This work was made possible thanks to support by: University of Pavia FRG 2016; University of Parma FIL 2014, 2016; and Kinesis for supplying the plasticware necessary to carry out this study. We thank Dr. Shane Windsor (Faculty of Engineering, University of Bristol, UK) for providing the Cell_PIV software.

All procedures involving animals were approved by the Institutional Animal Care and Use and Ethical Committees at University of Pavia. Animals were maintained under conditions of 22 &#176;C, 60% air humidity and a light/dark cycle of 12:12 h.
1.Ovary Isolation
<ol>
	<li>Inject intraperitoneally 2 four-to-eleven week-old CD1 female mice with 10 U of follicle stimulating hormone with a sterile 1 mL insulin syringe.</li>
	<li>Wait 46-48 h.</li>
	<li>Weigh the mouse and anesthetize with an intramuscular injection of 50 mg/kg of Zoletil (Tiletamina and Zolazepan cloridrate). Euthanize by cervical dislocation.</li>
	<li>Grasp the skin covering the abdominal wall using a pair of dissection forceps. Cut the skin and body wall with a pair of scissors and pull open the incision with forceps.</li>
	<li>Using a pair of tweezers, move the guts aside, pinpoint the uterine horns and localize the ovaries at their extremity.</li>
	<li>Gently hold the ovary using watchmaker forceps and use scissors to cut its ligament to the uterus. Repeat with the other ovary.</li>
	<li>Transfer the collected ovaries into a 35-mm cell culture Petri dish containing 1 mL of M2 isolation medium pre-warmed at 37 &#176;C, 5% CO2 in air.</li>
	<li>Remove the fat and the oviduct using fine scissors under a stereomicroscope.</li>
</ol>
2. Isolation of Cumulus Oocyte Complexes
<ol>
	<li>Puncture the ovarian surface using sterile tweezers and a 1G sterile insulin needle until antral cumulus oocyte complexes (COCs) are passively released.</li>
	<li>Collect COCs with more than three layers of cumulus cells using a mouth-controlled hand-pulled Pasteur micropipette (200 &#181;m in diameter) and transfer them into a 300 &#181;L drop of fresh M2 medium.</li>
	<li>Remove the cumulus cells surrounding the oocytes using a hand-pulled Pasteur micropipette (80 &#181;m in diameter) by gently pipetting in and out for few seconds,</li>
	<li>Repeatedly transfer oocytes from a 20 &#181;L drop of M2 medium to another one, until cumulus cells are completely eliminated.</li>
</ol>
3. Chromatin Organization-Based Oocyte Classification
<ol>
	<li>Prepare Hoechst 33342 staining solution at a final concentration of 0.05 &#181;g/mL in M2 medium starting from a mother solution of 5 mg/mL diluted in 1x PBS.</li>
	<li>Place 3.5 &#181;L micro droplets of Hoechst staining solution at the bottom of a 35 mm Petri dish lid.</li>
	<li>Transfer each single oocyte into a Hoechst staining drop, place the dish onto a pre-warmed (37 &#176;C) heating plate and cover it with a dark lid to avoid light exposition.</li>
	<li>After 10 min of incubation at room temperature, observe the oocytes with a fluorescence microscope at 10X magnification under UV light (330-385 nm), for no more than 1-2 s.</li>
	<li>Classify oocytes as a surrounded nucleolus (SN) (Figure 1A), if they present a ring of Hoechst-positive chromatin surrounding the nucleolus.</li>
	<li>Classify oocytes as a not surrounded nucleolus (NSN), if their nucleolus is not surrounded by Hoechst-positive chromatin and shows disperse heterochromatic spots within the nucleus (Figure 1B).</li>
</ol>
4. Neural Network-Based Oocyte Classification
<ol>
	<li>Prepare four 2 &#181;L droplets of &#945;-MEM medium supplemented with 5% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 5 mM taurine, 100 U/mL penicillin, 75 &#181;g/mL streptomycin and 23.5 &#181;g/&#181;L sodium pyruvate into a 35-mm glass bottom Petri dish and cover them with pre-warmed (37 &#176;C) and pre-equilibrated mineral oil to avoid medium evaporation. 
	NOTE: If using a live cell-screening system, keep the drops at a specific distance by using a drawn grid of 7 mm x 7 mm as a guideline.</li>
	<li>Transfer 3- 4 oocytes to each drop.</li>
	<li>Place the glass bottom Petri dish into a live cell-screening system, equipped with time-lapse recording software (see Table of Materials), which allows the observation of oocyte maturation in an environment with stable temperature (37 &#176;C) and CO2 concentration (5%).</li>
	<li>Open the time-lapse recording software. On the screen a window appears with a live shot of the oocyte on the left-hand side and all the setting buttons on the right-hand side.</li>
	<li>Select the center of the oocyte to position it at the screen center. Adjust focus if necessary.</li>
	<li>Select the Ph button and set the DIA Lamp to 192; the Exposure time to 1/125 s; the Gain to 1.99; and the Resolution to 1600 x 1200.</li>
	<li>Select FL2 and set the DIA Lamp to 5; the Exposure time to 3 s; the Gain to 2.37; and the Resolution to 1600 x 1200.</li>
	<li>Select the New point button in order to record the oocyte position within the culture drop. Repeat the same routine for each oocyte within the culture drop.</li>
	<li>Select Time and set the Acquisition cycle to 8 min and the Total time to 15 h.</li>
	<li>Select Start time-lapse to start the recording cycle.</li>
	<li>Open the MATLAB software and write &#62;&#62;cell_piv; and then select the PIV Tool.</li>
	<li>Choose the folder containing the recorded movie and select any of the .jpeg files to load the whole image sequence. For each oocyte, select the region of interest (ROI) by clicking the Select area tool.</li>
	<li>Select Process PIV in the File menu. Select the Viewer Tool button. 
	NOTE: The program will calculate the velocity vectors within the ROI and automatically produces a data file, saved as a .mat file.</li>
	<li>Select the .mat file to open. Select the button Select ROI tool and draw a circle around the outline of the oocyte. Click Save under the File menu. 
	NOTE: The vectors in this region are now displayed and the mean magnitude data in the graph window updated for this new ROI.</li>
	<li>Open the collected mean magnitude data of the whole time-lapse frames recorded into a spreadsheet, with, on the rows, the frame sequence and, on the columns, each single oocyte analyzed.</li>
	<li>Organize the data of both NSN and SN oocytes into 10 subgroups.</li>
	<li>Use 9 subgroups to train the feed-forward artificial neural network iteratively (FANN) and 1 subgroup for blind testing.
	<ol>
		<li>Repeat another 9 times, changing the blind testing sample (10-fold cross-validation).</li>
	</ol>
	</li>
	<li>Open MATLAB, run the custom-made script main and, upon request, insert the name of the file with the training data (train.txt), the number of NSN and SN oocytes used for training and the time-lapse interval to be analyzed (typically, from frame # 1-2 to frame # 112-113).</li>
	<li>Press enter to perform the training.</li>
	<li>Insert the name of the file with the testing data (test.txt).</li>
	<li>Open the vector test_outputs_cyt in MATLAB workspace. 
	NOTE: A window appears on the screen showing, in the first row, the probability to obtain True Positives (TP or True NSN) and False Positives (FP or False NSN) for NSN and SN oocytes, respectively; instead, in the second row, the probability to obtain False Negatives (FN or False SN) and True Negatives (TN or True SN) for NSN and SN oocytes, respectively.</li>
	<li>Use the formula: (TP+TN)/(TP+FP+FN+TN), to calculate the FANN accuracy, expressed as a percentage.</li>
</ol>

<ol>
	<li>Patrizio, P., Fragouli, E., Bianchi, V., Borini, A., Wells, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+methods+for+selection+of+the+ideal+oocyte.">Molecular methods for selection of the ideal oocyte.</a> Reprod. Biomed. Online. 15 (3), 346-353 (2007).</li><li>Rienzi, L., Vajta, G., Ubaldi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+value+of+oocyte+morphology+in+human+IVF:+a+systematic+review+of+the+literature.">Predictive value of oocyte morphology in human IVF: a systematic review of the literature.</a> Human. Reprod. Update. 17 (1), 34-35 (2011).</li><li>Tan, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+configurations+in+the+germinal+vesicle+of+mammalian+oocytes.">Chromatin configurations in the germinal vesicle of mammalian oocytes.</a> Mol. Hum. Reprod. 15 (1), 1-9 (2009).</li><li>Vigone, G., 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=Transcriptome+based+identification+of+mouse+cumulus+cell+markers+that+predict+the+developmental+competence+of+their+enclosed+antral+oocytes.">Transcriptome based identification of mouse cumulus cell markers that predict the developmental competence of their enclosed antral oocytes.</a> BMC Genomics. 14, 380 (2013).</li><li>Bui, T. 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=Cytoplasmic+movement+profiles+of+mouse+surrounding+nucleolus+and+not-surrounding+nucleolus+antral+oocytes+during+meiotic+resumption.">Cytoplasmic movement profiles of mouse surrounding nucleolus and not-surrounding nucleolus antral oocytes during meiotic resumption.</a> Mol. Reprod. Dev. 84 (5), 356-362 (2017).</li><li>Zuccotti, M., Piccinelli, A., Giorgi Rossi, P., Garagna, S., Redi, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+organization+during+mouse+oocyte+growth.">Chromatin organization during mouse oocyte growth.</a> Mol. Reprod. Dev. 41 (4), 479-485 (1995).</li><li>Zuccotti, M., Garagna, S., Merico, V., Monti, M., Redi, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+organisation+and+nuclear+architecture+in+growing+mouse+oocytes.">Chromatin organisation and nuclear architecture in growing mouse oocytes.</a> Mol. Cell. Endocrinol. 234 (1-2), 11-17 (2005).</li><li>Zuccotti, M., Merico, V., Cecconi, S., Redi, C. A., Garagna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+does+it+take+to+make+a+developmentally+competent+mammalian+egg?.">What does it take to make a developmentally competent mammalian egg?.</a> Hum. Reprod. Update. 17 (4), 525-540 (2011).</li><li>Inoue, A., Nakajima, R., Nagata, M., Aoki, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+oocyte+nucleus+and+cytoplasm+to+the+determination+of+meiotic+and+developmental+competence+in+mice.">Contribution of the oocyte nucleus and cytoplasm to the determination of meiotic and developmental competence in mice.</a> Hum. Reprod. 23 (6), 1377-1384 (2008).</li><li>Ajduk, 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=Rhythmic+actomyosin-driven+contractions+induced+by+sperm+entry+predict+mammalian+embryo+viability.">Rhythmic actomyosin-driven contractions induced by sperm entry predict mammalian embryo viability.</a> Nat. Commun. 2, 417 (2011).</li><li>Swann, 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=Phospholipase+C-&#950;-induced+Ca2++oscillations+cause+coincident+cytoplasmic+movements+in+human+oocytes+that+failed+to+fertilize+after+intracytoplasmic+sperm+injection.">Phospholipase C-&#950;-induced Ca2+ oscillations cause coincident cytoplasmic movements in human oocytes that failed to fertilize after intracytoplasmic sperm injection.</a> Fertil. Steril. 97 (3), 742-747 (2012).</li><li>Thakur, A., Mishra, V., Jain, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feed+forward+artificial+neural+network:+tool+for+early+detection+of+ovarian+cancer.">Feed forward artificial neural network: tool for early detection of ovarian cancer.</a> Sci. Pharm. 79 (3), 493-505 (2011).</li><li>Laudani, A., Lozito, G. M., Riganti Fulginei, F., Salvini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+Training+Efficiency+and+Computational+Costs+of+a+Feed+Forward+Neural+Network:+A+Review.">On Training Efficiency and Computational Costs of a Feed Forward Neural Network: A Review.</a> Comput. Intell. Neurosci. 2015, 818243 (2015).</li></ol>

The authors have nothing to disclose.

There are several critical steps one should take care of while performing this protocol with mouse oocytes as well as with those of other species. Once isolated from their follicles, oocytes should be immediately transferred into the recording drops, as the separation from the companion cumulus cells triggers the beginning of the GV-to-MII transition. A possible modification to the present protocol could be the addition of 3-isobutyl-1-methylxanthine (IBMX) to the M2 medium used for COCs isolation. IBMX prevents the immediate triggering of the GV-to-MII transition and allows a synchronization of the whole experimental group of oocytes.
The live cell-screening system used in our experiments (see Table of Materials) limits the number of maturation drops to 4 with 4 oocytes per drop, thus to a total of 16 oocytes per experiment. This limitation could be overcome using other time-lapse live cell-screening systems, commercially available and equipped with several culture chambers. 
Based on our experience, the recording interval between a frame and the following is a critical point. After a number of preliminary experiments, we took images every 8 min because this was the minimum time window that allowed the clear observation of the major events occurring during the GV-to-MII transition, such as the germinal vescicle breakdown and the extrusion of the first polar body. This time window might need to be revaluated when observing oocytes of other species.
A drawback of this method is the culture of oocytes in the absence of their surrounding cumulus cells, as the latter are known to further improve the GV-to-MII transition. To this regard, our laboratory is now testing a slight modification to the reported protocol by including the culture of cumulus-free oocytes upon a cumulus cells feeder layer.
By its nature, the FANN has a fixed number of both input (the number of analyzed CMV modules) and output (either a developmentally competent or incompetent oocyte) neurons, but the number of hidden neurons is chosen by the investigator through a trial and correction approach to obtain the best prediction performance. In our case, we experimented with different numbers of hidden neurons and found that three gave the best results. Also, FANN analyses require a large number of input data (in our experiments 112 CMV modules) to obtain a robust statistics. A possible improvement that could reduce the number of necessary input data is the use of alternative classification tools, such as Support Vector Machine, Tree-bag, or KNN classifiers. When the FANN analysis demonstrates its robustness, step 1 could be eliminated from the procedure.
This protocol, set up for the mouse, could be tested on oocytes of other species including humans. Furthermore, the combination of time-lapse recording with PIV and neural network analyses could be exploited for the observation of CMVs in zygotes10,11 and in preimplantation embryos for the evaluation of their further developmental potential.

Female infertility is a pathology that affects an increasing number of women. According to the World Health Organization, around 20% of couples are infertile, with a 40% due to female infertility. In addition, one third of women undergoing cancer treatments (300,000/year and 30,000/year in the USA or Italy, respectively) develop premature ovarian failure.
A strategy to prevent infertility in cancer patients is the isolation and cryopreservation of ovarian follicles before the oncological treatment, followed by in vitro maturation (IVM) of GV oocytes to the MII stage (GV-to-MII transition). The availability of non-invasive markers of oocyte developmental competence would improve the fertilization and developmental processes and the overall pregnancy success1,2.
Based on their chromatin configuration observed after staining with the supravital fluorochrome Hoechst 33342, mammalian fully-grown oocytes are classified either as a Surrounded Nucleolus (SN) or a Not Surrounded Nucleolus (NSN)3. Besides their different chromatin organization, these two types of oocytes display many morphological and functional differences3,4,5,6,7,8,9, including their meiotic and developmental competence. When isolated from the ovary and matured in vitro, both type of oocytes reach the MII stage, and after sperm insemination, develop to the 2-cell stage, but only those with an SN chromatin organization may develop to term9. Although good as a classification method for selecting competent vs. incompetent oocytes, the main drawback is the mutagenic effect that the fluorochrome itself and, above all, the UV light used for its detection might have on the cells.
For all these reasons, we searched for other non-invasive markers associated with the SN or NSN chromatin conformation that could substitute the use of Hoechst while maintaining the same high classification accuracy. The time-lapse observation of Cytoplasmic Movement Velocities (CMVs) is emerging as a feature distinctive of the cell status. For example, recent studies demonstrated the association between CMVs recorded at the time of fertilization and the capacity of mouse and human zygotes to complete preimplantation and full-term development10,11.
Based on these earlier studies, we describe here a platform for the recognition of developmentally competent or incompetent mouse fully-grown oocytes5,6,7,8. The platform is based on three main steps: 1) Oocytes isolated from antral follicles are first classified based on their chromatin configuration either as a surrounded nucleolus (SN) or a not-surrounded nucleolus (NSN); 2) Time-Lapse images of CMVs occurring during the GV-to-MII transition of each single oocyte are taken and analyzed with particle image velocimetry (PIV); and 3) the data obtained with PIV are analyzed with a Feed-forward Artificial Neural Network (FANN) for blind classification12,13. We give details of the most critical steps of the procedure designed for the mouse to make it ready available to be tested and used for other mammalian species (e.g., bovine, monkey and humans).

<table><tbody><tr><td>Folligon</td><td>Intervet</td><td>A201A02</td><td>Hormonal treatment</td></tr><tr><td>Hoechst 33342&amp;nbsp;</td><td>Sigma-Aldrich</td><td>B2261</td><td>For oocyte heterochromatin staining</td></tr><tr><td>Cell culture Petri-dish 35 mm x 10 mm&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>430165</td><td>For COCs isolation</td></tr><tr><td>EmbryoMax M2 Medium (1X), Liquid, with phenol red</td><td>Merck-Millipore</td><td>MR-015-D</td><td>For COCs isolation</td></tr><tr><td>MEM Alpha medium (1X) + Glutamax&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M4526</td><td>For oocyte in vitro maturation</td></tr><tr><td>Cell culture Petri-dish 35 mm glass-bottom&amp;nbsp;</td><td>WillCo&amp;nbsp;</td><td>GWSt-3522</td><td>For imaging experiments</td></tr><tr><td>BioStation IM-LM&amp;nbsp;</td><td>Nikon</td><td>MFA91001</td><td>Live cell screening system&amp;nbsp;</td></tr><tr><td>Pasteur pipette</td><td>Delchimica Scientific Glassware</td><td>6709230</td><td>For follicles manipulation</td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>M8410</td><td>To prevent contamination and medium evaporation</td></tr><tr><td>Penicillin / Streptomycin</td><td>Life Technologies</td><td>15070063</td><td>To prevent medium contamination&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>ML16141079</td><td>For making up &amp;alpha;MEM medium&amp;nbsp;</td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>Life Technologies</td><td>25030</td><td>For making up &amp;alpha;MEM medium&amp;nbsp;</td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T0625</td><td>For making up &amp;alpha;MEM medium&amp;nbsp;</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A3310</td><td>For making up &amp;alpha;MEM media</td></tr><tr><td>Sodium pyruvate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>&amp;nbsp;P4562</td><td>For making up &amp;alpha;MEM media</td></tr><tr><td>Zoletil (Tiletamina and Zolazepan cloridrate)</td><td>Virbac Srl</td><td>QN01AX9</td><td>For mice anesthesia</td></tr><tr><td>Cell_PIV sofware&amp;nbsp;</td><td>Kindly provided by Dr. Shane Windsor, University of Bristol, UK</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; &amp;nbsp;-</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; &amp;nbsp;-</td></tr><tr><td>MATLAB</td><td>The MathWorks, Natick, MA&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; &amp;nbsp;-</td><td>For multi-paradigm numerical computing</td></tr></tbody></table>

a neural network-based identification of developmentally competent or incompetent mouse fully-grown oocytes

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 &#176;C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Figure 2 shows a representative developmentally competent and incompetent oocyte, respectively at the beginning (GV) and at the end (MII) of the IVM procedure. IVM of fully-grown mouse oocytes occurs during 15 h culture. The Time-Lapse observation records the progression of meiosis and detects major meiotic events, including the GVBD and the extrusion of the first polar body.
The analysis and comparison of more than two-hundred developmentally competent vs. incompetent oocytes showed time differences in the progression of meiosis. Specifically, in NSN oocytes, GVBD is significantly delayed of 1-2 time-lapse frames, whereas the PB1 extrusion occurs with a 15 frames delay. Despite these differences, the variability is too high to allow single-oocyte classification. For this reason, the procedure was implemented with a mathematical classification tool.
Figure 3 shows a scheme of the mathematical classification tool used, named Feed-forward Artificial Neural Network (FANN). For each oocyte fed through, FANN simultaneously gives the probability that the gamete is a developmentally competent and the probability that it is an incompetent oocyte. In our experiments, the FANN classified mouse oocytes with a mean accuracy of 91.03%.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56668/56668fig1.jpg" /> 
Figure 1. Representative SN and NSN oocytes stained with Hoechst 33342. Following isolation and staining with the fluorochrome Hoechst 33342, fully-grown oocytes are classified either as surrounded nucleolus (SN) (A) or not surrounded nucleolus (NSN) (B), depending on the presence (arrow) or absence, respectively, of a ring of Hoechst-positive heterochromatin surrounding the nucleolus. Scale bar: 5 &#181;m <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56668/56668fig1large.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/56668/56668fig2.jpg" /> 
Figure 2. Particle Image Velocimetry analysis of Time-Lapse images. The results of the PIV analysis are shown below the bright-field image for each of the 113 time-lapse frames analyzed. The figure shows the beginning (GV) and the end (MII) of the recording process. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56668/56668fig2large.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/56668/56668fig3.jpg" /> 
Figure 3. Schematic representation of the Feed-forward Artificial Neural Network. FANN is the mathematical tool used to classify oocytes as developmentally competent or incompetent. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56668/56668fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes at JoVE.com

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

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6.6K Views.  University of Pavia. Hier presenteren we een protocol voor niet-invasieve beoordeling van ontwikkelingsstoornissen bekwaamheid oöcyt uitgevoerd tijdens hun rijping in vitro van het vesikel germinal aan de metafase-II. Deze methode combineert time-lapse imaging met particle image velocimetry (PIV) en neurale netwerk analyses.

Video: A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Genetics

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 ...

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

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

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

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

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...

Identifying DNA Damage in Prophase-Arrested Oocytes of Mice

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

Author Spotlight: Understanding DNA Damage Response in Mammalian Oocytes and Preimplantation Embryos 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Biology

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

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