We thank Dr. Naojiro Minami (Kyoto University) for providing the Oog1pro3.9 mice. This research was supported by the JSPS (KAKENHI # JP15H06275) and the Nitto Foundation.

Mice were housed in an environmentally controlled room at 23 &#177;1 &#176;C with a 12 h light/12 h dark cycle. Animal care protocols and experiments were conducted in accordance with the Guidelines for Animal Experimentation at Aichi Medical University and were approved by the incumbent Animal Care and Use Committee.
1. Preparation of Culture Medium and Dishes
<ol>
	<li>To prepare basic culture medium, add fetal bovine serum (FBS, 5% v/v), FSH (100 mIU/mL), LH (10 mU/mL), and penicillin-streptomycin (penicillin, 100 U/mL; streptomycin, 100 mg/mL) to minimum essential medium alpha (MEM-alpha) and mix in 50 mL tubes. 
	NOTE: Total volumes of required culture media vary between experiments, but 1 mL of culture medium is generally sufficient for 3.5 cm glass-bottom culture dishes.</li>
	<li>Pour 1 mL aliquots of mixed culture medium into 3.5 cm glass-bottom culture dishes and set place 30 mm cell culture inserts into dishes. Pre-warm media and dishes in an incubator (5% CO2 and 37 &#176;C).</li>
</ol>
2. Preparation of Ovarian Tissue
<ol>
	<li>Pre-warm phosphate buffered saline (PBS) (&#8722;) and MEM-alpha containing 5% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin in an incubator (5% CO2 and 37 &#176;C). 
	&#8203;NOTE: About 3 mL aliquots of PBS (&#8722;) per ovary are sufficient for use during ovary dissections, and 1 mL aliquots of MEM-alpha are sufficient for transient storage of single ovaries in 3.5 cm dishes.</li>
	<li>Excise ovaries from 4-week old female ICR (named after Institute of Cancer Research) mice and trim the tissues surrounding the ovary using scissors and tweezers under a stereoscopic microscope. Reserve the removed ovaries in culture medium (see step 2.1) until use.
	<ol>
		<li>Place single ovaries onto filter paper moistened with pre-warmed PBS (&#8722;) (see step 2.1) and slice into 4 pieces using a microtome blade under a stereoscopic microscope. 
		NOTE: Ovaries of 4-week old ICR mice are about 2 mm in diameter. The number of pieces depends on the ovary size; however, about 500 &#181;m thick pieces allow for proper follicle observations (in pieces thicker than 500 mm, tissue transparency is decreased; in pieces thinner than 500 mm, many antral follicles are broken and lost).</li>
		<li>After dissection, place the sliced ovary specimens into pre-warmed culture medium in 3.5 cm dishes (see step 2.1).</li>
	</ol>
	</li>
</ol>
3. Ovarian Tissue Culture
<ol>
	<li>Drop approximately 0.5 &#181;L of culture medium per sliced ovarian tissue on the cell culture insert where the ovarian tissue will be set using a micropipette.</li>
	<li>Place each sliced specimen into a drop of culture medium on the cell culture inserts using tweezers.</li>
	<li>Culture the ovary tissues in 5% CO2 at 37 &#176;C.</li>
	<li>Replace the culture medium with fresh pre-warmed medium every 2 days. 
	&#8203;NOTE: The schedule for medium changes for the culture of ovary sections from 4-week old ICR mice is presented in Figure 1.
	<ol>
		<li>To reproduce the LH surge, treat cultured 4-week old ICR mice ovaries with the medium containing 100 mU/mL FSH and LH for 12 h every 4 days (Figure 1).</li>
	</ol>
	</li>
</ol>
4. Microscope Images of Cultured Ovaries
<ol>
	<li>Start imaging the cultured ovaries after day 1 when the tissues have adhered onto the cell culture inserts, and perform confocal or inverted microscope analyses at 24 h intervals to allow sufficient follicle growth between time points. 
	NOTE: Confocal microscopy is superior to inverted microscopy for observing follicles in whole cultured ovaries.</li>
</ol>
5. Time-lapse Imaging of Cultured Ovaries
<ol>
	<li>Optimize the time-lapse imaging conditions, including laser intensities and exposure times, for the imaging system (Table 1).
	<ol>
		<li>Select paired ovary specimens from single mice for use as treatment and control groups. Vary laser intensities and exposure time to achieve the best images (Table 1).</li>
		<li>Compare follicle growth under each culture condition by measuring follicle areas in images of control samples at 24 h intervals and in time-lapse imaging samples (see step 6). Concurrently, count the number of ovulated oocytes in each ovary set and choose the optimal time-lapse imaging conditions.</li>
	</ol>
	</li>
	<li>Capture images at 30 min intervals using the time-lapse imaging system under the determined conditions (Table 1).</li>
</ol>
6. Follicle Growth Analysis
<ol>
	<li>To analyze the follicle development, measure the follicle areas in the captured images using ImageJ software (http://resbweb.nih.gov/ij/).
	<ol>
		<li>Initially, set the scale of the image by clicking &#34;Set Scale&#34; under &#34;Analyze&#34; in the tool bar. Enter the side lengths of the captured image and the corresponding numbers of pixels in the blank fields of &#34;Known distance&#34; and &#34;Distance in pixels&#34;, respectively.</li>
		<li>Click &#34;Free hand&#34; in the tool bar and outline the follicles in the captured bright field images.</li>
		<li>Click &#34;Measure&#34; under &#34;Analyze&#34; in the tool bar. 
		NOTE: If other measurement data are desired, click &#34;Set measurement&#34; under &#34;Analyze&#34; in the tool bar, and check the appropriate boxes in the list.</li>
	</ol>
	</li>
</ol>
7. Analysis of Follicle Development Using Transgenic Mice
<ol>
	<li>Collect ovaries from postnatal days 0 and 4 (P0 and P4) female transgenic mice containing the transgenes Oog1pro3.9 and R26-H2B-mCherr, and culture on inserts as described in steps 1.1&#8211;3.2, except do not slice the P0 and P4 ovaries.</li>
	<li>Replace the culture medium with fresh, pre-warmed medium in 3.5 cm dishes in an incubator containing 5% CO2 at 37 &#176;C every 2 days. 
	NOTE: The concentration of LH in the culture medium of P0 and P4 ovaries can remain constant because LH surges do not occur in P0 and P4 mice.</li>
	<li>Set the microscope to visualize only AcGFP1-positive primary follicles in cultured P4 ovaries (Table 1). 
	NOTE: Only primordial and primary follicles are present in P4 female mouse ovaries.</li>
	<li>Capture images of cultured P0 Oog1pro3.9/R26-H2B-mCherry transgenic mice ovaries at 30 min intervals using the settings used for P4 ovaries (see step 5.2).</li>
	<li>Trace and analyze follicle development using AcGFP1 and H2B-mCHerry signals.</li>
</ol>

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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. 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The authors have nothing to disclose.

In this study, we developed two new methods for studying follicle development in mouse ovaries. The first method involves culture of sliced ovarian adult mice tissues followed by analyses of follicle development, and the second involves the use of time-lapse imaging to visualize early follicle development during the gonadotropin-independent stage. Previously, we used the present ovary tissue culture method to assess the effect of leukemia inhibitory factor and progesterone on follicle development8,9, and showed that their effects vary with concentration and follicle stage. In the present study, sliced ovarian tissues exhibited follicle dynamics, warranting further analyses of ovarian follicle development mechanism using this model.
Ovaries from mice of more than 5 weeks of age contain corpus lutea that hinders microscopic observations. Hence, 4-week-old mouse ovaries are preferred for observations of follicle development and ovulation, and subsequent late primary, secondary, and antral follicles are more easily distinguished using bright field microscopy. Our methods offer comparisons of the effects of growth factors and drugs in culture media, with discernable changes in follicle development between differentially treated ovaries (Figure 3).
Transgenic mice expressing Cre or fluorescein proteins in oocytes have been described in previous studies18,19,20. However, follicular stage classification depends on histological characteristics including granulosa cell shapes, numbers of granulosa cell layers, and whether theca cells are formed. Thus, distinctions between primordial and primary follicles may be limited from observations of oocytes alone. Herein, we used transgenic mice containing the Oog1pro3.9 and R26-H2B-mCherry transgenes11,17 and visualize early follicle development from primordial to secondary follicle stages in cultured ovaries. Oog1 expression becomes detectable in oocytes during the primordial follicle stage, and is markedly increased in primary follicles. Hence, the AcGFP1 signal intensities in oocytes are associated with follicle stage (Figure 5) and were used to observe primordial-primary follicle transitions (Figure 4, Supplementary Movie 2). Cell nuclei are stained red in R26-H2B-mCherry transgenic mice (Figure 4, Supplementary Movie 2, and Supplementary Movie 3), allowing observations of follicle stages according to numbers of granulosa cells (Figure 5). Thus, collectively the present methods allow analyses of follicle development from primordial through to ovulation using Oog1pro3.9/R26-H2B-mCherry transgenic mice. Furthermore, because apoptotic cell chromatin is condensed, mCherry signals of atretic follicles are stronger than those of normal follicles (Supplementary Movie 3).
Among methodological subtleties, correct thicknesses of sliced tissues are required for successful culturing, with increased cell death in ovary slices that are too thick, and losses of large antral follicles in ovary slices that are too thin. The follicle growth speed is slow; hence, it is easy to trace and analyze the process of each follicle via 24-h interval observation. The present confocal microscope setting, including laser intensity, interval time, and digital gain, are dependent on the microscope mode, but are critical to consider when obtaining time-lapse images. In particular, strong laser intensities and long exposure times are required to capture clear images, but can affect cultured cells. Hence, moderation of these settings to avoid phototoxicity is critical. Sliced ovarian tissues of 4-week-old mice can be cultured for approximately 4 weeks, whereas primordial and primary follicles remain present subsequently, they no longer grow. In contrast, P0 and P4 ovaries can be cultured for approximately 10 days, during which primordial and primary follicles develop into primary or secondary follicles, respectively. However, follicles in cultured P0 and P4 ovaries do not develop into antral follicles. Therefore, the excision of ovaries is required at various stages for studies of the regulatory mechanisms associated with whole ovary follicle development.

Female mouse ovaries contain several thousand follicles1, and periodic ovulation matures approximately ten oocytes at each estrus cycle. Follicles are classified into several developmental stages: primordial, primary, secondary, antral, and Graafian follicles, depending on the form of the granulosa cellular layer surrounding each oocyte. Most primordial follicles are dormant, and some of them are activated and grow into primary follicles at each estrus cycle2. After the secondary follicular stage, follicle development is mainly regulated by gonadotropins, follicular stimulating hormone (FSH), and luteinizing hormone (LH). However, primordial and primary follicle development is independent of gonadotropin, and the regulatory mechanisms that govern these stages remain poorly inderstood3,4,5. In addition to growth factors and hormones, the primordial and primary follicle is regulated by the interactions among follicles6,7. Therefore, we performed analyses of follicle dynamics in mouse ovary tissues, and investigated the associated regulatory mechanisms using ovarian tissue cultures8,9,10.
Herein, we introduce two ovarian tissue culture model methods. The first is used to analyze follicle development by measurement of follicular areas, and the second is used to study the regulatory mechanism during early follicle development from primordial to secondary follicle stage with transgenic mice. For follicle development analysis, we mainly used ovaries of 4-week old female mice because they allow for easy visualization of follicles. To induce periodical ovulation and model in vivo follicle development, we reproduced LH surge and observed ovulation, follicle atresia, and secretion of estradiol under tissue culture conditions. Images of the cultured ovaries were captured, and the follicle development processes were analyzed by tracing changes in the follicular area. However, in bright field microscopy analyses, the distinction between primordial and early primary follicles was unclear. Thus, we developed a method to detect small follicles, and distinguish between the primordial, primary, and secondary follicle in cultured ovarian tissues using Oogenesin1 (Oog1) pro3.9 and R26-H2B-mCherry transgenic mice ovaries at days 0 and 4 after birth11. Oog1 expression is detectable in oocytes after entry into meiosis, and gradually increases with follicle development, allowing observation of the transition from primordial to primary follicles using time-lapse images of cultured ovary tissue11,12. Although morphological methods have been used to study factors that activate dormant primordial follicles13,14,15,16, physiological follicle development in ovaries is difficult to observe, and the effects of various factors remain uncharacterized. The present culture methods were designed to address this paucity in real time analyses of target factors.
In the present study, we tracked follicular development using a time-lapse imaging method and characterized the process of follicle development. Our novel methods offer an unprecedented tool for investigating the physiology of ovaries.

<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

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.

Figure 1&#160;shows the protocol for changes in media during ovarian tissue culture. Following this program, 4-week-old ICR mice ovaries were cultured and imaged at 24-h intervals using confocal microscopy (Figure 2). During culture of ovary tissues for 3 weeks, most antral and secondary follicles were degenerated by follicle atresia and some were ovulated (Figure 2D and Table 2). In analysis of follicle areas using ImageJ (Figure 3), some follicles developed in groups during each LH surge cycle (Figure 3B and Supplementary Movie 1). Moreover, ovulation occurred almost simultaneously in separate cultured ovaries from right and left ovaries of single mice. Whereas, the timing of ovulation and numbers of ovulated oocytes (Table 2) differed between mouse ovaries (data not shown), ovulation from cultured ovaries predominantly occurred within 48 h of LH surges (Figure 3, and Supplementary Movie&#160;1). However, not all ovulated oocytes released first polar bodies after ovulation (Figure 2E and Table 2), and although ovulated oocytes could be fertilized, development ceased at the 4-cell stage (data not shown). To elucidate mechanisms by which dormant primordial follicles are activated in mouse ovaries, we detected primordial follicle activation in cultured tissues from transgenic mice carrying Oog1pro3.9 and R26-H2B-mCherry transgenes (Figure 4, Figure 5, and Supplementary Movie 2). Oocytes express very low levels of the Oog1 gene from the primordial follicle stage, and time-lapse imaging distinguished low AcGFP1-expressing primordial follicles from the high AcGFP1-expressing primary follicles. Primordial and primary follicles are present simultaneously in P4 mouse ovaries, whereas only primordial follicles are present in P0 ovaries (Figure 4A, B). Thus, we optimized time-lapse imaging conditions to detect only primary follicles in cultured P4 ovaries (Figure 4C). Subsequently, we captured images of cultured P0 ovaries for 10 days in the same conditions (Figure 4D&#8211;G). AcGFP1 can be used as an index of primary follicles in these transgenic mice and AcGFP1-positive primary follicles were detectable in cultured P0 ovaries after 30&#8211;40 h culture (Figure 4, Figure 5A-F, and Supplementary Movie 2). Primordial and primary follicles in cultured ovaries were also distinguished by mCherry-positive nuclei of granulosa cells (Figure 5B, E). Specifically, mCherry signals indicate forms of follicles and allow observation of follicle stages in cultured ovaries (Figure 5B, E, and H). Therefore, this culture system using ovaries from Oog1pro3.9/R26-H2B-mCherry mice revealed the process of early follicle development from primordial to secondary follicle stages. These methods will facilitate studies of the regulatory mechanisms of gonadotropin-independent follicle development.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57794/57794fig1.jpg" /> 
Figure 1: Time course of medium changes. Medium A contains 5% FBS, 100-mU FSH, 10-mU LH, 100-U/mL penicillin, and 100 mg/mL streptomycin in MEM-alpha. Medium B contains 5% FBS, 100-mU FSH, 100-mU LH, 100-U/mL penicillin, and 100 mg/mL streptomycin in MEM-alpha. Medium B was used to produce LH surges every 4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/57794fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57794/57794fig2.jpg" /> 
Figure 2: Images of cultured ovarian tissues. Images were captured at 24 h intervals using confocal microscopy. (A) Culture day 1; (B) Culture day 5; (C) Culture day 10; (D) Culture day 13; (E) Magnified image of the region indicated by the square in (D); oocytes in D were ovulated from the follicles indicated by arrows in B, C, and D. (F) Oocyte releasing a polar body after ovulation; Scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/57794fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57794/57794fig3.jpg" /> 
Figure 3: Measurements of follicle areas in cultured ovaries. (A) Image of cultured ovary; dotted lines represent the area measured by ImageJ. (B) Follicular areas in cultured ovary after 3 weeks; gray lines represent the culture period from the addition of 100-mM LH (LH surge). Lines show the development stages in each follicle in the cultured ovary; Scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/57794fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57794/57794fig4.jpg" /> 
Figure 4: Hematoxylin and Eosin (H&#38;E) staining images of P0 and P4 ovaries and time-lapse imaging. (A) H&#38;E staining of P0 ovary; (B) H&#38;E staining of P4 ovary; (C) Image of a P4 ovary from an Oog1pro3.6 transgenic mouse after 10 h culture. The arrow shows an AcGFP1-positive primary follicle. (D&#8211;G) Images of P0 ovaries from transgenic mice expressing Oog1pro3.9 and R26-H2B-mCherry; green and red signals in D and F represent AcGFP1 and mCherry, respectively. White signals in E and G represent AcGFP1 in D and F, respectively. D and E are images of cultured ovaries after 0.5 h culture. F and G show ovaries after 180 h culture; Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/57794fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57794/57794fig5.jpg" /> 
Figure 5:&#160;Images of primordial, primary, and secondary follicles in cultured ovaries from oog1pro3.9/R26-H2B-mCherry mice. (A-C) Images of primordial follicles in cultured P0 ovaries; (D-F) Images of primary follicles in P0 cultured ovary; (G-H) Images of secondary follicles in cultured 4-week-old mouse ovaries. A, D, and G are merged images of B and C, E and F, and H and I, respectively. Green, AcGFP1; red, mCherry; Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/57794fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>microscope</td>
			<td>objective lens</td>
			<td>laser</td>
			<td>exposure (ms)</td>
			<td>Gain</td>
			<td>time-interval</td>
			<td>z-stack</td>
			<td>others</td>
			<td>Figures and Movies</td>
		</tr>
		<tr>
			<td>CV1000</td>
			<td>X10</td>
			<td>488 nm: 30, 561 nm: 20</td>
			<td>488 nm: 700, 561 nm: 600</td>
			<td>488 nm: 90, 561nm: 20</td>
			<td>30 min</td>
			<td>5 mm</td>
			<td></td>
			<td>Figure 4C-G, Movie 1 and 2</td>
		</tr>
		<tr>
			<td>BZ-X700</td>
			<td>X20 (with collection collar)</td>
			<td>40%</td>
			<td>auto</td>
			<td>X4</td>
			<td>30 min</td>
			<td>10 mm</td>
			<td>binning 3x3</td>
			<td>Movie 3</td>
		</tr>
		<tr>
			<td>LSM710</td>
			<td>X10</td>
			<td>488 nm: 6%, 564 nm: 5%</td>
			<td>camera speed: 4</td>
			<td>488 nm: 850, 564 nm: 850</td>
			<td>24 h</td>
			<td>10 mm</td>
			<td>digital gain, 488 nm: 2, 564 nm:1</td>
			<td>Figure 2, 3 and 5</td>
		</tr>
	</tbody>
</table>
Table 1: Time-lapse imaging conditions. Time-lapse imaging condition for confocal and bright field microscopes.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Ovary no.</td>
			<td>Number of total ovulated oocytes</td>
			<td>Number of MII oocytes</td>
		</tr>
		<tr>
			<td>1</td>
			<td>7</td>
			<td>2</td>
		</tr>
		<tr>
			<td>2</td>
			<td>19</td>
			<td>9</td>
		</tr>
		<tr>
			<td>3</td>
			<td>14</td>
			<td>6</td>
		</tr>
		<tr>
			<td>4</td>
			<td>7</td>
			<td>3</td>
		</tr>
		<tr>
			<td>5</td>
			<td>12</td>
			<td>4</td>
		</tr>
		<tr>
			<td>6</td>
			<td>15</td>
			<td>6</td>
		</tr>
		<tr>
			<td>7</td>
			<td>25</td>
			<td>11</td>
		</tr>
		<tr>
			<td>8</td>
			<td>13</td>
			<td>3</td>
		</tr>
		<tr>
			<td>9</td>
			<td>11</td>
			<td>7</td>
		</tr>
		<tr>
			<td>10</td>
			<td>19</td>
			<td>8</td>
		</tr>
		<tr>
			<td>11</td>
			<td>18</td>
			<td>4</td>
		</tr>
		<tr>
			<td>12</td>
			<td>7</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
Table 2: Numbers of ovulated oocytes in cultured ovaries. Numbers of ovulated oocytes from twelve cultured ovaries; MII indicates the numbers of ovulated oocytes that released first polar bodies after ovulation.
<img alt="Supplementary Movie 1" src="/files/ftp_upload/57794/57794movie1.jpg" /> 
Supplementary Movie 1: Time-lapse movie of a cultured ovary. Ovaries were collected from 4-week old mice and were sliced and cultured for 3 weeks. The movie spans a culture period of 0&#8211;200 h at 10 frames/s. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/movie_1.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Supplementary Movie 2" src="/files/ftp_upload/57794/57794movie2.jpg" /> 
Supplementary Movie 2: Time-lapse movie of a cultured P0 ovary from a transgenic mouse. Green, AcGFP1; red, mCherry. The movie spans a culture period of 0&#8211;180 h at 10 frames/s. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/Movie_2.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Supplementary Movie 3" src="/files/ftp_upload/57794/57794movie3.jpg" /> 
Supplementary Movie 3:&#160;Time-lapse movie of a cultured ovary from a 4-week old mouse. Green, AcGFP1; red; mCherry; the movie spans a culture period of 9&#8211;13 culture days at 10 frames/s. The arrows in the first image indicate follicles in which atresia occurred during culture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/Movie_3.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>

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

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

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

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

13.6K Views.  Aichi Medical University. Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Video: Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

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.

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

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.

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.

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

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

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.

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 ...

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.

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

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.

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.

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.

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.

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