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...

<ol>
	<li>Lonergan, P., Fair, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+Oocytes+in+Vitro.">Maturation of Oocytes in Vitro.</a> Annual Review of Animal Biosciences. 4, 255-268 (2016).</li><li>Luciano, A. M., Franciosi, F., Modina, S. C., Lodde, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junction-mediated+communications+regulate+chromatin+remodeling+during+bovine+oocyte+growth+and+differentiation+through+cAMP-dependent+mechanism(s).">Gap junction-mediated communications regulate chromatin remodeling during bovine oocyte growth and differentiation through cAMP-dependent mechanism(s).</a> Biology of Reproduction. 85 (6), 1252-1259 (2011).</li><li>McLaughlin, M., 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=Oocyte+development+in+bovine+primordial+follicles+is+promoted+by+activin+and+FSH+within+a+two-step+serum-free+culture+system.">Oocyte development in bovine primordial follicles is promoted by activin and FSH within a two-step serum-free culture system.</a> Reproduction. 139 (6), 971-978 (2010).</li><li>Galli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achievements+and+unmet+promises+of+assisted+reproduction+technologies+in+large+animals:+a+per-sonal+perspective.">Achievements and unmet promises of assisted reproduction technologies in large animals: a per-sonal perspective.</a> Animal Reproduction. 14 (3), 614-621 (2017).</li><li>Luciano, A. M., Sirard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+in+vitro+maturation+of+oocytes:+a+matter+of+follicular+differentiation.">Successful in vitro maturation of oocytes: a matter of follicular differentiation.</a> Biology of Reproduction. 98 (2), 162-169 (2018).</li><li>Lonergan, P., Fair, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-produced+bovine+embryos:+dealing+with+the+warts.">In vitro-produced bovine embryos: dealing with the warts.</a> Theriogenology. 69 (1), 17-22 (2008).</li><li>Clement, M. D. F., Dalbies-Tran, R., Estienne, A., Fabre, S., Mansanet, C., Monget, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ovarian+reserve+of+primordial+follicles+and+the+dynamic+reserve+of+antral+growing+follicles:+what+is+the+link.">The ovarian reserve of primordial follicles and the dynamic reserve of antral growing follicles: what is the link.</a> Biology of Reproduction. 90 (4), 85 (2014).</li><li>Lussier, J. G., Matton, P., Dufour, 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=Growth+rates+of+follicles+in+the+ovary+of+the+cow.">Growth rates of follicles in the ovary of the cow.</a> Journal of Reproduction and Fertility. 81 (2), 301-307 (1987).</li><li>Fair, T., Hulshof, S. C., Hyttel, P., Greve, T., Boland, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+ultrastructure+in+bovine+primordial+to+early+tertiary+follicles.">Oocyte ultrastructure in bovine primordial to early tertiary follicles.</a> Anatomy and Embryology (Berlin). 195 (4), 327-336 (1997).</li><li>Pavlok, A., Lucas-Hahn, A., Niemann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+and+developmental+competence+of+bovine+oocytes+derived+from+different+categories+of+antral+follicles.">Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles.</a> Molecular Reproduction and Development. 31 (1), 63-67 (1992).</li><li>Blondin, P., Sirard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+and+follicular+morphology+as+determining+characteristics+for+developmental+competence+in+bovine+oocytes.">Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes.</a> Molecular Reproduction and Development. 41 (1), 54-62 (1995).</li><li>Harada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+oocytes+from+early+antral+follicles+grow+to+meiotic+competence+in+vitro:+effect+of+FSH+and+hypoxanthine.">Bovine oocytes from early antral follicles grow to meiotic competence in vitro: effect of FSH and hypoxanthine.</a> Theriogenology. 48 (5), 743-755 (1997).</li><li>Hirao, Y., 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=In+vitro+growth+and+development+of+bovine+oocyte-granulosa+cell+complexes+on+the+flat+substratum:+effects+of+high+polyvinylpyrrolidone+concentration+in+culture+medium.">In vitro growth and development of bovine oocyte-granulosa cell complexes on the flat substratum: effects of high polyvinylpyrrolidone concentration in culture medium.</a> Biology of Reproduction. 70 (1), 83-91 (2004).</li><li>Alm, H., Katska-Ksiazkiewicz, L., Rynska, B., Tuchscherer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+meiotic+competence+of+bovine+oocytes+originating+from+early+antral+ovarian+follicles.">Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles.</a> Theriogenology. 65 (7), 1422-1434 (2006).</li><li>Taketsuru, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+oocytes+in+secondary+follicles+grow+in+medium+containing+bovine+plasma+after+vitrification.">Bovine oocytes in secondary follicles grow in medium containing bovine plasma after vitrification.</a> Journal of Reproduction and Development. 57 (1), 99-106 (2011).</li><li>Endo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estradiol+supports+in+vitro+development+of+bovine+early+antral+follicles.">Estradiol supports in vitro development of bovine early antral follicles.</a> Reproduction. 145 (1), 85-96 (2013).</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=Steroid+hormones+promote+bovine+oocyte+growth+and+connection+with+granulosa+cells.">Steroid hormones promote bovine oocyte growth and connection with granulosa cells.</a> Theriogenology. 82 (4), 605-612 (2014).</li><li>Yamamoto, 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=Development+to+live+young+from+bovine+small+oocytes+after+growth,+maturation+and+fertilization+in+vitro.">Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro.</a> Theriogenology. 52 (1), 81-89 (1999).</li><li>Alam, M. H., Lee, J., 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=Inhibition+of+PDE3A+sustains+meiotic+arrest+and+gap+junction+of+bovine+growing+oocytes+in+in+vitro+growth+culture.">Inhibition of PDE3A sustains meiotic arrest and gap junction of bovine growing oocytes in in vitro growth culture.</a> Theriogenology. 118, 110-118 (2018).</li><li>Lodde, V., Modina, S., Galbusera, C., Franciosi, F., Luciano, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+chromatin+remodeling+in+germinal+vesicle+bovine+oocytes:+interplay+with+gap+junction+functionality+and+developmental+competence.">Large-scale chromatin remodeling in germinal vesicle bovine oocytes: interplay with gap junction functionality and developmental competence.</a> Molecular Reproduction and Development. 74 (6), 740-749 (2007).</li><li>Lodde, V., 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=Oocyte+morphology+and+transcriptional+silencing+in+relation+to+chromatin+remodeling+during+the+final+phases+of+bovine+oocyte+growth.">Oocyte morphology and transcriptional silencing in relation to chromatin remodeling during the final phases of bovine oocyte growth.</a> Molecular Reproduction and Development. 75 (5), 915-924 (2008).</li><li>Dieci, C., 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=Differences+in+cumulus+cell+gene+expression+indicate+the+benefit+of+a+pre-maturation+step+to+improve+in-vitro+bovine+embryo+production.">Differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production.</a> Molecular Human Reproduction. 22 (12), 882-897 (2016).</li><li>Soares, A. C. S., 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=Steroid+hormones+interact+with+natriuretic+peptide+C+to+delay+nuclear+maturation,+to+maintain+oocyte-cumulus+communication+and+to+improve+the+quality+of+in+vitro-produced+embryos+in+cattle.">Steroid hormones interact with natriuretic peptide C to delay nuclear maturation, to maintain oocyte-cumulus communication and to improve the quality of in vitro-produced embryos in cattle.</a> Reproduction, Fertililty and Development. 29 (11), 2217-2224 (2017).</li><li>Soares, A. C. S., 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=Characterization+and+control+of+oocyte+large-scale+chromatin+configuration+in+different+cattle+breeds.">Characterization and control of oocyte large-scale chromatin configuration in different cattle breeds.</a> Theriogenology. 141, 146-152 (2020).</li><li>Franciosi, F., 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=Natriuretic+peptide+precursor+C+delays+meiotic+resumption+and+sustains+gap+junction-mediated+communication+in+bovine+cumulus-enclosed+oocytes.">Natriuretic peptide precursor C delays meiotic resumption and sustains gap junction-mediated communication in bovine cumulus-enclosed oocytes.</a> Biology of Reproduction. 91 (3), 61 (2014).</li><li>Luciano, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+different+levels+of+intracellular+cAMP+on+the+in+vitro+maturation+of+cattle+oocytes+and+their+subsequent+development+following+in+vitro+fertilization.">Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization.</a> Molecular Reproduction and Development. 54 (1), 86-91 (1999).</li><li>Bilodeau-Goeseels, S., Panich, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+oocyte+quality+on+development+and+transcriptional+activity+in+early+bovine+embryos.">Effects of oocyte quality on development and transcriptional activity in early bovine embryos.</a> Animal Reproduction Science. 71 (3-4), 143-155 (2002).</li><li>Dieci, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cilostamide+on+gap+junction+communication+dynamics,+chromatin+remodeling,+and+competence+acquisition+in+pig+oocytes+following+parthenogenetic+activation+and+nuclear+transfer.">The effect of cilostamide on gap junction communication dynamics, chromatin remodeling, and competence acquisition in pig oocytes following parthenogenetic activation and nuclear transfer.</a> Biology of Reproduction. 89 (3), 68 (2013).</li><li>Shu, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cilostamide+and+forskolin+on+the+meiotic+resumption+and+embryonic+development+of+immature+human+oocytes.">Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes.</a> Human Reproduction. 23 (3), 504-513 (2008).</li><li>Lodde, V., 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=Zinc+supports+transcription+and+improves+meiotic+competence+of+growing+bovine+oocytes.">Zinc supports transcription and improves meiotic competence of growing bovine oocytes.</a> Reproduction. 159 (6), 679-691 (2020).</li><li>Henderson, K. M., McNeilly, A. S., Swanston, I. 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>

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 border="0" cellpadding="0" cellspacing="0" style="width:635px;" width="633">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:215px;">4-well dishes</td>
			<td style="width:127px;">Nunclon</td>
			<td style="width:123px;">179830</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
			<td></td>
		</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 ...

In this video protocol, we demonstrate in detail, a culture system for growing bovine oocytes. The system supports the oocytes viability in culture for up to five days and allows for their differentiation. Using this culture system, oocytes collected from small antral follicles, which are normally not used in standard protocols for in vitro in the production, can be grown to increase the availability of fertilizable gametes.This exploitation of the wider reserve, can provide new options for the genetic salvage of threatened species, of the bovine family, or of local breeds at risk of genetic erosion. To begin, prepare 15 milliliters of basic culture medium and supplement it according to manuscript directions. Next, prepare three milliliters of holding medium by adding five micromolar cilostamide to the basic culture medium and pour it in a 35 millimeter Petri dish.Prepare long IVCO medium by supplementing basic culture medium according to manuscript directions. Add 200 microliters of long IVCO medium to each well of a 96-well coated plate. Then fill the four edges with sterile water to compensate for evaporation and to maintain appropriate humidity during culture.Incubate the 96-well plate and the holding medium at 38.5 degrees celsius and 5%carbon dioxide with maximum humidity. Wash the ovaries four times in sterile saline, maintained at 26 degrees celsius. Then aspirate all follicles that are more than two millimeter in diameter, with an 18 gauge needle connected to an aspiration pump.Lace the aspirated ovaries in a beaker with sterile saline maintained at 26 degrees celsius. Place one ovary at a time on a sterile PTFE cutting board, and use a surgical number 22 blade to cut slices of ovarian cortex that are 1.5 to two millimeters thick, and parallel to the major axis of the organ. Lace the slices of ovarian cortex in a sterile glass Petri dish, with dissecting medium on a warm plate, at 38.5 degrees celsius.Lace one ovarian cortex slice in a 60 millimeter glass Petri dish, with two to three milliliters of M199D under a dissection microscope, select the follicles that are between 5 and two millimeters. Identify the healthy non-atretic follicles under the stereo microscope, by observing morphological parameters, such as a uniformly bright translucent appearance, extensive vascularization, and a regular granulosa layer. While atretic follicles have a gray opaque appearance, and are poorly vascularized, with a dark COC inside.Discard the atretic follicles and process all the others. Use the surgical blade to remove the ovarian tissue surrounding the follicle on one side, until it is exposed. Then use a 26 gauge needle to carefully make a slit in the exposed follicle wall, which will release the follicular content comprising the COC, follicular fluid, and clumps of cells.Identify the COC and examine it for cumulus integrity, zona pellucida integrity and homogeneity of the cytoplasm. If these criteria are fulfilled, aspirate the COC with a P20 pipette. Lace the isolated COC in M199D cilostamide, and continue the isolation procedure for 30 minutes.After selecting COCs, as previously described, prepare 16 drops with 20 microliters of M199D cilostamide in a 60 millimeter Petri dish, and place one healthy COC into each drop. Use an inverted microscope attached to a camera to measure the oocyte diameter, excluding the zona pellucida. With a clear visualization of the oocyte, make two perpendicular measurements, assure that the mean of the two measurements is within a range of 100 to 110 micrometers.It can be difficult to precisely measure the oocytes diameter, due to the companion cumulus cells. COCs that have oocytes with smaller or larger diameter, or that don't have a rounded oocyte, or those with oocytes that are not measurable, should be discarded. Transfer the selected COCs into a 35 millimeter dish containing M199H medium, and keep them in the incubator at 38.5 degrees celsius and 5%carbon dioxide with maximum humidity, make sure that the overall working time does not exceed two hours.Once the selection and collection procedures are completed, transfer one COC into the center of each well of the previously prepared 96-well plate. Incubate the plate for five days at 38.5 degrees celsius and 5%carbon dioxide with maximum humidity, refresh the medium every other day. To renew the medium, replace 100 microliters of old medium with 100 microliters of fresh long IVCO medium, do this under the stereo microscope to avoid moving the COCs.At the end of the long IVCO, analyze the COCs morphology. If they have a compact cumulus cell investment with no sign of cumulus expansion, or cell degeneration, classify them as class one. If the COCs have a compact cumulus cell investment with no sign of cumulus expansion or cell degeneration, and with one or more antrum-like formations, classify them as class two.Class three COCs, show several layers of cumulus cells with no sign of cumulus expansion, some disaggregated cells in the outer layer of the cumulus cells and no antrum-like formation. Classify the COCs as class four if they show abundant loss of cumulus cells extending for more than 50%of the oocyte surface, as well as signs of cell degeneration and cell debris. At the end of long IVCO the gross morphology of the COC is changed.And four classes were identified based on the appearance of the cumulus cells. Overall, 74 oocytes in five biological replicates were analyzed, of which, 9.45%were discarded from further evaluation. Classes one, two, and three were judged healthy, while class four showed clear signs of degeneration, such as the absence of complete layers of cumulus cells surrounding the oocytes.These COCs were considered unsuitable to undergo downstream procedures in a prospective IVP setting. Assessment of the meiotic stage at the end of the long IVCO showed that a significantly higher percentage of the oocytes remained arrested at the immature stage, with the chromatin still enclosed within the GV.When chromatin condensation within the GV was investigated as a marker of gain of competence, the transition of the chromatin configuration to more condensed stages, namely GV two and GV three, was observed in 59%of the oocytes. A small percentage resumed meiosis reaching metaphase one stage, or degeneration.These results demonstrate that L-IVCO culture supports the oocyte viability, while preventing meiotic resumption for five days. At the end of the long IVCO, the COCs can be used in downstream procedures of in vitro embryo production, namely, in vitro maturation, fertilization, and embryo culture. These steps are necessary to provide proof of concept that the oocyte have acquired a higher developmental competence.Besides holding the potential of increasing the amount of fertilizable oocytes, the long IVCO culture system is a tool for scientists who are interested in dissecting the cellular and molecular processes that regulate the formation of a competent gamete.

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

Research

JoVE Journal

Developmental Biology

8.3K vistas.  University of Milan. En este protocolo de vídeo, demostramos en detalle un sistema de cultivo para el cultivo de ovocitos bovinos. El sistema soporta la viabilidad de los ovocitos en la cultura durante un máximo de cinco días y permite su diferenciación. Usando este sistema de cultivo, los ovocitos recogidos de pequeños folículos antrales, que normalmente no se utilizan en protocolos estándar para in vitro en la producción, pueden ser cultivados para aumentar la disponibilidad de gametos fertilizables.