Name: in vitro growth of mouse preantral follicles under simulated microgravity
Uploaded: 2017-12-17T13:00:00.000+00:00
Duration: 10 min 48 s
Description: Watch this Scientific Journal Video about In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity at JoVE.com

The authors would like to thank Yilong Wang and Yanlin Zhao for their assistance in maintaining the animals, Chan Zhang for her assistance in preparing the histological samples, and Dr. Songtao He for critically reading this manuscript. This work was supported by the Natural Science Foundation of Zhejiang Province (grant number: LY13C120002).

Ethical approval for this study was obtained from the Animal Research Ethics Committee of Wen Zhou Medical College.
1. Preparation of Culture Media and Alginate Hydrogel
<ol>
	<li>Prepare ovarian tissue and follicle handling medium using L-15 medium with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 &#181;g/mL streptomycin. Store at 4 &#176;C for two weeks.</li>
	<li>Prepare ovarian tissue and follicle culture medium consisting of alpha-minimal essential medium [&#945;-MEM] supplemented with 5% fetal bovine serum [FBS], 1% insulin-selenium-transferrin, 10 mIU/ml rFSH, 100 IU/mL penicillin, and 100 &#181;g/mL streptomycin. Store at 4 &#176;C for two weeks.</li>
	<li>Prepare alginate hydrogel using 0.8% (w/v) sodium alginate solution in sterile PBS, then drop 10 &#181;L of this solution into the encapsulation solution (140 mM NaCl /50 mM CaCl2) to create alginate beads. 
	NOTE: The alginate bead size was about 3 mm in diameter.</li>
</ol>
2. Mouse Follicle Isolation and Encapsulation
<ol>
	<li>Cull 14 day-old ICR female mice by cervical dislocation, remove ovaries aseptically using scissors and forceps, and put ovaries into 2 mL of handling medium.</li>
	<li>Cut ovaries in half using a scalpel. The half-sized ovarian tissue was about 1 mm thickness for culture in the culture medium.</li>
	<li>Manually isolate follicles from ovaries using 26 1/2-guage needles under a 2-4X stereomicroscope, and collect the selected follicles for culture in the culture medium. 
	NOTE: Follicle selection criteria: 1) follicles were round with oocytes in the middle and 2-3 layers of granulosa cells surrounding the oocytes, 2) follicles had an intact basal membrane attached with some theca cells, and 3) follicle size was 90-100 &#181;m in diameter</li>
	<li>Create alginate beads per step 1.3. Wash follicles with alginate solution and then pipette a single follicle into the middle of each alginate bead to make certain every bead contains a single follicle under a stereomicroscope. After rinsing in culture medium, culture the alginate beads in 150 &#181;L of culture medium in a 35&#215;10 mm culture dish or a RWV device.</li>
</ol>
3. Ovarian Tissue or Follicle Culture Under Simulated Microgravity
<ol>
	<li>Use a RWV device for generating simulated microgravity. Fill the vessel with light paraffin oil (~10 mL), place 150 &#181;L of culture medium in a droplet into the oil, then transfer one piece of ovarian tissue or three alginate beads with encapsulated follicles into the medium droplet. Close the vessel port cap and fix the vessel onto the rotating base of the device.</li>
	<li>In control groups, place alginate beads containing follicles or ovarian tissue into 150 &#181;L of culture medium in a droplet covered with light paraffin oil in 35&#215;10 mm culture dishes.</li>
	<li>Incubate both the culture dishes and RWV device in a humidified incubator at 37 &#176;C with 5% CO2 in air.</li>
	<li>To change culture medium in RWV device: pour the oil and medium from the vessel into a 150&#215;20 mm culture dish, transfer the ovarian tissue or alginate beads to a culture dish with culture medium. Re-establish the vessel culture system per step 3.1.</li>
</ol>
4. Decapsulation and Follicle Retrieval
<ol>
	<li>After 4 days of culturing, pour the oil and medium from the vessel into a 150&#215;20 mm culture dish. Pick up alginate beads and decapsulate follicles from the alginate beads by incubating in handling medium supplemented with 10 U/mL alginate lyase for 30 min at 37 &#176;C.</li>
	<li>Collect follicles and then wash them in handling medium under a stereomicroscope.</li>
</ol>
5. Ovarian Tissue and Follicle Assessment
<ol>
	<li>Cell Viability Assay
	<ol>
		<li>Wash the decapsulated follicles three times in D-PBS, and then incubate them in 150 &#181;L of the combined Cell Viability Assay reagents (2 &#181;M calcein AM and 4 &#181;M EthD-1) at room temperature for 45 min.</li>
		<li>Transfer follicles to a clean microscope slide with 10 &#181;L D-PBS, cover with a coverslip, and then seal with clear fingernail polish.</li>
		<li>Count green and red cells in the slides under a confocal fluorescence microscope (400X). 
		NOTE: In this viability assay, green cells are live cells, and red cells are dead cells. Follicles with less than 10% red cells (dead) are considered as survived follicles.</li>
	</ol>
	</li>
	<li>Hematoxylin and Eosin (H&#38;E) Staining of Ovarian Tissue and Follicle
	<ol>
		<li>Fix fresh 14-day-old mouse ovaries and preantral follicles isolated from 14-day-old mice in Bouin&#39;s solution as Day 0 samples. Fix the cultured ovarian tissue after either 2-day or 4-day culture, and individual follicles inside the alginate beads after 4-day culture as experimental samples.</li>
		<li>Embed all samples in paraffin, section serially at 5 &#181;m thickness, and then stain with H&#38;E according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Follicle Counting and Diameter Measurement
	<ol>
		<li>In ovarian tissue sections stained with H&#38;E, count all follicles and healthy preantral follicles within the tissue section area under a microscope (magnification 400X). Calculate follicle density by dividing the number of preantral follicles by the total number of follicles. 
		NOTE: the healthy preantral follicles are those with at least two layers of granulosa cells.</li>
		<li>Measure follicle and oocyte diameter of the Day 0 follicles and Day 4 cultured follicles.</li>
	</ol>
	</li>
	<li>Immunohistochemistry
	<ol>
		<li>Fix ovarian tissue in 4% paraformaldehyde, embed in paraffin, section at 5-&#181;m-thickness, and mount tissue sections on slides. Dewax the sections, rehydrate, and incubate them in citrate buffer for 15 min. Block the endogenous peroxidase in 3% hydrogen peroxide, unmask antigen in 5% goat serum, and then incubate with primary antibodies at room temperature for 3 h.
		<ol>
			<li>Wash in PBS for 3 min, then incubate the slides with 1:500 diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) for 1 h at room temperature. 
			NOTE: The primary antibodies used in the study were rabbit anti-GDF-9 antibody (1:500 dilution) and rabbit anti-PCNA antibody (1:200 dilution).</li>
		</ol>
		</li>
		<li>Incubate ovarian section slides with 3,3&#39;-Diaminobenzidine [DAB] to detect the peroxidase activity and mount on glass slides with aqueous mounting medium.</li>
		<li>Observe ovarian section slides under a microscope at 400X magnification.</li>
	</ol>
	</li>
	<li>Transmission Electron Microscopy [TEM]
	<ol>
		<li>Fix follicles in 2.5% glutaraldehyde for 3 h at room temperature, then post-fix in 1% osmium tetroxide for 1 h at 37 &#176;C. Wash with PBS, then treat the follicles with 1% phosphotungstic acid and 1% sodium uranyl acetate for 1 h at 37 &#176;C. Dehydrate follicles using an acetone series: 25, 50, 75, 95, and 100% (3 times), and epoxy resin-acetone mixture after the final 100% acetone step at 37 &#176;C, and then embed in epoxy resin at 45 &#176;C.</li>
		<li>Cut the embedded blocks into 1.0 &#181;m semi-thin sections, and stain the sections with 1% methylene blue for 1-2 min to localize the follicles in the blocs. Cut the embedded blocks into 50 nm ultrathin sections and mount the sections on grids for TEM.</li>
	</ol>
	</li>
</ol>
6. Statistical Analysis
<ol>
	<li>Present all data in mean &#177; SEM format. Determine the differences between groups by one-way analysis of variance (ANOVA) followed by Tukey multiple comparison test. The &#945; threshold for test result significance was set at p &#60; 0.05.</li>
</ol>

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The authors declare that they have no competing financial interests.

The critical steps for successful culturing of preantral follicles are: 1) correctly selecting the preantral follicles according to the selection criteria that were described in Protocol Text 2.1); 2) preforming all procedures in a skeptical condition and maintaining a sterile culture; and 3) correctly setting up the RWV culture system.
The safeguarding of consistency of experimental observations in the replicate experiments is to use similar or identical materials. The criteria for the selection of mouse preantral follicles revealed that the follicles had 1) healthy oocytes at the germinal vesicle breakdown (GVBD) stage surrounded by a thin zona pellucida; 2) 2-3 layers of granulosa cells enclosed by a basal membrane; and 3) some thecal cells attached to the basal membrane. Thus, the follicles selected had not only all three functional compartments of an ovarian follicle for supporting oocyte growth, but also a similar morphological identification with a diameter of 90 - 100 &#181;m.
This is the first attempt to grow mouse preantral follicles in the manner of 3D culturing under the simulated microgravity. Several culture systems have been developed for mouse preantral follicles. Oocytes obtained from the follicle cultured on a flat surface of petri dishes, the so-called 2D culture, could be fertilized and produced live offspring. Moreover, 3D cultures, such as cultures of follicles in alginate beads, maintain a tissue structure similar to what the body produces in high percentage meiotically competent oocytes. However, the development process of ovarian follicles/oocytes in a zero-gravity field is unknown. RWV, a device which is attracting substantial attention in tissue engineering, can generate a simulated microgravity environment and provide an ideal environment for investigating the effects of simulated microgravity on the ovarian follicle/oocytes growth in vitro.
It is important to set up the RWV apparatus properly for cell culture. The air bubbles in the vessel must be removed. The method to remove air bubbles, if generated in the vessel, is as follows: Place two syringes with 0.5 mL of oil in each of the two syringe ports. Open the valves controlling the syringe ports. Maneuver air bubbles underneath the syringe port. Pull the bubbles into the syringe while injecting approximately the same volume of media through the other syringe port. After all bubbles are removed, close the valves, remove the syringes, and replace the syringe port caps. It is also critical to find out the correct rotation speed, which prevents cells from settling via a constant rotation. Optimization of the rotation speed is determined by the specific weight of the cells, the fluid density, and the viscosity.
The expression profiles of PCNA and GDF-9, which were similar to those in GDF-9-deficient mouse follicles16, revealed that simulated microgravity had detrimental effects on the development of granulosa cells and oocytes. The vessel of the RWV device spun around a horizontal axis inside a 5% CO2 incubator, allowing for permeation of oxygen and carbon dioxide across a membrane in the back, providing efficient oxygenation and very low shear stress, which was unlikely to be the cause of follicle/oocyte injury. Optimization of the experimental setup and further analyses are needed to investigate the mechanism of the detrimental effects.
Further studies are needed to investigate the developmental potential of the in vitro oocytes derived from the RWV culture system. In vitro maturation of oocytes to MII stage is presently being carried out. The final validation of this culture system will be examined by the fertilization capacity of the retrieved oocytes.

In vitro ovarian follicle development and oocyte maturation have been achieved using traditional methods for 2-dimensional culture, such as on the surface of petri dishes, and three dimensional (3D) matrix, such as alginate and hydrogel1,2,3. A 3D culture system effectively maintained follicles in a tissue-like structure and kept the similar gene expression profiles as in vivo4 follicles, and produced meiotically competent oocytes5,6. 3D culture systems can also be established in a matrix-free condition, for example, hanging drop suspension and rolling vessels. The rotating wall vessel (RWV) developed by the National Aeronautics and Space Administration (NASA) generates a simulated microgravity (s-&#181;g) for the cells cultured inside the vessel7. This simulated microgravity condition provides a unique culture system for cell proliferation and differentiation because of the lack of sedimentation for convection. It has been shown that simulated microgravity promoted endothelial vessel formation from endothelial cells8,9, thyroid tissue assembling from thyroid cells10, and chondrogenesis from adipose-derived mesenchymal stem cells in RWV devices11. However, other studies have shown that simulated microgravity induced apoptosis in gastric cells and B lymphoblasts12,13,14, suggesting the effects of simulated microgravity on cellular injury may be cell type-dependent. Simulated microgravity could also cause changes at the ultrastructural level, as reported in the disturbed spindle organization and induced cytoplasmic blebbing in oocytes15. The optimized simulated microgravity conditions and mechanisms that induce tissue engineering or cellular injuries under the system require further investigation. In vitro experiments of growing ovarian follicles/oocytes using RWV devices can provide valuable information on oocyte gene expressions and ultrastructures of oocyte organelles. In this study, ovarian tissue containing preantral follicles and isolated preantral follicles was used to investigate the effects of simulated microgravityon follicle development and oocyte maturation.
In this study, ovarian tissue containing preantral follicles and isolated preantral follicles were used to investigate the effects of simulated microgravity on follicle development and oocyte maturation. In our study, the RWV, a simulated microgravity device, spins around a horizontal axis inside a 5% CO2 incubator, providing a simulated microgravity condition with efficient oxygenation and very low shear stress. We analyzed the gene expressions of proliferating cell nuclear antigen (PCNA) and growth differentiation factor 9 (GDF-9) as indicators for the development of granulosa cells and oocytes, respectively. We demonstrated that PCNA and GDF-9 expressions were suppressed in the granulosa cells and oocytes, respectively, when cultured in the RWV device, and we showed the withdrawal of oocyte microvilli from the zona pellucida in the follicles cultured under the s-&#181;g condition, which was similar to that in GDF-9-deficient mouse follicles16.

<table><tbody><tr><td>ICR mice</td><td>SLRC Laoratory</td><td></td><td>14-day-old female mice</td></tr><tr><td>L-15 medium</td><td>Sigma</td><td>L1518</td><td>With L-glutamine, liquid, sterile-filtered, suitable for cell culture</td></tr><tr><td>fetal bovine serum</td><td>Gibco</td><td>10100147</td><td>Origin: Australia</td></tr><tr><td>Penicillin V potassium</td><td>Sigma</td><td>1504503</td><td>United States Pharmacopeia (USP) Reference Standard (USP)</td></tr><tr><td>Streptomycin sesquisulfate hydrate</td><td>Sigma</td><td>46754</td><td>Analytical standard</td></tr><tr><td>Universal&amp;nbsp;Click&amp;nbsp;Pen Needles</td><td>Penfine</td><td></td><td>12&amp;times;1/2&quot; 0.33&amp;times;12 mm</td></tr><tr><td>Alginate</td><td>Sigma</td><td>W201502</td><td></td></tr><tr><td>alpha-minimal essential medium</td><td>Sigma</td><td>M0894</td><td>Alpha Modification, with L-glutamine, without ribonucleosides, deoxyribonucleosides and sodium bicarbonate, powder, suitable for cell culture</td></tr><tr><td>Insulin-selenium-transferrin</td><td>Invitrogen</td><td>51500056</td><td>Liquid, sterile-filtered, BioReagent, suitable for cell culture</td></tr><tr><td>GONAL-F</td><td>Merck Serono</td><td></td><td>Recombinant follitropin alfa for injection</td></tr><tr><td>Disposable high-aspect-ratio rotating vessel</td><td>Synthecon</td><td></td><td>10 mL</td></tr><tr><td>Culture oil</td><td>Vitrolife</td><td></td><td>Sterile light paraffin oil</td></tr><tr><td>Alginate Lyase</td><td>Sigma</td><td>A1603</td><td>powder, &gt;10,000 units/g solid</td></tr><tr><td>26 1/2-guage needle</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>35&amp;times;10mm dish</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Viability/Cytotoxicity assay Kit</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>Bouin&amp;rsquo;s solution</td><td>Sigma</td><td>HT10132</td><td></td></tr><tr><td>Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L)</td><td>Jackson Immuno-Research Laboratories</td><td>111-035-003</td><td></td></tr><tr><td>3,3N-Diaminobenzidine Tertrahydrochloride</td><td>Beyotime</td><td>P0203</td><td></td></tr><tr><td>Aqueous mounting medium</td><td>Dako</td><td>C0563</td><td></td></tr><tr><td>Rabbit anti-GDF-9 antibody</td><td>Beijing Biosynthesis Biotechnology</td><td>BS-175R</td><td></td></tr><tr><td>Rabbit anti-PCNA antibody</td><td>Proteintech</td><td>24036-1-AP</td><td></td></tr><tr><td>Methylene blue</td><td>Sigma</td><td>M9140</td><td></td></tr><tr><td>Transmission electron microscope</td><td>Hitachi</td><td>H-7500</td><td></td></tr></tbody></table>

in vitro growth of mouse preantral follicles under simulated microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

Figure 1A shows a RWV with oil inside the vessel. A droplet of 150 &#181;L medium containing one piece of ovarian tissue or three alginate beads with encapsulated follicles was put into the oil. The medium droplet was kept in a state of terminal velocity due to the fluid drag on the droplets within the rotating oil at rate of 25 rotations per minute, which generated a simulated microgravity (s-&#181;g) condition. In a control experiment, ovarian tissue or capsulated follicles were cultured in a droplet of 150 &#181;L medium covered with mineral oil in 35&#215;10mm dishes (Figure 1B), which generated a 1-g condition.
To study the effects of simulated microgravity on preantral follicle development in vitro, we cultured the 14 day-old mouse ovarian tissue under 1-g (control experiment) or s-&#181;g conditions. We counted the number of healthy follicles in the H&#38;E stained sections in two conditions after 0, 2, and 4 days of culture (Figure 2). We found that the follicle survival in ovarian tissue treated in the s-&#181;g condition was significantly lower than that under the 1-g condition at day 2 and day 4 of culturing (p &#60;0.05, ANOVA). Moreover, we found that no PCNA positive signals were detected in the ovarian tissue under the s-&#181;g condition (Figure 3). In the culture of isolated preantral follicles encapsulated in alginate beads, we revealed that significantly more follicles survived under 1-g condition (76.8% &#177; 5.3%, n = 227) than s-&#181;g condition (54.4% &#177; 6.7%, n = 249) on day 4 of culturing. In addition, oocyte size had no significant increase after 4 days of culturing, though the follicle size increased significantly under both gravity conditions (Figure 4). However, follicles with less than 10% dead granulosa cells (Figure 5) were significantly lower under the 1-g condition (81.5 &#177; 5%, n = 10) than under s-&#181;g condition (90 &#177; 8%, n = 10) (p &#60;0.05).
To investigate the effects of simulated microgravity on oocyte functionality, we examined the expression of the oocyte-specific marker GDF-9. We demonstrated that GDF-9 expression was remarkably down-regulated in the ovarian tissue under s-&#181;g condition (Figure 6). In addition, we demonstrated frequent ultrastructural abnormalities of oocyte organelles in the encapsulated follicles at day 4 of culture under the s-&#181;g condition (Figure 7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55641/55641fig1.jpg" /> 
Figure 1: Setup of s-&#181;g and 1-g culture conditions. (A) s-&#181;g was created by the rotating wall vessel and keeping the culture subjects in a state of constant free-fall within the moving oil. (B) A 1-g condition was created by culturing ovarian tissue or follicles on the surface of a petri dish covered with mineral oil. The arrow indicates a droplet of medium. Scale bar = 1 cm. This figure has been modified from Zhang et al.17 <a href="https://cloudflare.jove.com/files/ftp_upload/55641/55641fig1large.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/55641/55641fig2.jpg" /> 
Figure 2: Effects of s-&#181;g on the ovarian tissue culture. (A) Sections of ovarian tissue cultured under 1-g or s-&#181;g conditions for 0, 2, and 4 days. Scale bar = 50 &#181;m. (B) Follicle density in the ovarian tissue sections. Error bar represents 1 SEM, *: p &#60;0.05. This figure has been modified from Zhang et al.17 <a href="https://cloudflare.jove.com/files/ftp_upload/55641/55641fig2large.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/55641/55641fig3.jpg" /> 
Figure 3: Immunohistochemistry of PCNA. PCNA protein expression was examined in the granulosa cells in the ovarian tissue cultured under 1-g or s-&#181;g conditions for 0, 2, and 4 days. Scale bar = 50 &#181;m. This figure has been modified from Zhang et al.17 <a href="https://cloudflare.jove.com/files/ftp_upload/55641/55641fig3large.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/55641/55641fig4.jpg" /> 
Figure 4: Effects of s-&#181;g on the culture of encapsulated preantral follicles in alginate beads. (A) Morphology of preantral follicles cultured under 1-g or s-&#181;g conditions for 0 days and 4 days. Scale bar = 50 &#181;m. (B) Oocyte diameter and (C) follicle diameter were compared between 1 g and s-&#181;g conditions. Error bar represents 1 SEM, *: p &#60;0.05. This figure has been modified from Zhang et al.17 <a href="https://cloudflare.jove.com/files/ftp_upload/55641/55641fig4large.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/55641/55641fig5.jpg" /> 
Figure 5: Cell viability assay. Representative images were taken under a microscope at 400X magnification. The assay uses Calcein AM to visualize live cells stained in green and EthD-1 to visualize the nuclei of dead cells stained in red. (A): A follicle with ~100% live granulosa cells (green). (B): A follicle with live granulosa cells (green), as well as more than 10% dead cells (red). Scale bar = 50 &#181;m. This figure has been modified from Zhang et al.17 <a href="https://cloudflare.jove.com/files/ftp_upload/55641/55641fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55641/55641fig6.jpg" /> 
Figure 6: Immunohistochemistry of GDF-9. GDF-9 protein expression was examined in the oocytes in the ovarian tissue cultured under 1-g or s-&#181;g conditions for 0, 2, and 4 days. Scale bar = 50 &#181;m. This figure has been modified from Zhang et al.17 <a href="https://cloudflare.jove.com/files/ftp_upload/55641/55641fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55641/55641fig7.jpg" /> 
Figure 7: Ultrastructural analysis of isolated preantral follicles cultured under 1-g or s-&#181;g conditions on day 4 of culture. (A) An oocyte with microvilli (arrows) extending into the zona pellucida (1-g condition). (B-F) Oocytes with large vacuoles (*), multilamellar bodies (#), lipid droplets (arrows), vacuolated mitochondria lacking cristae (arrow), and dispersing Golgi apparatus (arrow), respectively (s-&#181;g condition). O: oocyte; ZP: zona pellucida. This figure has been modified from Zhang et al.17 <a href="https://cloudflare.jove.com/files/ftp_upload/55641/55641fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity at JoVE.com

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

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Research

JoVE Journal

Developmental Biology

8.3K Views.  The First Affiliated Hospital of Wenzhou Medical University. Een zeer veelbelovende techniek voor het genereren van weefsel construeert zonder met behulp van matrix aan cultuur cellen in een gesimuleerde microzwaartekracht voorwaarde is. Met behulp van een roterende cultuur systeem, onderzochten we ovariële follikel groei en oöcyt rijping in termen van overleving van de follikel, morfologie, groei en oöcyt functie onder de voorwaarde van de gesimuleerde microzwaartekracht.

Video: In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

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 ...

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Bioengineering

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.

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

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.

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

Gene Expression Analyses in Human Follicles

The ovary is a heterogeneous organ composed of different cell types. To study the molecular mechanisms occurring during folliculogenesis, the localization of proteins and gene expression can be performed on fixed tissue. However, to properly assess gene expression levels in a human follicle, this complex and delicate structure must be isolated. Hence, an adapted protocol previously described by Woodruff&#39;s laboratory has been developed to separate follicles (the oocyte and the granulosa cells) from their surrounding environment. The ovarian cortical tissue is first manually processed to obtain small fragments using two tools: a tissue slicer and a tissue chopper. The tissue is then enzymatically digested with 0.2% collagenase and 0.02% DNase for at least 40 min. This digestion step is performed at 37 &#176;C and 5% CO2 and is accompanied by mechanical pipetting of the medium every 10 min. After incubation, the isolated follicles are collected manually using a calibrated microcapillary pipette under microscope magnification. If follicles are still present in the pieces of tissue, the procedure is completed with manual microdissection. The follicles are collected on ice in a culture medium and are rinsed twice in droplets of phosphate-buffered saline solution. This digestion procedure must be carefully controlled to avoid follicle deterioration. As soon as the structure of the follicles appears to be compromised or after a maximum of 90 min, the reaction is stopped with a 4 &#176;C blocking solution containing 10% fetal bovine serum. A minimum of 20 isolated follicles (sized under 75 &#181;m) should be collected to obtain an adequate amount of total RNA after RNA extraction for real-time quantitative polymerase chain reaction (RT-qPCR). After extraction, the quantification of total RNA from 20 follicles reaches a mean value of 5 ng/&#181;L. The total RNA is then retrotranscribed into cDNA, and the genes of interest are further analyzed using RT-qPCR.

Here, we describe a protocol outlining how to isolate human ovarian follicles from frozen-thawed cortical tissue to perform gene expression analyses.

The ovary is a heterogeneous organ composed of different cell types. To study the molecular mechanisms occurring during folliculogenesis, the localization ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System

Given the current limitations of conducting biological research in space, a few options exist for subjecting cell culture to simulated microgravity (SMG) on Earth. These options vary in their methods, principles, and suitability for use with suspension cell culture. Here, a cell culture method is described for subjecting lymphocytes to simulated microgravity using a commercially available rotary cell culture system, also known as a 2D clinostat or a rotating wall vessel (RWV) device. This cell culture method utilizes the principle of time-averaged gravity vector nullification to simulate microgravity by rotating the cells on a horizontal axis. The cells cultured in this system can be harvested and utilized in many different experimental assays to assess the effects of simulated microgravity on cellular function and physiology. The culturing technique may vary slightly depending on the cell type or line that is used, but the method described here may be applied to any suspension-type cell culture.

This is a step-by-step guide for using a commercially available rotary cell culture system to culture lymphocytes in simulated microgravity using specialized disposable culture vessels. This culturing method may be applied to any suspension-type cell culture.

Given the current limitations of conducting biological research in space, a few options exist for subjecting cell culture to simulated microgravity (SMG) ...

Immunology and Infection

Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and endangered species. Research has been mostly limited to antral and large preantral follicles due to difficulty in the isolation of smaller preantral follicles, especially in large mammals such as bovine species. This work presents an efficient approach to retrieve large numbers of small preantral follicles from a single bovine ovary. The cortex of individual bovine ovaries was sliced into 500 &#181;m cubes using a tissue chopper and homogenized for 6 min at 9,000-11,000 rpm using a 10 mm probe. Large debris was separated from the homogenate using a cheese cloth, followed by serial filtration through 300 &#181;m and 40 &#181;m cell strainers. The contents retained in the 40 &#181;m strainer were rinsed into a search dish, where follicles were identified and collected into a drop of medium. The viability of the collected follicles was tested via trypan blue staining. This method enables the isolation of a large number of viable small preantral follicles from a single bovine ovary in approximately 90 min. Importantly, this method is entirely mechanical and avoids the use of enzymes to dissociate the tissue, which may damage the follicles. The follicles obtained using this protocol can be used for downstream applications such as isolation of RNA for RT-qPCR, immunolocalization of specific proteins, and in vitro culture.

Advancing the study of preantral folliculogenesis requires efficient methods of follicle isolation from single ovaries. Presented here is a streamlined, mechanical protocol for follicle isolation from bovine ovaries using a tissue chopper and homogenizer. This method allows collection of a large number of viable preantral follicles from a single ovary.

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and ...

Biology

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...

Developing an Ovariectomized Mouse Model with Elevated FSH Level

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.

Author Spotlight: Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model 

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.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

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