenomena in mouse ovary

포유류 암컷은 정기적으로 각 발 정기 주기 동안 oocytes의 거의 일정 한 수 배 란. 이러한 규칙과 주기 유지, 규정 hypothalamic-뇌 하 수 체-gonadal 축 수준 및 난소에서 난 포 개발에 발생 합니다. 활성 연구에도 불구 하 고 여러 단계 휴면 원시 여 포 활성화에서 배 란을 및 각 소 낭 모양 단계에서 다른 규제 복잡성 때문에 여 포 개발 메커니즘이 명확 하지 않다. 여 포 발달의 기계 장치 그리고 발 정기 주기 낭의 역학 조사, 우리는 관찰 하는 현미경을 사용 하 여 여 포 개발 하는 데 사용할 수 있는 마우스 난소 조직 문화 모델을 개발 했다. 체계적인 여 포 개발, 정기 배 란 및 여 포가 폐쇄 증 모두 재생 될 수 있다 경작된 난소 모델에 그리고 문화 조건을 실험적으로 변조 될 수 있습니다. 여기, 우리 여 포 개발의 규제 메커니즘 및 다른 난소 현상의 연구에서이 방법의 유용성을 보여 줍니다.

그림 1 난소 조직 문화 중 미디어에서 변경에 대 한 프로토콜을 보여 줍니다. 이 프로그램, 4 주 된 ICR 마우스 난소 배양 되었고 24 시간 간격으로 confocal 현미경 검사 법 (그림 2)를 사용 하 여 몇 군데. 3 주 동안 난소 조직의 문화, 동안 가장 antral 및 보조 모 공 여 포가 폐쇄 증으로 전락 했다 고 일부 ovulated (그림 2<...

Watch this Scientific Journal Video about Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary at JoVE.com

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

난소 조직 문화 현상을 마우스 난소에서 시각화 하

난소 조직 문화 여 포 개발, 배 란, 그리고 여 포가 폐쇄 증의 모델로 사용할 수 있습니다 하 고 동적 난소 프로세스의 규제 메커니즘을 나타냅니다.

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

This method can help answer key questions in the ovary research field about follicle development, follicle atresia and operation. The main advantage of this technique is that we can reproduce the ovarian phenomena under controlled culture conditions for the observation in real time. To prepare the ovaries from a four week old Institute of Cancer Research mouse for culture:Use scissors and tweezers to trim the surrounding tissue from the ovaries under a stereo microscope, and place the cleaned ovaries into a dish of fresh culture medium.When all of the ovaries have been trimmed, transfer the individual ovaries onto a piece of filter paper moistened with 37 degrees Celsius PBS, and use a microtome blade to slice each ovary into four pieces. Transfer the slices into a 3.5 centimeter dish of 37 degrees Celsius culture medium, and use a micropipette to add an about 0.5 micrometer droplet of culture medium onto one cell culture insert per ovarian tissue section. In a second 3.5 centimeter glass bottom cell culture dish containing one millimeter of medium:Using tweezers, transfer one tissue specimen into each droplet and place the plate in a cell culture incubator.Replace the supernatant in the dish with fresh 37 degrees Celsius culture medium every two days and treat the ovaries with 100 milliunits per milliliter of FSH and LH for 12 hours every fourth day. Begin imaging the cultured ovaries every 24 hours after one day of culture to allow sufficient follicle growth between imaging sessions;capturing images at 30 minute intervals using a time-lapse imaging system under the appropriate experimental conditions. To analyze ovarian follicle development:Open the images in ImageJ and open the analyze menu to select set scale.Enter the side lengths of the captured images and the corresponding numbers of pixels in the known distance and distance in pixels fields, respectively. Then, use the free-hand tool to outline the follicles and click measure. During a three week ovarian tissue slice culture period, most antral and secondary follicles are degenerated by follicle atresia, and some are ovulated.Some follicles develop in groups during each LH surge cycle. While the timing of ovulation and the number of ovulated oocytes differs between mouse ovaries, ovulation from cultured ovaries predominantly occurs within 48 hours of each LH surge. Note that not all ovulated oocytes release first polar bodies after ovulation.Primordial and primary follicles are present simultaneously in postnatal day four mouse ovaries. Whereas, only primordial follicles are present in postnatal day zero ovaries. However, primary follicles become detectable in cultured postnatal day zero ovaries after 30 to 40 hours of culture as demonstrated.Primordial and primary follicles can also be distinguished in the cultured ovaries isolated from mCherry transgenic animals by the mCherry positive nuclei of granulosa cells;allowing the tracking of the follicle stages in the ovary slice cultures. I hope that this technique paves the way for researchers in the field of reproductive technology to explore new techniques for fertility treatment.

ovarian-tissue-culture-to-visualize-phenomena-in-mouse-ovary

Research

JoVE Journal

Developmental Biology

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.

문화 공동 문화 마우스의 난소와 난소

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

연구

발생학

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

마우스 해마 조직의 예 자궁 Electroporation에와 Organotypic 슬라이스 문화

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

착상 전의 마우스 배아의 초기 세포 운명 결정 요인을 조사 할 Loss- 및 이득 -의 - 기능 접근

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

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.

혈관 내피 줄기 세포의 혈관 생성을 시각화하기위한 유방 팻 패드 이식 기술

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

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Mouse Secondary Palate Fusion의 라이브 이미징

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is being used to investigate the health-protective effects of young ovaries in young females. The current procedures employ an orthotopic transplantation method, where allogenic ovaries are transplanted back to their original position in the ovarian bursa. This is in contrast to the more commonly used heterotopic transplantation of ovaries/ovarian tissue subcutaneously or under the kidney capsule. All three locations provide efficient revascularization of the transplanted tissues. However, orthotopic transplantation provides the ovary with the most natural signaling environment and is the only procedure that provides the opportunity for the animal to reproduce naturally post-operatively. One must take care to remove all endogenous ovarian tissue during the ovariectomy procedure. If any endogenous tissue remains or if only one ovary is removed, the transplanted tissue will remain dormant until the existing tissue becomes senescent. While revascularization of the transplanted ovaries occurs very quickly, the transplant recipient can take a considerable amount of time to adapt to a new hypothalamic/pituitary/gonadal/adrenal (HPG/A) axis signaling regime associated with the transplanted tissue. This normally takes about 100 days in the mouse. Therefore, transplantation experiments should be designed to accommodate this adaptation period. Typical results with ovarian transplantation will include changes in the health of the recipient that reflect the age of the transplanted ovary, rather than the chronological age of the recipient.

Described here are orthotopic ovarian transplantation protocols for modifying ovarian influence on the physiology of the recipient mouse. This surgical procedure is used to expose female mice to various types of ovarian influence and may include heterochronic transplantations and transplantation of ovaries that have undergone various ex vivo treatments.

여성 쥐에서 난소 노화의 수명 및 건강 영향 조사를 Orthotopic 난소 이식 절차

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

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Polycystic 난소 증후군 연구 Hyperandrogenic 마우스 모델

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

자외선-C (UV-C) 손상에 응하여 1 차 마우스 안구 표면 세포/줄기 세포에 있는 산화 손상의 평가

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

신경 문장 원래 지방 유래 줄기 세포의 격리, 문화 및 지방 유도는 periaortic 지방 조직에서

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

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.