This work was supported by Regione Lombardia PSR INNOVA n.201801061529 and UNIMI n.PSR 2019_DIP_027_ALUCI_01

Ovaries were collected from 4 to 8 years old &#160;Holstein dairy cows recovered at the local abattoir (INALCA S.p.A., Ospedaletto Lodigiano, LO, IT 2270M CE, Italy).
1. Media preparation
NOTE: All media must be prepared at least four hours before use. Sodium bicarbonate buffered media are incubated at 38.5 &#176;C and 5% CO2 in air, maximum humidity. HEPES-buffered media are maintained at 38.5 &#176;C in thermostatic oven.
<ol>
	<li>&#160;Long in vitro culture of oocytes (L-IVCO) medium
	<ol>
		<li>Prepare 15 mL of the basic culture medium (M199-B): Supplement M199 with 2 mM glutamine, 0.4% fatty acid free bovine serum albumin (BSA), 0.2 mM sodium pyruvate, 25 mM sodium bicarbonate, 0.1 mM cysteamine, 21.3 &#181;g/mL of phenol red, 75 &#956;g/mL of kanamycin and 4% Polyvinylpyrrolidone (PVP; 360 k molecular weight).</li>
		<li>Prepare 3 mL of the holding medium (M199-H): To M199-B add 5 &#181;M cilostamide and pour it in a 35 mm Petri dish.</li>
		<li>Prepare the L-IVCO medium (M199-L): Supplement M199-B with 0.15 &#956;g/mL Zn sulphate, 10-4 IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone and 5 &#181;M Cilostamide.</li>
		<li>Place 200 &#181;L of M199-L medium in each well of the 96 well coated plate. Fill the wells in the four edges of the plate with sterile culture water to compensate for evaporation and to maintain appropriate humidity during culture.</li>
		<li>Incubate the 96 well plate and the M199-H medium in the incubator at 38.5 &#176;C and 5% CO2 in air, maximum humidity.</li>
	</ol>
	</li>
	<li>Dissection medium
	<ol>
		<li>Prepare the dissection medium (M199-D): Supplement M199 with 0.4% BSA fraction V, 0.164 mM penicillin, 0.048 mM streptomycin, 1790 units/L heparin. M199-D can be prepared in bulk, dispensed in 20 mL aliquots and stored at 4 &#176;C for 6 months. When needed, warm and supplement 1 aliquot.</li>
		<li>Prepare 20 mL of M199-D supplemented with 5 &#181;M cilostamide (M199-D cilostamide).</li>
	</ol>
	</li>
</ol>
2. Ovary collection and processing
NOTE: All procedures are conducted at room temperature (26 &#176;C) unless otherwise indicated.
<ol>
	<li>Recover the ovaries at the abattoir from cows.</li>
	<li>Place the ovaries in sterile saline (NaCl, 9 g/L) at 26 &#8211; 28 &#176;C added with penicillin 100 U/mL and streptomycin 0.1 mg/mL.</li>
	<li>Transport the organs to the laboratory in warm sterile saline within 4 h.</li>
	<li>Wash the ovaries 4x in sterile saline maintained at 26 &#176;C.</li>
	<li>Remove all mid-to-large antral follicles by aspirating all follicles more than 2 mm in diameter using a 18 G needle connected to an aspiration pump with vacuum pressure set at -28 mmHg and place the aspirated ovaries in a beaker with sterile saline at 26 &#176;C. 
	NOTE: Removal of the content of follicles &#62; 2 mm is a critical step to remove as much as possible the source of fully-grown oocytes that would &#8216;contaminate&#8217; the experiment.</li>
	<li>Under a horizontal laminar flow hood, place one ovary at the time on a sterile polytetrafluoroethylene cutting board. Using a surgical blade No. 22 mounted on a scalpel handle, cut slices of ovarian cortex (the outer portion of the ovary, which contains the follicles), 1.5 &#8211; 2 mm thick and parallel to the major axis of the organ.</li>
	<li>Place the slices of ovarian cortex in a sterile glass Petri dish covered with dissecting medium on a warm plate at 38.5 &#176;C. 
	NOTE: From now on all the procedures are performed at 38.5 &#176;C using a warm plate.</li>
</ol>
3. Selection and isolation of the follicles and retrieval of the COCs
<ol>
	<li>Place one ovarian cortex slice in a 60 mm glass Petri dish with 2-3 mL of M199-D.</li>
	<li>Using a dissection microscope, select the follicles between 0.5 &#8211; 2 mm using a micrometer-equipped eyepiece.</li>
	<li>Identify the healthy, non-atretic follicles under the stereomicroscope. Assess follicle atresia by observing morphological parameters, such as very clear translucent appearance, with a dark COC inside. Discard the atretic follicles and process all the others.</li>
	<li>Using a surgical blade&#160; No. 22 mounted on a scalpel handle remove the ovarian tissue surrounding the follicle on one side until the follicle is exposed on one edge.</li>
	<li>Using a 26G needle mounted on a syringe, carefully make a slit in the exposed follicle wall. This action will release the follicular content, comprising the COC, follicular fluid and clumps of cells.</li>
	<li>Identify the COC under the microscope and examine for cumulus integrity, zona pellucida integrity and homogeneity of the cytoplasm. If these criteria are fulfilled, aspirate the COC using a P20 pipette.</li>
	<li>Place the isolated COC in M199-D cilostamide.</li>
	<li>Continue the isolation procedure for 30 min.</li>
</ol>
4. Selection of COCs to be subjected to in vitro culture
<ol>
	<li>Under the dissection microscope, select healthy COCs based on the criteria in step 3.6.</li>
	<li>In a 60 mm Petri dish, prepare 16 drops of 20 &#181;L of M199-D cilostamide and place one healthy COC per drop (Figure 1A).</li>
	<li>Using an inverted microscope attached to a camera measure the oocyte diameter, excluding the zona pellucida, using the software provided with the camera.</li>
	<li>With a clear visualization of the oocyte, make two perpendicular measurements excluding the zona pellucida (Figure 1B).</li>
	<li>Assure whether the mean of the two oocyte&#8217;s measurement, excluding the zona pellucida, is within a range of 100 &#8211; 110 &#181;m. Discard COCs with not-rounded shaped oocyte or with oocytes that are not measurable.</li>
	<li>Transfer the selected COCs in a 35 mm dish containing M199-H medium and keep them in the incubator at 38.5 &#176;C and 5% CO2 in air, maximum humidity until step 5.1.</li>
	<li>Repeat steps 3 and 4 up to 4x. The overall working time must not exceed 2 h.</li>
</ol>
5. Long in vitro culture of the oocytes (L-IVCO)
<ol>
	<li>Transfer one COC per well in the center of a well of the 96 well plate to be prepared in step 1.1.5.</li>
	<li>Incubate the plate for 5 days at 38.5 &#176;C and 5% CO2 in air, maximum humidity.</li>
	<li>Every other day (day 2 and day 4) prepare fresh M199-L as described in step 1.</li>
	<li>Renew half of the medium by removing 100 &#181;L of medium and replacing with 100 &#181;L of freshly prepared M199-L. Perform the medium renewal under the stereomicroscope and avoid moving the COCs into the well.</li>
</ol>
6. COC classification after the culture
<ol>
	<li>At the end of the L-IVCO, analyze the COCs&#8217; morphology under the dissection microscope.</li>
	<li>Classify as depicted in Figure 2.
	<ol>
		<li>Classify as Class 1 if COCs show a compact cumulus cell investment with no sign of cumulus expansion and cell degeneration.</li>
		<li>Classify as Class 2 if COCs show a compact cumulus cell investment with no sign of cumulus expansion and cell degeneration and with antrum-like formation in the cumulus mass.</li>
		<li>Classify as Class 3 if COCs show several layers of cumulus cell with no sign of cumulus expansion and some disaggregated cells in the outer layer of cumulus cells and no antrum-like formation.</li>
		<li>Classify as Class 4 if COCs shows abundant loss of cumulus cells extending for more than 50% of the oocyte surface, and signs of cell degeneration and cell debris.</li>
	</ol>
	</li>
</ol>
7. Evaluation of meiotic progression after culture
<ol>
	<li>Oocyte denudation
	<ol>
		<li>Place each COC in a single well of a four-well plate containing 400 &#181;L of 199D per well.&#160;</li>
		<li>Under a dissection microscope gently remove the cumulus cells mechanically by repeated pipetting using a pipette set at 130-140 &#181;L.</li>
		<li>Once the oocytes are free of&#160;the cumulus investment, transfer them to another well containing 199D.</li>
		<li>Repeat the process until all the oocytes are completely denuded.</li>
	</ol>
	</li>
	<li>Oocyte nuclear staining 
	NOTE: From now on all the procedures are performed at room temperature. Reagents are at room temperature.
	<ol>
		<li>Fix the oocytes in paraformaldehyde 4% in phosphate buffer saline (PBS) for 1 h. 
		CAUTION: Wear personal protective equipment when handling paraformaldehyde and dispose of contaminated materials in accordance with hazardous waste disposal guidelines.</li>
		<li>Wash the oocytes 3x for 5 min each in PBS containing 1% polyvinylalcohol (PVA). 
		NOTE: The samples can be processed right away or stored at 4 &#176;C for maximum one week.</li>
		<li>Place the oocytes in PBS containing 0.1% Triton X for 10 min.</li>
		<li>Wash the oocytes 3x for 5 min each in PBS containing 1% PVA.</li>
		<li>Place the oocytes singularly in drops of 5 &#181;L of antifade medium supplemented with 4&#8217;,6-diamidino-2-phenylindole (DAPI) dilactate (1 &#181;g/mL) over a slide.</li>
		<li>Place two strips of double-sided tape along the long sides of the slide, to avoid excessive flattening of the oocytes when putting the cover slip on top.</li>
		<li>Place the cover slip on top, make it adhere to the tape and keep in the dark while processing all the samples.</li>
		<li>Analyze the oocytes using a conventional epifluorescence microscope equipped with DAPI filters (Excitation/Emission: 358&#8260;461) to assess the meiotic progression of the oocytes.</li>
		<li>Classify the oocytes according to their meiotic progression: GV - oocytes with different degrees of chromatin condensation within the GV; MI &#8211; oocytes from GV breaking down to metaphase I; and degenerate &#8211; oocytes that could be not identified as being at any of the previous stages.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+hormone+concentrations+in+the+fluid+of+bovine+follicles+relative+to+size,+quality+and+stage+of+the+oestrus+cycle.">Steroid hormone concentrations in the fluid of bovine follicles relative to size, quality and stage of the oestrus cycle.</a> Theriogenology. 24 (4), 395-408 (1985).</li><li>Sakaguchi, 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=Relationships+between+the+antral+follicle+count,+steroidogenesis,+and+secretion+of+follicle-stimulating+hormone+and+anti-Mullerian+hormone+during+follicular+growth+in+cattle.">Relationships between the antral follicle count, steroidogenesis, and secretion of follicle-stimulating hormone and anti-Mullerian hormone during follicular growth in cattle.</a> Reproductive Biology and Endocrinology. 17 (1), 88 (2019).</li><li>Makita, M., Miyano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Androgens+promote+the+acquisition+of+maturation+competence+in+bovine+oocytes.">Androgens promote the acquisition of maturation competence in bovine oocytes.</a> Journal of Reproduction and Development. 61 (3), 211-217 (2015).</li><li>Walters, K. A., Allan, C. M., Handelsman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Androgen+actions+and+the+ovary.">Androgen actions and the ovary.</a> Biology of Reproduction. 78 (3), 380-389 (2008).</li><li>Luciano, A. M., Pappalardo, A., Ray, C., Peluso, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+inhibits+large+granulosa+cell+apoptosis+by+stimulating+progesterone+synthesis+and+regulating+the+distribution+of+intracellular+free+calcium.">Epidermal growth factor inhibits large granulosa cell apoptosis by stimulating progesterone synthesis and regulating the distribution of intracellular free calcium.</a> Biology of Reproduction. 51 (4), 646-654 (1994).</li><li>Gordon, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Production of Cattle Embryos, 2nd edn. , (2003).</li><li>Telfer, E. E., McLaughlin, M., Ding, C., Thong, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-step+serum-free+culture+system+supports+development+of+human+oocytes+from+primordial+follicles+in+the+presence+of+activin.">A two-step serum-free culture system supports development of human oocytes from primordial follicles in the presence of activin.</a> Human Reproduction. 23 (5), 1151-1158 (2008).</li><li>McLaughlin, M., Albertini, D. F., Wallace, W. H. B., Anderson, R. A., Telfer, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaphase+II+oocytes+from+human+unilaminar+follicles+grown+in+a+multi-step+culture+system.">Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system.</a> Molecular Human Reproduction. 24 (3), 135-142 (2018).</li><li>Fair, T., Hyttel, P., Greve, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+oocyte+diameter+in+relation+to+maturational+competence+and+transcriptional+activity.">Bovine oocyte diameter in relation to maturational competence and transcriptional activity.</a> Molecular Reproduction and Development. 42 (4), 437-442 (1995).</li></ol>

The authors have nothing to disclose.

Here we describe a culture system for growing oocytes that promotes oocyte development for 5 days by supporting their viability and preventing meiotic resumption. This latter aspect is of the outmost importance to allow the continued growth and differentiation necessary to confer the oocyte with meiotic and embryonic developmental competence2,20, that would be otherwise blocked by a premature resumption of the meiotic division.
When de...

During the reproductive life span, only a minimal fraction of the oocytes that are present in an ovary mature, are released in the fallopian tubes upon ovulation, and are available for being fertilized and develop into a viable embryo1. On the other hand, most of the oocytes within an ovary undergo atresia and are never ovulated. In vitro embryo production (IVP) technologies have attempted to increase the exploitation of the ovarian reserve2,3. Thus far, such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. Nevertheless, most protocols use oocytes that have basically completed the growth phase within the antral ovarian follicle, and hence are referred to as fully-grown oocytes. In cattle, where IVP technologies are widely used, fully grown oocytes reach a final diameter of approximately 120 &#181;m and are collected from follicles that span from 2 to 8 mm in diameter (medium antral follicles)1. Upon isolation from the follicles, such oocytes are in vitro matured and fertilized. The zygotes are then cultured up to the blastocyst stage and either transferred into a recipient or cryopreserved. In cattle, as well as in many other species, despite the potential offered by IVP, the number of in vitro produced embryo per cow did not largely improve for the last 40 years. This is in part due to the limited number of fully grown oocytes that populate an ovary at a given time which can be retrieved and subjected to standard IVP techniques4,5,6.
The oocytes enclosed within early antral follicles, i.e., those follicles that are less than 2 mm in diameter, represent a potential source to be used in fertility preservation programs7 , as an ovary roughly contains 10 times more early antral follicles than medium antral8. However, these oocytes are still in the growth phase and have not yet reached the fully-grown stage9. As such, they are still transcriptionally active, producing mRNAs that will be stored for later developmental steps, and have not yet undergone all the differentiation process required to confer the oocytes with the ability of spontaneously resuming and completing meiosis I once isolated from the follicular compartment10,11. Therefore, they cannot be directly submitted to standard in vitro maturation (IVM) protocols, but they require an additional period of culture that would allow them to complete the growth phase and properly differentiate.
The transition from the growing to the fully-grown stage, which in cattle occurs when the follicle develops from the early antral to the medium antral stage, is one of the critical steps during oocyte development. In cattle, several studies attempted to recapitulate these events in vitro2,12,13,14,15,16,17,18,19. However, to date no reliable protocols have been developed and only limited success has been reported. According to previous studies20, these growing oocytes constitute a homogeneous population. Besides being transcriptionally active, their chromatin is dispersed in the germinal vesicle (GV), in a configuration that is named GV02,21. Conversely, the population of fully-grown oocytes obtained from medium antral follicles is more heterogeneous, a condition that is mirrored by the various degrees of chromatin compaction (GV1, GV2 and GV3) that can be observed20. Among these, previous data have shown that GV2 and GV3 oocytes are overall characterized by a better quality and higher embryonic developmental competence20,21,22,23,24.
Starting from the above observations, here we describe a 5-days long culture system of oocytes (L-IVCO) that allows the differentiation of oocytes isolated as cumulus-oocyte complexes (COCs) from early antral follicles. This culture strategy has evolved from 10 years long studies conducted in our lab and roots its ground on the previously developed 24-48 hours in vitro oocyte culture (IVCO)2, prematuration systems23,25 and zinc supplementation during oocyte culture . A combination of follicle stimulating hormone (FSH) and a phosphodiesterase-3 (PDE3) inhibitor, able to enhance cumulus-oocyte communication2, prevent untimely meiotic resumption2, and support oocyte growth2 was used.

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			<td height="20" style="height:21px;width:215px;">4-well dishes</td>
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			<td style="width:171px;"></td>
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			<td height="42" style="height:43px;width:215px;">96-well dish</td>
			<td style="width:127px;">Becton Dickinson Biosciences</td>
			<td style="width:123px;">356649</td>
			<td>BioCoat&#8482; Collagen I</td>
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		<tr height="41">
			<td height="41" style="height:42px;width:215px;">Bovine Serum Albumin (Fatty acid free)</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">A8806</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Bovine Serum Albumin (Fraction V)</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">A3311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Cell culture water</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">W3500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Cilostamide</td>
			<td style="width:127px;">Sigma</td>
			<td>C7971</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Cysteamine</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">M9768</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Digital camera</td>
			<td style="width:127px;">Nikon Corp</td>
			<td>Camera DS-5M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Disodium phosphate</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">S5136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Estradiol</td>
			<td>Sigma</td>
			<td>E2758</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Glutamax Supplement</td>
			<td style="width:127px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Gonal F</td>
			<td style="width:127px;">Merck Serono</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Heparin</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">H3149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Hepes</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">H3784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Vacuum pump</td>
			<td>Cook-IVF</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Incubator</td>
			<td style="width:127px;">Sanyo</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Kanamycin sulfate from Streptomyces kanamyceticus</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">K1377</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:45px;width:215px;">Medium 199</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">M3769</td>
			<td style="width:171px;">Powder for hepes-buffered TCM199</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:26px;width:215px;">Medium 199</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">M2520</td>
			<td>Powder for M199-D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Microscope</td>
			<td>Nikon Corp</td>
			<td>Nikon Diaphot</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Microscope</td>
			<td style="width:127px;">Nikon Corp</td>
			<td style="width:123px;">Eclipse E 600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Monopotassium phosphate</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P5655</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Paraformaldehyde</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">158127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Penicilin</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P3032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Phenol Red</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P5530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Polyvinyl alcohol</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P8137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Polyvinylpyrrolidone</td>
			<td style="width:127px;">Sigma</td>
			<td>P5288</td>
			<td>360k molecular weight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Potassium chloride</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P5405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Progesterone</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P8783</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sodium bicarbonate</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sodium choride</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">P5886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sodium pyruvate</td>
			<td style="width:127px;">Sigma</td>
			<td>P4562</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Streptomycin</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">S9137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Testosterone</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">86500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Triton X</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">T9284</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Vectashield with DAPI</td>
			<td style="width:127px;">Vector Laboratories</td>
			<td style="width:123px;">H1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Water</td>
			<td>Sigma</td>
			<td style="width:123px;">W3500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Zinc sulfate heptahydrate</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:123px;">Z0251</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro culture strategy for oocytes from early antral follicle in cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

At the end of the L-IVCO, the gross morphology of the COCs changed and 4 classes were identified based on the appearance of the cumulus cells, as shown in Figure 2. Based on the morphological criteria commonly adopted to select healthy COCs11,26,27, the class 1, 2 and 3 were judged healthy, while the class 4, which showed clear signs of degeneration such as the absence of complete layers of cumulus ...

Watch this Scientific Journal Video about In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle at JoVE.com

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

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8.4K Views.  University of Milan. We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage.  

Video: In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

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 ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

This protocol describes the isolation of epithelial cells from different anatomical regions of the human amniotic membrane to determine their heterogeneity and functional properties for possible application in clinical and physiopathological models.

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that ...

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.