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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotrophin+and+steroid+concentrations+in+bovine+follicular+fluid+and+their+relationship+to+follicle+size.">Gonadotrophin and steroid concentrations in bovine follicular fluid and their relationship to follicle size.</a> Journal of Reproduction and Fertility. 65 (2), 467-473 (1982).</li><li>Kruip, T. A., Dieleman, S. 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.

<table><tbody><tr><td>4-well dishes</td><td>Nunclon</td><td>179830</td><td></td></tr><tr><td>96-well dish</td><td>Becton Dickinson Biosciences</td><td>356649</td><td>BioCoat&amp;trade; Collagen I</td></tr><tr><td>Bovine Serum Albumin (Fatty acid free)</td><td>Sigma</td><td>A8806</td><td></td></tr><tr><td>Bovine Serum Albumin (Fraction V)</td><td>Sigma</td><td>A3311</td><td></td></tr><tr><td>Cell culture water</td><td>Sigma</td><td>W3500</td><td></td></tr><tr><td>Cilostamide</td><td>Sigma</td><td>C7971</td><td></td></tr><tr><td>Cysteamine</td><td>Sigma</td><td>M9768</td><td></td></tr><tr><td>Digital camera</td><td>Nikon Corp</td><td>Camera DS-5M</td><td></td></tr><tr><td>Disodium phosphate</td><td>Sigma</td><td>S5136</td><td></td></tr><tr><td>Estradiol</td><td>Sigma</td><td>E2758</td><td></td></tr><tr><td>Glutamax Supplement</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>Gonal F</td><td>Merck Serono</td><td></td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3149</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3784</td><td></td></tr><tr><td>Vacuum pump</td><td>Cook-IVF</td><td>KMAR-5100</td><td></td></tr><tr><td>Incubator</td><td>Sanyo</td><td></td><td></td></tr><tr><td>Kanamycin sulfate from Streptomyces kanamyceticus</td><td>Sigma</td><td>K1377</td><td></td></tr><tr><td>Medium 199</td><td>Sigma</td><td>M3769</td><td>Powder for bicarbonate-buffered media</td></tr><tr><td>Medium 199</td><td>Sigma</td><td>M2520</td><td>Powder for HEPES-buffered media</td></tr><tr><td>Microscope</td><td>Nikon Corp</td><td>Nikon Diaphot</td><td></td></tr><tr><td>Microscope</td><td>Nikon Corp</td><td>Eclipse E 600</td><td></td></tr><tr><td>Monopotassium phosphate</td><td>Sigma</td><td>P5655</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td></td></tr><tr><td>Penicilin</td><td>Sigma</td><td>P3032</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma</td><td>P5530</td><td></td></tr><tr><td>Polyvinyl alcohol</td><td>Sigma</td><td>P8137</td><td></td></tr><tr><td>Polyvinylpyrrolidone</td><td>Sigma</td><td>P5288</td><td>360k molecular weight</td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P5405</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Sodium choride</td><td>Sigma</td><td>P5886</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma</td><td>P4562</td><td></td></tr><tr><td>Streptomycin</td><td>Sigma</td><td>S9137</td><td></td></tr><tr><td>Testosterone</td><td>Sigma</td><td>86500</td><td></td></tr><tr><td>Triton X</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>Vectashield with DAPI</td><td>Vector Laboratories</td><td>H1200</td><td></td></tr><tr><td>Water</td><td>Sigma</td><td>W3500</td><td></td></tr><tr><td>Zinc sulfate heptahydrate</td><td>Sigma</td><td>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 ...

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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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Research

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Developmental Biology

8.6K 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

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

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

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 ...

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart. 
Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily ...

Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) exist within the reconstructed embryo: one within the recipient ooplasm and one within the donor somatic cell. This mitochondrial heteroplasmy can lead to developmental issues in the embryo and the fetus. Handmade cloning protocols include oocyte bisection, which can be used to decrease the mtDNA copy number, reducing the degree of mitochondrial heteroplasmy in a reconstructed embryo. Centrifugation of denuded, mature bovine oocytes produced a visible mitochondria-dense fraction at one pole of the oocyte. Oocytes&#39; zonae pellucidae were removed by exposure to a pronase solution. Bisection was performed using a microblade to remove the visible mitochondria fraction. qPCR was used to quantify the mtDNA present in DNA samples extracted from whole oocytes and bisected ooplasts, providing a comparison of mtDNA copy numbers before and after bisection. Copy numbers were calculated using cycle threshold values, a standard curve&#39;s regression line formula, and a ratio that included the respective sizes of mtDNA PCR products and genomic PCR products. One bovine oocyte had an average mtDNA copy number (&#177; standard deviation) of 137,904 &#177; 94,768 (n = 38). One mitochondria-depleted ooplast had an average mtDNA copy number of 8,442 &#177; 13,806 (n = 33). Average mtDNA copies present in a mitochondria-rich ooplast were 79,390 &#177; 58,526 mtDNA copies (n = 28). The differences between these calculated averages indicate that the centrifugation and subsequent bisection can significantly decrease the mtDNA copy numbers present in the mitochondria-depleted ooplast when compared to the original oocyte (P &#60; 0.0001, determined by one-way ANOVA). The reduction in mtDNA should decrease the degree of mitochondrial heteroplasmy in a reconstructed embryo, possibly fostering standard embryonic and fetal development. Supplementation with mitochondrial extract from the somatic donor cell may also be essential to achieve successful embryonic development.

Here, we present a protocol to significantly reduce the mitochondrial DNA copy numbers in a bovine oocyte (P &lt; 0.0001). This method utilizes centrifugation and bisection to substantially reduce oocyte mitochondria and may allow for an increased chance of development in the reconstructed interspecies somatic cell nuclear transfer embryos.

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Biology

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

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

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

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

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

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

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

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

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